Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Ringo, Herieth Heriel
Master of Science - MSc
University of British Columbia
Non-tuberculous mycobacteria (NTM) are comprised of more than 190 species, broadly characterized as slowly or rapidly growing mycobacteria (SGM/RGM). In the RGM group, Mycobacterium abscessus (MAB) is the leading cause of NTM pulmonary diseases among individuals with pre-existing lung diseases like cystic fibrosis. The mode of transmission is not fully understood. MAB is extremely resistant to antibiotics, which leads to a long and often unsuccessful treatment. Even worse, there are no FDA approved drugs against MAB. Hence, these challenges highlight the urgent need to develop effective MAB antibiotics. Here I describe a collaborative work involving phenotypic screening of natural extracts and pure compounds libraries against MAB in broth and in human macrophages. The libraries were Traditional Chinese Medicine (TCM), marine extracts (ME), pure marine compounds (PMC), and Global Health Priority Box (GHP). We identified seven pure active compounds in broth which displayed a dose-dependent mechanism of inhibition. Also, we found three intracellularly active fractions, which are currently undergoing bioassay guided fractionation to isolate pure active compounds. The seven active compounds in broth had cytotoxicity profiles with varying selectivity to MAB compared to HEK293T cells and three of these compounds indicated a selective growth inhibition against mycobacteria and Gram-positive bacteria. Further work needs to be done to improve the antimycobacterial activities and cytotoxicity profiles of these active compounds. The present study contributes to the ongoing drug discovery initiatives to reduce the burden of MAB infections among individuals most in need. Advanced studies of our active compounds have the potential to discover novel tools for the effective treatment against MAB infections.
IDENTIFICATION OF ACTIVE COMPOUNDS AGAINST MYCOBACTERIUM ABSCESSUS by Herieth Heriel Ringo B.Sc., Quest University Canada, 2018 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2024 © Herieth Heriel Ringo, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Identification of active compounds against Mycobacterium abscessus submitted by Herieth Heriel Ringo in partial fulfilment of the requirements for the degree of Master of Science in Microbiology and Immunology Examining Committee: Dr. Yossef Av-Gay, Professor, Microbiology and Immunology, UBC Supervisor Dr. Elitza I. Tocheva, Assistant Professor, Microbiology and Immunology, UBC Supervisory Committee Member Dr. Jim Sun, Assistant Professor, Microbiology and Immunology, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Lindsay Eltis, Professor, Microbiology and Immunology, UBC Supervisory Committee Member iii Abstract Non-tuberculous mycobacteria (NTM) are comprised of more than 190 species, broadly characterized as slowly or rapidly growing mycobacteria (SGM/RGM). In the RGM group, Mycobacterium abscessus (MAB) is the leading cause of NTM pulmonary diseases among individuals with pre-existing lung diseases like cystic fibrosis. The mode of transmission is not fully understood. MAB is extremely resistant to antibiotics, which leads to a long and often unsuccessful treatment. Even worse, there are no FDA approved drugs against MAB. Hence, these challenges highlight the urgent need to develop effective MAB antibiotics. Here I describe a collaborative work involving phenotypic screening of natural extracts and pure compounds libraries against MAB in broth and in human macrophages. The libraries were Traditional Chinese Medicine (TCM), marine extracts (ME), pure marine compounds (PMC), and Global Health Priority Box (GHP). We identified seven pure active compounds in broth which displayed a dose-dependent mechanism of inhibition. Also, we found three intracellularly active fractions, which are currently undergoing bioassay guided fractionation to isolate pure active compounds. The seven active compounds in broth had cytotoxicity profiles with varying selectivity to MAB compared to HEK293T cells and three of these compounds indicated a selective growth inhibition against mycobacteria and Gram-positive bacteria. Further work needs to be done to improve the antimycobacterial activities and cytotoxicity profiles of these active compounds. The present study contributes to the ongoing drug discovery initiatives to reduce the burden of MAB infections among individuals most in need. Advanced studies of our active compounds have the potential to discover novel tools for the effective treatment against MAB infections. iv Lay Summary Non-tuberculous mycobacteria (NTM) cause serious human diseases especially among individuals with pre-existing lung diseases such as cystic fibrosis. NTM can be grouped as slow or fast growing and within the latter group, Mycobacterium abscessus (MAB) is the leading cause of lung diseases. MAB transmission in humans is unclear. This pathogen is highly resistant to antibiotics, which makes treatment long and often unable to clear the infection. Currently, there are no FDA approved drugs for MAB treatment. Ongoing efforts seek to discover new and effective antibiotics against MAB. We tested natural products and pure compounds libraries including Traditional Chinese Medicine (TCM), marine extracts (ME), and pure marine compounds (PMC) and Global Health Priority (GHP) box. This thesis reports seven active compounds with promising inhibition against MAB. These compounds have the potential to be developed as new therapeutics to minimize the burden of infections caused by MAB within nations and worldwide. v Preface This thesis delivers work that I both performed on my own and collaborated with others under the supervision of Dr. Yossef Av-Gay (Principal Investigator) and Dr. Henok Sahile (Postdoctoral Fellow). I planned, conducted, and analyzed data from all experiments that utilized TCM and GHP box libraries. Dr. Sahile identified active marine extracts against MAB in broth from the 400 natural products of the marine extract (ME) library and collaborated with the Andersen Lab for bioassay guided fractionation. David Williams, from the Andersen Lab, fractionated the active marine extracts to obtain marine fractions. I joined the Av-Gay Lab at this point and continued this project by screening marine fractions. David isolated the active pure marine compounds and determined their chemical structures. I planned, conducted, and analyzed data from all the marine fractions and pure active compounds’ experiments which I worked on. Further, I supervised Trushaan Bhundoo, who was a senior undergraduate student undertaking directed studies in the Av-Gay Lab. We planned, conducted, and analyzed data from the 251 pure compounds (PMC) of the marine extract library. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Figures .................................................................................................................................x List of Abbreviations .................................................................................................................... xi Acknowledgements .................................................................................................................... xiii Chapter One ...................................................................................................................................1 1. Introduction ................................................................................................................................1 1.1 Mycobacterium abscessus ......................................................................................................1 1.1.1 MAB Pathogenesis......................................................................................................... 3 1.1.2 Current MAB treatment and drug resistance ................................................................. 6 1.1.3 MAB drug discovery...................................................................................................... 8 1.2 Natural products .....................................................................................................................9 1.3 Phenotypic screening of compound libraries .......................................................................10 1.4 Rationale 1 ........................................................................................................................... 11 1.4.1 Aim 1 ............................................................................................................................ 12 1.5 Rationale 2 ...........................................................................................................................12 1.5.1 Aim 2 ............................................................................................................................ 13 Chapter Two .................................................................................................................................14 2. Materials and methods ............................................................................................................14 2.1 Natural product libraries ......................................................................................................14 2.1.1 Natural products from a library of Traditional Chinese Medicine extracts ................. 14 2.1.2 Natural products from a library of marine extracts ...................................................... 16 2.1.2.1 Preparation of marine extracts, bioassay guided fractionation, and isolation of pure active compounds against MAB ............................................................................... 16 2.2 Pure compounds from Global Health Priority Box Library.................................................18 2.3 Bacteria and host cells .........................................................................................................25 2.3.1 MAB cells and culture media ...................................................................................... 25 2.3.2 THP-1 cells and culture media ..................................................................................... 25 2.3.3 RAW 264.7 cells and culture media ............................................................................. 26 2.3.4 HEK293T cells and culture media ............................................................................... 26 vii 2.4 Single concentration activity determination of libraries against MAB in broth ..................27 2.5 THP-1 infection model ........................................................................................................27 2.6 Minimum inhibitory concentration determination assay .....................................................29 2.6.1 Minimum inhibitory concentration of active compounds in broth .............................. 29 2.6.2 Minimum inhibitory concentration of active compounds intracellularly .................... 29 2.7 Cytotoxicity assay using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) ........................................................................................................................................32 2.8 Antibacterial activity determination of the active compounds ............................................32 Chapter Three ..............................................................................................................................34 3. Results .......................................................................................................................................34 3.1 Single concentration activity determination in broth ...........................................................34 3.1.1 Seven compounds were active against MAB in broth ................................................. 34 3.1.1.1 TCM Library ......................................................................................................... 36 3.1.1.2 ME Library............................................................................................................ 36 3.1.1.3 PMC Library ......................................................................................................... 37 3.1.1.4 GHP Box ............................................................................................................... 37 3.1.2 Compounds 1 - 7 displayed a dose dependent antimycobacterial activity against MAB in broth .................................................................................................................................. 39 3.1.2.1 Cytotoxicity profiles of active compounds indicated varying selectivity ............. 39 3.1.2.2 Compounds 5, 6, and 7 exhibited antibacterial selectivity against mycobacteria and Gram-positive bacteria in broth ................................................................................. 43 3.2 Single concentration activity determination using the THP-1 infection model ...................44 3.2.1 TCM Fractions 53, 56, and 73 were active against MAB intracellularly .................... 44 3.2.1.1 TCM Library ......................................................................................................... 45 3.2.1.2 ME Library............................................................................................................ 48 3.2.1.3 PMC Library ......................................................................................................... 48 3.2.1.4 GHP Box ............................................................................................................... 48 Chapter Four ................................................................................................................................50 4.1 Discussion................................................................................................................................50 4.1.1 Compounds 1 – 7 were active and displayed a dose dependent activity against MAB in broth ...................................................................................................................................... 50 4.1.1.1 Cytotoxicity profiles of active compounds indicated varying selectivity ............. 53 4.1.1.2 Compounds 5, 6, and 7 exhibited antibacterial selectivity against mycobacteria and Gram-positive bacteria in broth ................................................................................. 54 4.1.2 TCM Fractions 53, 56, and 73 were active against MAB intracellularly .................... 56 4.2 Conclusion ..............................................................................................................................57 viii 4.3 Significance .............................................................................................................................58 4.4 Limitations ..............................................................................................................................58 4.5 Future directions ....................................................................................................................59 References .....................................................................................................................................61 ix List of Tables Table 1. Common NTM species causing NTM-PD globally. ......................................................... 2 Table 2. MAB treatment recommendations according to the 2020 ATS/ERS/ESCMID guidelines.......................................................................................................................................................... 6 Table 3. List of TCM library used for phenotypic screening. ....................................................... 15 Table 4. List of ME and PMC libraries used for phenotypic screening. ....................................... 17 Table 5. List GHP library used for phenotypic screening. ............................................................ 19 Table 6. Bacterial species that were used to determine antibacterial activity of active compounds........................................................................................................................................................ 33 Table 7. Distribution of the percentages of growth inhibition from TCM, ME, PMC, and GHP against MAB growth in broth ....................................................................................................... 36 Table 8. Antimycobacterial and cytotoxicity activities from compounds 1 - 7 against THP-1, RAW 264.7, and HEK293T cells. ........................................................................................................... 40 Table 9. Antibacterial activities of active compounds against mycobacteria, Gram-positive, and Gram-negative bacterial species. .................................................................................................. 44 Table 10. Distribution of percentages of THP-1 cell survival from TCM, PMC, and GHP against MAB growth intracellularly. ......................................................................................................... 45 Table 11. Percentages of THP-1 cell survival and MAB growth inhibition of 21 intracellularly active TCM extracts. ..................................................................................................................... 47 x List of Figures Figure 1. MAB cell envelope assembly .......................................................................................... 5 Figure 2. Broth microdilution method .......................................................................................... 31 Figure 3. Phenotypic screening of natural extracts and pure compounds libraries against MAB in broth .............................................................................................................................................. 35 Figure 4. Chemical structures of the seven active compounds in broth ....................................... 38 Figure 5. Dose response curves and MIC90 determination of compounds 1 - 7 against MAB in broth....................................................................................................................................................... 41 Figure 6. Dose-dependent cytotoxic concentrations of active compounds against HEK293T cells....................................................................................................................................................... 42 Figure 7. Phenotypic screening of natural extracts and pure compounds libraries against MAB intracellularly ................................................................................................................................ 46 Figure 8. Phenotypic screening of 80 TCM fractions against MAB intracellularly ..................... 49 xi List of Abbreviations ADS Albumin, Dextrose, Sodium Chloride ATS American Thoracic Society BCG Mycobacterium bovis Bacillus Calmette-Guérin BDQ Bedaquiline CF Cystic Fibrosis CFU Colony Forming Units CLSI Clinical and Laboratory Standards Institute DMEM Dulbecco’s Modified Eagles Medium DMSO Dimethyl Sulfoxide ERS European Respiratory Society ESCMID European Society of Clinical Microbiology and Infectious Diseases FDA Food and Drug Administration GHP Global Health Priority Box GPL Glycopeptidolipids HEK Human Embryonic Kidney Cells CC Cytotoxic Concentration MAB Mycobacterium abscessus MAC Mycobacterium avium complex ME Marine Extracts MIC Minimum Inhibitory Concentration MmpS/L Mycobacteria membrane protein Small/Large MMV Medicines for Malaria Venture xii MOM Mycolic acid Outer Membrane Mtb Mycobacterium tuberculosis MTT 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide NP Natural Products NTM Non-tuberculous Mycobacteria OD Optical Density PBS Phosphate-buffered saline PIM Phosphatidyl-myo-inositol dimannoside PMA Phorbol 12-myristate-13-acetate PMC Pure Marine Compounds PD Pulmonary Diseases RAW Murine macrophage cell line RFP Red Fluorescence Protein RGM Rapidly Growing Mycobacteria SGM Slowly Growing Mycobacteria SI Selectivity Index TCM Traditional Chinese Medicine THP Human monocytic cell line TMM Trehalose monomycolate TDM Trehalose-6,6-dimycolate TPP Trehalose polypheleates xiii Acknowledgements I am incredibly grateful to my supervisor Dr. Yossef Av-Gay for his mentorship and guidance throughout my graduate studies. Dr. Av-Gay has been instrumental in ensuring that I received the support that I needed as a trainee to foster the necessary skills, experience, and knowledge. Also, I would like to extend my sincere gratitude to my graduate thesis committee supervisors, Dr. Elitza Tocheva and Dr. Lindsay Eltis for their support, feedback, and encouragement during my studies. I would like to thank the past and present members of the Av-Gay Lab for helping me to integrate into the lab easily, assisting me to find the resources I needed, and providing me with feedback pertaining to my work. I appreciate your contribution in making my time in the lab memorable and impactful. I am especially thankful for the support from Dr. Henok Sahile, Dr. Tirosh Shapira, Mary Ko, Joseph Chao, Trushaan Bhundoo, Virginia Pichler, Leah Rankine-Wilson, Asana Imanpour, Matthew Christopherson, Maddy Riddler, and Xingji Zheng. I had the opportunity to collaborate with research groups at the University of British Columbia (UBC) and internationally. In the Department of Chemistry at UBC, I am very grateful to Dr. Raymond Andersen for his collaboration and provision of natural products from their marine extract library. Also, my sincere appreciation goes to David William (Andersen Lab) who prepared the marine samples for our phenotypic screening assay and collaborated with us in performing bioassay guided fractionation to isolate the active pure marine compounds. Moreover, I would like to acknowledge Angal Biotechnology (Suzhou) Ltd. for providing us with the Traditional Chinese Medicine (TCM) extracts. We utilized TCM extracts for our phenotypic screening assay and collaborated with the company in performing bioassay guided fraction that yielded active fractions xiv for further fractionation. Also, I would like to extend my appreciation to Medicines for Malaria Venture who supplied us with pure compounds from the Global Health Priority Box. I am extremely grateful to the government of British Columbia, Canada for financial support through a scholarship award offered during my graduate studies at UBC. I would like to extend my heartfelt appreciation to my family, particularly my loving parents, Mr & Mrs. Heriel Ringo, sister, Happiness Ringo, and brother, Harryson Ringo for their encouragement and prayers throughout my master’s program. Thank you for always believing in me and inspiring me to aim higher. Also, I am thankful for the care and support that I received during my graduate studies from my close friends who have become like family in Squamish and Vancouver, Canada. Above all, I give God glory for the work that I have accomplished throughout my graduate studies at UBC. 1 Chapter One 1. Introduction Non-tuberculous mycobacteria (NTM) are microorganisms also referred to as atypical mycobacteria or mycobacteria other than Mycobacterium tuberculosis.1,2 NTMs are environmental microorganisms which were first found to contribute to human infections in the mid-twentieth century.1,3,4 Pulmonary, lymphatic, and skin diseases are common among NTM patients, but pulmonary diseases (PD) are the most prevalent.5–7 For instance, in North America3, Europe6, and Australia6 the number of NTM-PD cases in the early 2000s were more than double those in the late twentieth century. A rise in NTM-PD incidence and prevalence have been attributed to improved life expectancy, frequent use of immunosuppressants, increased knowledge about NTM diseases, and better diagnostic tools.8–10 Over 190 NTM species have been characterized and are broadly classified as slowly or rapidly growing mycobacteria (SGM/RGM).5,11 SGM involves species that take more than seven days to grow visible colonies on solid media, while RGM species grow in less than a week.5 Important species causing NTM-PD globally include both SGM and RGM (Table 1).4,11 The work presented hereafter is focused on Mycobacterium abscessus (MAB), an emerging RGM that currently creates a burden in the medical field and demands effective interventions. 1.1 Mycobacterium abscessus In the mid-twentieth century, the first case of a Mycobacterium abscessus infection was reported from a patient suffering from a knee injury.12,13 MAB used to be classified as a subspecies of Mycobacterium chelonae.13,14 In 1992, this assignment was changed due to advancements in genetic techniques and MAB was thereafter recognized as its own species.13,14 2 Table 1. Common NTM species causing NTM-PD globally. Non-tuberculous mycobacteria 1. Slowly growing mycobacteria A. Mycobacterium avium complex (indicating opportunistic species only) i. Mycobacterium avium ii. Mycobacterium intracellulare iii. Mycobacterium chimaera B. Mycobacterium kansasii C. Mycobacterium xenopi D. Mycobacterium malmoense 2. Rapidly growing mycobacteria A. Mycobacterium abscessus i. Mycobacterium abscessus subspecies abscessus ii. Mycobacterium abscessus subspecies bolletii iii. Mycobacterium abscessus subspecies massiliense B. Mycobacterium fortuitum The table summarizes NTM species that are reported worldwide to cause NTM-PD. The details presented here were adapted from Honda et al., (2018)4 and Johansen et al., (2020)11. In the RGM group, MAB is most frequently associated with NTM-PD especially among individuals with pre-existing respiratory diseases including cystic fibrosis (CF) and bronchiectasis.15,16 A study conducted recently analyzed 3,771 NTM pulmonary CF patient records in the United States from 2010 - 2019 and found that over a quarter of the cases were caused by MAB.17 MAB is comprised of three subspecies, Mycobacterium abscessus subspecies abscessus (M. abscessus), Mycobacterium abscessus subspecies massiliense (M. massiliense), and Mycobacterium abscessus subspecies bolletii (M. bolletii).11,16 A research group analyzed 6,295 CF patient data in Germany from 2015 - 2020 whereby a third of these patients underwent NTM diagnosis and M. abscessus was the most isolated species.18 M. massiliense subspecies are known to cause infections among patients recovering from medical operations13 and M. bolletii is the least common subspecies isolated from patients.15 For example, of the 220 MAB isolates collected in 3 2012 – 2017 from patients in Japan and Taiwan, 50.9 %, 47.7 %, and 1.4 % were identified as M. abscessus, M. massiliense, and M. bolletii, respectively.19 MAB has been found in soil, water supply and reservoirs, medical tools, fomites, and domestic animals.16,20,21 It’s even been shown to possess symbiotic life in amoeba.22,23 Human interaction with environments that are colonized with this species is thought to be a plausible source of infections.11. However, the mechanism by which MAB infections spread is still unclear. Research has so far presented inconsistent findings with regards to human to human transmission.24 One study performed whole genome sequencing (WGS) using 70 clinical samples from CF and non-CF patients in Ireland but did not observe similarities within the sequenced clinical strains to suggest transmission between patients.25 Likewise, another study performed WGS using 145 clinical samples from CF patients in Spain and UK but did not find evidence of MAB spreading from one patient to another.26 However, a different study that applied WGS using 104 clinical specimens from non-CF patients in Japan reported that the tested samples were genetically comparable and indicative of a possible patient to patient transmission.27 Even though findings from one study to another may be contradictory, research groups acknowledge that there is a need to conduct further research to understand the mode of transmission of MAB infections. 1.1.1 MAB Pathogenesis MAB possesses an intricate cell envelope including an inner membrane, peptidoglycan and arabinogalactan layers, and a mycolic acid outer membrane (MOM) (Figure 1).11,28 The cell envelope harbors a myriad of lipids and glycolipids that form distinct phenotypes which determine the course of MAB infections. MAB morphology is categorized into two variants, smooth (S) and rough (R) phenotypes, both of clinical significance.11,28,29 The S variant is identified by high levels of glycopeptidolipids (GPLs) while the R variant is known to lack GPLs.11,21 GPLs are unique to 4 NTMs especially RGM, both pathogenic and non-pathogenic strains.30,31 The structure of GPLs include a peptide base with acyl groups added by 3-hydroxy or 3-methoxyl-fatty acids (C26-33) and further addition of sugar molecules from O-methylated and O-acetylated deoxy hexoses.30,31 The S strain is often detected from wounds and environmental samples while the R strain is frequently observed among patients with severe MAB pulmonary disease (MAB-PD).14,32 These morphotypes behave differently when inside a host, where the S strain is reported to be less virulent than its R counterpart.11,28 It is hypothesized that the S variant invades the lungs first and then, spontaneous mycobacterial mutations that decrease the level of GPLs facilitate the transition to a virulent R strain resulting in MAB-PD.14,32 Alveolar macrophages serve as the host first line protective immune cells that engulf invading pathogen.14,33 GPLs are thought to benefit the S strain by shielding the underlying immune-detectable lipids such as phosphatidyl-myo-inositol dimannoside (PIM) and trehalose-6,6-dimycolate (TDM).14,21 PIM contain phospholipids, mannose (1-6 molecules), and acyl groups and these components have been reported to incite primary immunity within host macrophages.32,34,35 TDM is also known as a cord factor, highlighting its virulent role in mycobacterial pathogenesis.36,37 Spontaneous mutations on the gpl site hamper the production and transport of GPLs and likely promote the switch from an S to R phenotype.16,21,28 For example, modifications at Tyr842 and Tyr854 of the mycobacterial membrane protein large (MmpL) genes, mmpl4a and mmpl4b, respectively disrupt GPLs transport.11,21 A reduced level of GPLs on the MOM allows PIMs to interact with toll-like receptor 2 (TLR-2) which in turn facilitates inflammation through the release of potent cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-8 (IL-8).14,16,28 Inflammation also causes the establishment of granuloma, which promotes the survival of the R strain.16,29 As opposed to the S variant, the R variant readily allows 5 for phagosome maturation and progression to apoptosis.11,14 Apoptosis then becomes an escape pathway for MAB dissemination, multiplication and establishment of networks of complex serpentine-like cords that damage host tissues.11,14 Lipids such as trehalose polyphleates (TPP) are reported to facilitate bacterial clumping, which hinders proper bacterial degradation within macrophages, leaving room for bacterial spread that contributes to the virulent cording feature of R strains.38 Figure 1. MAB cell envelope assembly. Cytoplasmic membrane anchors mycobacterial membrane protein small/large (MmpS/L) for antibiotics and glycopeptidolipids (GPLs) transfer. Also, bacterial secretion systems (ESX-4 and ESX-3) facilitate the release of effector proteins. The peptidoglycan layer supports the structure of a glycolipid lipoarabinomannan (LAM) that extends through the arabinogalactan layer to the MOM. MOM contains various lipids, glycolipids, and lipoproteins that promote MAB virulence. For example, phosphatidyl-myo-inositol dimannoside (PIM), trehalose polyphleates (TPP), trehalose-6,6-dimycolate (TDM) and trehalose monomycolate (TMM). Image was adapted from Parmar and Tocheva (2023).28 6 1.1.2 Current MAB treatment and drug resistance To date, there aren’t any specific and effective MAB antimicrobials approved by the Food and Drug Administration (FDA).15 Treatment regimens used by health professionals vary but the duration of these therapies remains long (~2 years).15,39 Common antibiotic classes prescribed to patients include macrolides, aminoglycosides, beta lactams, and tetracyclines (Table 2).39,40 Table 2. MAB treatment recommendations according to the 2020 ATS/ERS/ESCMID guidelines. Drug Antibiotic class Target 1. Early-stage treatment (~3 months) Oral macrolide with intravenous amikacin, imipenem and tigecycline Azithromycin or clarithromycin Macrolide Protein synthesis on 23S ribosomal RNA Amikacin Aminoglycoside Protein synthesis on 16S ribosomal RNA Imipenem Beta-lactam Peptidoglycan and penicillin-binding protein synthesis Tigecycline Tetracycline Aminoacyl-transfer RNA binding to the ribosomes 2. Follow-up treatment (~14 months) Oral macrolide and nebulized amikacin with one to three oral antibiotics Azithromycin or clarithromycin Macrolide Protein synthesis on 23S ribosomal RNA Nebulized amikacin Aminoglycoside Protein synthesis on 16S ribosomal RNA Linezolid Oxazolidinone Protein synthesis on 50S subunit Clofazimine Riminophenazine Reactive oxygen species Minocycline Tetracycline Aminoacyl-transfer RNA binding to the ribosomes Moxifloxacin Fluoroquinolone DNA gyrase for maintaining DNA supercoiling List of drugs as recommended in the 2020 ATS/ERS/ESCMID guidelines for early-stage and follow-up treatment of MAB-PD. Details in this table were adapted from Daley et al., (2020)41, Johansen et al., (2020)11, Quang and Jang (2021)15. 7 Patients undergo multidrug treatment and the choice of antibiotics to use is individually determined by drug susceptibility testing.42,43 In 2020, the American Thoracic Society (ATS), European Respiratory Society (ERS), and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) revised the NTM-PD treatment guidelines.41 Currently, in Canada, MAB-PD treatment guidelines are adapted from the 2020 ATS/ERS/ESCMID guidelines. The guidelines are categorized into initial and then continuation phases (Table 2).5,20 Early-stage treatment involves three months of oral macrolides such as azithromycin and clarithromycin with intravenous drugs including amikacin, tigecycline and imipenem.42,44 Follow-up treatment can take up to 14 months of oral macrolides, nebulized amikacin, and oral antibiotics including clofazimine, minocycline and moxifloxacin.6,44 However, treatment outcome for MAB-PD is relatively unimpressive with only ~40 - 50 % of patients managing to clear the infection and avoid relapsing.11,42 This challenge is mostly due to MAB’s extreme resistance to antibiotics.39,43 MAB’s inner membrane components such as efflux pumps often block the entrance of antibiotics into the bacteria.28,45 MmpL group is an example of efflux pumps used by the bacteria to shuttle molecules across the cell envelope to the outer space.11,28 A previous study reported that genetic changes in an important MAB transcriptional regulator (TetR) triggered overexpression of mmpS and mmpL, which led to clofazimine resistance.46 Further, drug-inactivating enzymes such as beta-lactamase render MAB’s resistance specifically to beta-lactams.16,21 For instance, gradual chemical modification of beta-lactams such as imipenem and cefoxitin via MAB class A beta-lactamase (BlaMAB) reduces the drug potency and promotes resistance.16,21 Modification of drug targets is another mechanism that contributes to MAB antibiotic resistance. MAB treatment regimen relies heavily on the use of macrolides due to their potency.5,15 However, this class of antibiotics can trigger the production of an enzyme that 8 modifies the target and stop the drug from binding.5,20 Erythromycin ribosomal methyltransferase (erm) is expressed upon the use of macrolides and it ushers the addition of a methyl group to 23S ribosomal RNA (rRNA), which reduces drug affinity.5,20 Resistance to macrolides is very common among clinical isolates, which is likely due to the presence of an erm encoding gene in two MAB subspecies (M. abscessus and M. bolletii).16,47 Overall, MAB drug resistance mechanisms need to be studied further to enhance treatment outcomes and shed light on drug targets that could be exploited in the development of effective therapies. 1.1.3 MAB drug discovery Effective drugs need to be discovered to address MAB antibiotic resistance and improve treatment outcome. After screening compound libraries against a whole cell or specific target, compounds that display the projected outcome, such as inhibiting a certain level of bacteria growth are commonly referred to as potential hits.48,49 Hits undergo validation studies and therapeutically relevant tests, which are aimed to verify compounds with pharmacological potential, then referred to as leads.48,49 Preclinical research has so far identified some hits against MAB including epetraborole (small-molecule inhibitor) and a natural product, etamycin (marine actinomycete-derived15), both found to block bacterial protein synthesis in a zebrafish model.40,44 Also, cyclipostins and cyclophostin analogs (natural products derived from Streptomyces sp.50) inhibited MAB growth and their mechanism of action was linked to cell wall synthesis.5,11 In a severe combined immunodeficiency disease (SCID) mouse model, benzimidazole SPR719 (synthetic inhibitor51) showed inhibitory activity against M. bolletii.11,15 Further, MAB drug discovery use lead compounds that are active against the Mycobacterium genus including Mycobacterium tuberculosis (Mtb), as shared traits increase the likelihood of cross-activity.40,44,45 For example, 9 Piperidinol (PIPD1) and indole-2-carboxamides, which disrupt lipid transport through the MmpL3 complex, were reported to inhibit MAB growth in zebrafish and mouse models.5,11 Also, two novel oxazolidinones (LCB01-0371 (delpazolid) and tedizolid) against Mtb have been reported to inhibit MAB growth by hindering protein synthesis.15,45 In clinical studies, there are even fewer lead compounds against MAB growth than those in preclinical stages.40,45 Drugs that have progressed to the end of clinical phase II trials include tigecycline (2010)52, nitric oxide (2019)53, and liposomal amikacin for inhalation (LAI) (2019).54 However, from these studies, little progress has been made and therefore highlighting the continual need to develop effective therapies against MAB infections. 1.2 Natural products Natural products (NPs) such as plants, fungi, microbes, and animal isolates have provided essential resources in the discovery of potent drugs applied in the medical field today, for instance antibiotics (~75 % were developed from NPs).55–57 For example, aspirin also known as acetylsalicylic acid, which is currently used as a pain reliever and an anti-inflammatory, was derived from salicin, a product extracted from a willow bark tree.55,58 Another well-known NP discovery was made serendipitously by Dr. Alexander Fleming, who from a fungus Penicillium notatum, identified penicillin, an important antibiotic currently used against Gram-positive and Gram-negative bacteria.58,59 Also, artemisinin, a traditional Chinese medicine extracted from Artemisia annua plant was found to exhibit anti-malarial properties.55,58 Artemisinin is now used worldwide to combat multidrug-resistant malaria caused by Plasmodium falciparum.55,58,60 The ability of NPs to modify cellular activities related to immune response and pathogenesis has been attributed to nature’s way of generating effective and chemically rich molecules.44,61 10 The use of NPs to identify active compounds against MAB is advantageous in accelerating MAB drug discovery.15,20 NPs from various sources including marine and land have been exploited where marine nature reserves many of the products yet to be discovered.62 The presence of novel chemicals with unique structures in NPs have led to the isolation of active compounds.61,63,64 For example, marine red algae Laurencia johnstonii was utilized by a research group that identified laurinterol, an active natural product against MAB from patient samples.57 The antimycobacterial activity of laurinterol was found to surpass that of imipenem, an antibiotic that is currently used to treat MAB infections.57 Another study reported the identification of two active compounds from testing 500 NPs (fermented fungi and bacteria) for their activity against antibiotic-resistant MAB strains.44 Antimicrobial activity against clarithromycin resistant MAB patient samples was also shown in two compounds derived from Thai medicinal plants and fungi.65 Along with the benefits of utilizing NPs, challenges are also observed. Limited supply of crude extracts, difficulties isolating active compounds, and roadblocks in seeking intellectual property remain among many challenges associated with the use of NPs.61,66 However, an increase in technological innovations play a vital role in overcoming some of these challenges. For example, improved techniques of analyzing NPs data with high accuracy and sensitivity, which ultimately consumes less time and raw materials.61 1.3 Phenotypic screening of compound libraries Drug discovery can be conducted through two main methods, namely phenotypic or target-based screening. Phenotypic screening pertains to testing compound libraries to identify active compounds whose use results in the predicted outcome, for example; growth inhibition of whole-cell microorganisms such as bacterial cells.61,67 Target-based screening, as its name suggests, is a method that relies on testing compound libraries to identify active compounds against a specific drug target.67 These two methods have been supposed to work in a “complementary” fashion, 11 however each has its own benefits and drawbacks.61,68 Phenotypic screening is commonly used in testing NPs61 and one of the benefits of applying this method is its potential to identify active compounds acting on novel host/pathogen targets or mechanisms.69,70 Also, since this approach uses whole-cell microorganisms, there is a greater chance to identify active compounds that can readily be taken up by cells through travelling across the cell envelope.68,71 However, the challenge with using phenotypic screening assays is often the lack of knowledge about the drug target and how the drug produces an effect in the host/pathogen.68,69 In target-based screening, the target is known prior to testing compound libraries and therefore, this method avoids the hurdles of drug target identification. Nonetheless, the outcome from this approach tends to translate poorly in whole-cell microorganisms due to issues such as membrane impermeability, toxicity, and decreased target binding capacity.67,69 Phenotypic screening assays are essential in the current search for novel MAB therapeutics since there are only a few targets known to date. For example, in the past decade, research has managed to uncover ~5 targets associated with membrane proteins, DNA maintenance, and transcription enzymes.67 Phenotypic screening of large libraries has the potential to accelerate the search for effective MAB therapeutics and also to discover new drug targets. 1.4 Rationale 1 The current work applied phenotypic screening to test the inhibitory activities of natural extracts (Traditional Chinese Medicine and marine extracts) and pure compounds (pure marine compounds and Global Health Priority Box) libraries against MAB. Traditional Chinese Medicine is a broad term for traditional medicinal practices (herbal medicine, acupuncture, and body exercises) done in China and other eastern and south-eastern Asian countries.72–74 However, Traditional Chinese herbal medicine (TCM) use is the most fundamental and has been applied for many centuries.72,75 Due to their ongoing consumption by humans, TCM could be a potential 12 resource for the search of antimycobacterial drugs. Marine extracts and pure marine compounds libraries were obtained from a research group with a wealth of experience in marine research work, involving the characterization of marine products collected for more than four decades.56 Marine nature continues to be a rich source of extracts with diverse chemical composition to test against mycobacterial species. Global Health Priority Box library contained pure compounds with varying inhibitory activities including antitubercular, antimalarial, and antiparasitic. Since these pure compounds were previously found to be active against other pathogens, they could potentially display growth inhibition against MAB. Phenotypic screening of our libraries against MAB in broth and intracellularly allows for the identification of active compounds that inhibit growth directly against the bacteria or indirectly by acting on the MAB-infected human macrophages. It is necessary to conduct both assays since MAB can cause both intracellular and extracellular infections. Macrophages are host immune cells that act as a first line of defense against pathogens and therefore, the use of MAB-infected macrophages has been relevant in testing for intracellular inhibitory activity of libraries.39 We hypothesized that phenotypic screening of natural extracts and pure compounds libraries will result in the identification of active compounds against MAB in broth and in human macrophages, hereafter described as “intracellularly”. 1.4.1 Aim 1 Identify compounds that are active against MAB in broth and in human macrophages through phenotypic screening of natural extracts and pure compounds libraries. 1.5 Rationale 2 After identifying active compounds, further studies are often conducted to validate the hits, for example minimum inhibitory concentration (MIC), cytotoxicity, and antibacterial selectivity.56 13 MIC90 is the lowest concentration of active compounds required to inhibit 90 % of the growth of the pathogen, such as bacteria. The lower the MIC the more potent the active compounds are against the bacteria. Cytotoxicity assays are performed to determine the active compounds’ concentration ratio of host cell death to bacterial inhibitory activity, also known as the selectivity index (SI). The higher the SI of active compounds, the more selective the compounds are to the bacteria than to host cells, which is a desirable outcome in drug discovery. Further, active compounds can display selective inhibitory activities against specific bacteria, which is also a suitable outcome when searching for novel molecules. 1.5.1 Aim 2 Conduct validation assays of the identified active compounds including determination of MIC90 and cytotoxicity, as well as antibacterial activity against Mycobacterium smegmatis, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Salmonella enterica serovar Typhimurium. 14 Chapter Two 2. Materials and methods 2.1 Natural product libraries 2.1.1 Natural products from a library of Traditional Chinese Medicine extracts TCM library was supplied by our collaborator, Angal Biotechnology (Suzhou) Ltd. and it comprised of a total 356 extracts and 38 pure compounds (we received the pure compounds in two separate batches) (Table 3). We first received 356 extracts together with 15 pure TCM compounds, which we used in our phenotypic screening assay against MAB. Active TCM extracts underwent bioassay guided fractionation done by our collaborator and we received 80 TCM fractions for further testing against MAB. Alongside these fractions, our collaborators sent 23 pure TCM compounds for phenotypic screening against MAB. TCM library was received in a lyophilized form and in preparation for screening, dimethyl sulfoxide (DMSO) was used as a solvent. 15 Table 3. List of TCM library used for phenotypic screening. Receiving date TCM TCM ID Stock (mg/mL) June 24, 2022 Extracts 1-356 10 Pure compound KJH-1 10 Pure compound KJH-2 10 Pure compound KJH-3 10 Pure compound KJH-4 10 Pure compound ZQX-92-5 10 Pure compound ZQX-92-8 10 Pure compound ZQX-99-7 10 Pure compound ZQX-99-27 10 Pure compound ZQX-99-28 10 Pure compound ZQX-188-14 10 Pure compound ZQX-196-2 10 Pure compound ZQX-196-4 10 Pure compound ZQX-199-12 10 Pure compound ZQX-199-13 10 Pure compound 357 10 September 25, 2023 Fractions Fraction 1-80 10 Pure compound Quercetin 10 Pure compound Methylnissolin 10 Pure compound Isoastragaloside II 10 Pure compound calycosin-7-O-β-D-glucoside 10 Pure compound Ononin 10 Pure compound Hyperoside 10 Pure compound Formononetin 10 Pure compound Rutin 10 Pure compound Apigenin 10 Pure compound Kaempferol 10 Pure compound Isorhamnetin-3-O-neohespeidoside 10 Pure compound Methylnissolin 10 Pure compound Isorhamnetin-3-glucoside 10 Pure compound Hesperetin 10 Pure compound Astragaloside II 10 Pure compound Soyasaponin II 10 Pure compound Calycosin 10 Pure compound Astragaloside A 10 Pure compound Astragalin 10 Pure compound Diosmetin 10 Pure compound Baicalein 10 Pure compound Chlorogenic acid 10 Pure compound Hesperidin 10 16 2.1.2 Natural products from a library of marine extracts 2.1.2.1 Preparation of marine extracts, bioassay guided fractionation, and isolation of pure active compounds against MAB Marine extracts library was supplied by our collaborator, Dr. Raymond Andersen Laboratory at the University of British Columbia (UBC) and it comprised of a total of 400 marine extracts (ME) and 251 pure marine compounds (PMC) (Table 4). Samples of marine invertebrates including sponges, soft corals, tunicates, and nudibranchs were harvested by hand from Indo-pacific and the Caribbean oceans using a self-contained underwater breathing apparatus (SCUBA). Collection of specimens was followed by their immediate freezing and preparation for shipping. The Andersen laboratory at UBC was the destination where the voucher samples were deposited. Crude extracts were harnessed at room temperature from sliced up samples which were soaked in MeOH and then extracted multiple times. MeOH was removed from the extracted products (in vacuo) and the concentrated residues were partitioned using a separating funnel with EtOAc/BuOH and H2O. Since these solutions are immiscible, residues were collected separately in EtOAc/BuOH and H2O. Evaporation of EtOAc/BuOH and H2O left behind extracts which were used for phenotypic screening against MAB. The marine library was received in a dry form and in preparation for screening, dimethyl sulfoxide (DMSO) was used as a solvent. Phenotypic screening of ME was followed by bioassay guided fractionation which was done in collaboration with the Andersen research group. The identified active extracts underwent fractionation using Silica gel flash chromatography which produced fractions for further testing against MAB. The resulting active fractions were further fractionated through high-performance liquid chromatography until pure active compounds were isolated. Nuclear magnetic resonance (1D and 2D) and HRTOFESIMS spectra analysis was applied to elucidate the chemical structures of the isolated active compounds.56,76 17 Table 4. List of ME and PMC libraries used for phenotypic screening. Receiving date Marine products Marine ID Stock (mg/mL) February, 2021 80 Extracts RJA UBC 08 1008 Anzia B 10 80 Extracts RJA UBC 76047 -76129 10 80 Extracts RJA UBC 02 203-286 10 80 Extracts RJA UBC 44709 - 55480 10 80 Extracts RJA UBC 76295 - 44708 10 July 07, 2022 Fraction 76057: BuOH-A 10 Fraction 76057: BuOH-B 10 Fraction 76057: BuOH-C 10 Fraction 76057: BuOH-D 10 Fraction 76057: BuOH-E 10 Fraction 76309: BuOH-A 10 Fraction 76309: BuOH-B 10 Fraction 76309: BuOH-C 10 Fraction 76309: BuOH-D 10 Fraction PNG 03 264: BuOH-A 10 Fraction PNG 03 264: BuOH-B 10 Fraction PNG 03 264: BuOH-C 10 Fraction PNG 03 264: BuOH-D 10 March 28, 2023 72 Pure compounds Plate E (B1-G12) 2 72 Pure compounds Plate F (B1-G12) 2 72 Pure compounds Plate G (B1-G12) 2 35 Pure compounds Plate H (B1-D11) 2 18 2.2 Pure compounds from Global Health Priority Box Library Global Health Priority (GHP) box was supplied by Medicines for Malaria Venture (MMV) and it comprised of a total 240 pure compounds (Table 5). This box featured three groups of compounds and their chemical structures were made available from the MMV website.77 One group had 80 active compounds compiled by MVV and partners against drug-resistant malaria (MB2). Another group contained 80 active compounds provided by Bristol-Myers Squibb® against zoonotic and neglected diseases (ZND). The third group also had 80 active compounds and compiled with the help of Innovative Vector Control Consortium (IVCC) against vector species (VEC). GHP box was received in a dry form and in preparation for screening, dimethyl sulfoxide (DMSO) was used as a solvent. 19 Table 5. List GHP library used for phenotypic screening. Receiving date GHP Box GHP Box ID Original disease indication Stock (µM) August 23, 2022 MB2 plate MMV001197 Drug-resistant malaria 10 MB2 plate MMV006187 Drug-resistant malaria 10 MB2 plate MMV006219 Drug-resistant malaria 10 MB2 plate MMV006344 Drug-resistant malaria 10 MB2 plate MMV006430 Drug-resistant malaria 10 MB2 plate MMV006597 Drug-resistant malaria 10 MB2 plate MMV006621 Drug-resistant malaria 10 MB2 plate MMV006931 Drug-resistant malaria 10 MB2 plate MMV007007 Drug-resistant malaria 10 MB2 plate MMV007282 Drug-resistant malaria 10 MB2 plate MMV007300 Drug-resistant malaria 10 MB2 plate MMV007501 Drug-resistant malaria 10 MB2 plate MMV011471 Drug-resistant malaria 10 MB2 plate MMV014853 Drug-resistant malaria 10 MB2 plate MMV019053 Drug-resistant malaria 10 MB2 plate MMV019177 Drug-resistant malaria 10 MB2 plate MMV019421 Drug-resistant malaria 10 MB2 plate MMV019547 Drug-resistant malaria 10 MB2 plate MMV020033 Drug-resistant malaria 10 MB2 plate MMV020192 Drug-resistant malaria 10 MB2 plate MMV020196 Drug-resistant malaria 10 MB2 plate MMV020203 Drug-resistant malaria 10 MB2 plate MMV020207 Drug-resistant malaria 10 MB2 plate MMV020335 Drug-resistant malaria 10 MB2 plate MMV020445 Drug-resistant malaria 10 MB2 plate MMV020456 Drug-resistant malaria 10 MB2 plate MMV020508 Drug-resistant malaria 10 MB2 plate MMV020709 Drug-resistant malaria 10 MB2 plate MMV022487 Drug-resistant malaria 10 MB2 plate MMV023190 Drug-resistant malaria 10 MB2 plate MMV023401 Drug-resistant malaria 10 MB2 plate MMV023523 Drug-resistant malaria 10 MB2 plate MMV023573 Drug-resistant malaria 10 MB2 plate MMV023951 Drug-resistant malaria 10 MB2 plate MMV024309 Drug-resistant malaria 10 MB2 plate MMV024638 Drug-resistant malaria 10 MB2 plate MMV024825 Drug-resistant malaria 10 MB2 plate MMV024850 Drug-resistant malaria 10 MB2 plate MMV025013 Drug-resistant malaria 10 MB2 plate MMV026458 Drug-resistant malaria 10 20 GHP Box GHP Box ID Original disease indication Stock (µM) MB2 plate MMV026953 Drug-resistant malaria 10 MB2 plate MMV027331 Drug-resistant malaria 10 MB2 plate MMV028822 Drug-resistant malaria 10 MB2 plate MMV029587 Drug-resistant malaria 10 MB2 plate MMV085494 Drug-resistant malaria 10 MB2 plate MMV1103183 Drug-resistant malaria 10 MB2 plate MMV1167451 Drug-resistant malaria 10 MB2 plate MMV1262756 Drug-resistant malaria 10 MB2 plate MMV1266067 Drug-resistant malaria 10 MB2 plate MMV1267536 Drug-resistant malaria 10 MB2 plate MMV1435700 Drug-resistant malaria 10 MB2 plate MMV1542706 Drug-resistant malaria 10 MB2 plate MMV1704710 Drug-resistant malaria 10 MB2 plate MMV1726212 Drug-resistant malaria 10 MB2 plate MMV1752548 Drug-resistant malaria 10 MB2 plate MMV1760745 Drug-resistant malaria 10 MB2 plate MMV1791425 Drug-resistant malaria 10 MB2 plate MMV1795555 Drug-resistant malaria 10 MB2 plate MMV1795557 Drug-resistant malaria 10 MB2 plate MMV1796046 Drug-resistant malaria 10 MB2 plate MMV1797658 Drug-resistant malaria 10 MB2 plate MMV1798946 Drug-resistant malaria 10 MB2 plate MMV1804275 Drug-resistant malaria 10 MB2 plate MMV1804559 Drug-resistant malaria 10 MB2 plate MMV1805007 Drug-resistant malaria 10 MB2 plate MMV1808809 Drug-resistant malaria 10 MB2 plate MMV1841740 Drug-resistant malaria 10 MB2 plate MMV1845852 Drug-resistant malaria 10 MB2 plate MMV660057 Drug-resistant malaria 10 MB2 plate MMV670631 Drug-resistant malaria 10 MB2 plate MMV674132 Drug-resistant malaria 10 MB2 plate MMV753519 Drug-resistant malaria 10 MB2 plate MMV936405 Drug-resistant malaria 10 MB2 plate MMV959706 Drug-resistant malaria 10 MB2 plate MMV962005 Drug-resistant malaria 10 MB2 plate MMV974630 Drug-resistant malaria 10 MB2 plate MMV978042 Drug-resistant malaria 10 MB2 plate MMV979319 Drug-resistant malaria 10 MB2 plate MMV979930 Drug-resistant malaria 10 MB2 plate MMV981619 Drug-resistant malaria 10 VEC plate MMV002231 Vector control 10 VEC plate MMV002287 Vector control 10 VEC plate MMV002371 Vector control 10 21 GHP Box GHP Box ID Original disease indication Stock (µM) VEC plate MMV002519 Vector control 10 VEC plate MMV003872 Vector control 10 VEC plate MMV003891 Vector control 10 VEC plate MMV003958 Vector control 10 VEC plate MMV003972 Vector control 10 VEC plate MMV003974 Vector control 10 VEC plate MMV003975 Vector control 10 VEC plate MMV003977 Vector control 10 VEC plate MMV004017 Vector control 10 VEC plate MMV027339 Vector control 10 VEC plate MMV078538 Vector control 10 VEC plate MMV083774 Vector control 10 VEC plate MMV1501398 Vector control 10 VEC plate MMV1577452 Vector control 10 VEC plate MMV1577453 Vector control 10 VEC plate MMV1577454 Vector control 10 VEC plate MMV1577455 Vector control 10 VEC plate MMV1577456 Vector control 10 VEC plate MMV1577458 Vector control 10 VEC plate MMV1577459 Vector control 10 VEC plate MMV1577460 Vector control 10 VEC plate MMV1577461 Vector control 10 VEC plate MMV1577462 Vector control 10 VEC plate MMV1577463 Vector control 10 VEC plate MMV1577464 Vector control 10 VEC plate MMV1577465 Vector control 10 VEC plate MMV1577466 Vector control 10 VEC plate MMV1577467 Vector control 10 VEC plate MMV1577468 Vector control 10 VEC plate MMV1577469 Vector control 10 VEC plate MMV1577470 Vector control 10 VEC plate MMV1577471 Vector control 10 VEC plate MMV1577472 Vector control 10 VEC plate MMV1577473 Vector control 10 VEC plate MMV1577474 Vector control 10 VEC plate MMV1577889 Vector control 10 VEC plate MMV1577890 Vector control 10 VEC plate MMV1578924 Vector control 10 VEC plate MMV1633823 Vector control 10 VEC plate MMV1633824 Vector control 10 VEC plate MMV1633825 Vector control 10 VEC plate MMV1633826 Vector control 10 VEC plate MMV1633827 Vector control 10 22 GHP Box GHP Box ID Original disease indication Stock (µM) VEC plate MMV1633828 Vector control 10 VEC plate MMV1633829 Vector control 10 VEC plate MMV1634079 Vector control 10 VEC plate MMV1634080 Vector control 10 VEC plate MMV1634081 Vector control 10 VEC plate MMV1634082 Vector control 10 VEC plate MMV1634083 Vector control 10 VEC plate MMV1634506 Vector control 10 VEC plate MMV1634507 Vector control 10 VEC plate MMV1793194 Vector control 10 VEC plate MMV1794204 Vector control 10 VEC plate MMV1794205 Vector control 10 VEC plate MMV1794206 Vector control 10 VEC plate MMV1794207 Vector control 10 VEC plate MMV1794209 Vector control 10 VEC plate MMV1794210 Vector control 10 VEC plate MMV1794211 Vector control 10 VEC plate MMV1794212 Vector control 10 VEC plate MMV1794213 Vector control 10 VEC plate MMV1794214 Vector control 10 VEC plate MMV1794215 Vector control 10 VEC plate MMV1794216 Vector control 10 VEC plate MMV1796254 Vector control 10 VEC plate MMV1796401 Vector control 10 VEC plate MMV637085 Vector control 10 VEC plate MMV637136 Vector control 10 VEC plate MMV637769 Vector control 10 VEC plate MMV672931 Vector control 10 VEC plate MMV673126 Vector control 10 VEC plate MMV688934 Vector control 10 VEC plate MMV689404 Vector control 10 VEC plate MMV689409 Vector control 10 VEC plate MMV689416 Vector control 10 VEC plate MMV689586 Vector control 10 ZND plate MMV1542798 Zoonotic & Neglected Diseases 10 ZND plate MMV1542799 Zoonotic & Neglected Diseases 10 ZND plate MMV1542820 Zoonotic & Neglected Diseases 10 ZND plate MMV1542822 Zoonotic & Neglected Diseases 10 ZND plate MMV1545674 Zoonotic & Neglected Diseases 10 ZND plate MMV1545675 Zoonotic & Neglected Diseases 10 ZND plate MMV1545678 Zoonotic & Neglected Diseases 10 ZND plate MMV1558322 Zoonotic & Neglected Diseases 10 ZND plate MMV1558323 Zoonotic & Neglected Diseases 10 23 GHP Box GHP Box ID Original disease indication Stock (µM) ZND plate MMV1558324 Zoonotic & Neglected Diseases 10 ZND plate MMV1558949 Zoonotic & Neglected Diseases 10 ZND plate MMV1579822 Zoonotic & Neglected Diseases 10 ZND plate MMV1579936 Zoonotic & Neglected Diseases 10 ZND plate MMV1592761 Zoonotic & Neglected Diseases 10 ZND plate MMV1593278 Zoonotic & Neglected Diseases 10 ZND plate MMV1795508 Zoonotic & Neglected Diseases 10 ZND plate MMV1795510 Zoonotic & Neglected Diseases 10 ZND plate MMV1828026 Zoonotic & Neglected Diseases 10 ZND plate MMV1828056 Zoonotic & Neglected Diseases 10 ZND plate MMV1828058 Zoonotic & Neglected Diseases 10 ZND plate MMV1828705 Zoonotic & Neglected Diseases 10 ZND plate MMV1828728 Zoonotic & Neglected Diseases 10 ZND plate MMV1828766 Zoonotic & Neglected Diseases 10 ZND plate MMV1828776 Zoonotic & Neglected Diseases 10 ZND plate MMV1828810 Zoonotic & Neglected Diseases 10 ZND plate MMV1828814 Zoonotic & Neglected Diseases 10 ZND plate MMV1828816 Zoonotic & Neglected Diseases 10 ZND plate MMV1828832 Zoonotic & Neglected Diseases 10 ZND plate MMV1828843 Zoonotic & Neglected Diseases 10 ZND plate MMV1828855 Zoonotic & Neglected Diseases 10 ZND plate MMV1828860 Zoonotic & Neglected Diseases 10 ZND plate MMV1829088 Zoonotic & Neglected Diseases 10 ZND plate MMV1829111 Zoonotic & Neglected Diseases 10 ZND plate MMV1829114 Zoonotic & Neglected Diseases 10 ZND plate MMV1829150 Zoonotic & Neglected Diseases 10 ZND plate MMV1829290 Zoonotic & Neglected Diseases 10 ZND plate MMV1829345 Zoonotic & Neglected Diseases 10 ZND plate MMV1829350 Zoonotic & Neglected Diseases 10 ZND plate MMV1829361 Zoonotic & Neglected Diseases 10 ZND plate MMV1829377 Zoonotic & Neglected Diseases 10 ZND plate MMV1829412 Zoonotic & Neglected Diseases 10 ZND plate MMV1829460 Zoonotic & Neglected Diseases 10 ZND plate MMV1829523 Zoonotic & Neglected Diseases 10 ZND plate MMV1829556 Zoonotic & Neglected Diseases 10 ZND plate MMV1829574 Zoonotic & Neglected Diseases 10 ZND plate MMV1829575 Zoonotic & Neglected Diseases 10 ZND plate MMV1829658 Zoonotic & Neglected Diseases 10 ZND plate MMV1829659 Zoonotic & Neglected Diseases 10 ZND plate MMV1848726 Zoonotic & Neglected Diseases 10 ZND plate MMV673982 Zoonotic & Neglected Diseases 10 ZND plate MMV674056 Zoonotic & Neglected Diseases 10 ZND plate MMV674593 Zoonotic & Neglected Diseases 10 24 GHP Box GHP Box ID Original disease indication Stock (µM) ZND plate MMV674787 Zoonotic & Neglected Diseases 10 ZND plate MMV674884 Zoonotic & Neglected Diseases 10 ZND plate MMV675568 Zoonotic & Neglected Diseases 10 ZND plate MMV688723 Zoonotic & Neglected Diseases 10 ZND plate MMV689461 Zoonotic & Neglected Diseases 10 ZND plate MMV689463 Zoonotic & Neglected Diseases 10 ZND plate MMV689625 Zoonotic & Neglected Diseases 10 ZND plate MMV689626 Zoonotic & Neglected Diseases 10 ZND plate MMV689635 Zoonotic & Neglected Diseases 10 ZND plate MMV691936 Zoonotic & Neglected Diseases 10 ZND plate MMV691942 Zoonotic & Neglected Diseases 10 ZND plate MMV692107 Zoonotic & Neglected Diseases 10 ZND plate MMV692115 Zoonotic & Neglected Diseases 10 ZND plate MMV692623 Zoonotic & Neglected Diseases 10 ZND plate MMV692630 Zoonotic & Neglected Diseases 10 ZND plate MMV692857 Zoonotic & Neglected Diseases 10 ZND plate MMV692861 Zoonotic & Neglected Diseases 10 ZND plate MMV693130 Zoonotic & Neglected Diseases 10 ZND plate MMV693133 Zoonotic & Neglected Diseases 10 ZND plate MMV884814 Zoonotic & Neglected Diseases 10 ZND plate MMV892517 Zoonotic & Neglected Diseases 10 ZND plate MMV892523 Zoonotic & Neglected Diseases 10 ZND plate MMV893256 Zoonotic & Neglected Diseases 10 ZND plate MMV893278 Zoonotic & Neglected Diseases 10 ZND plate MMV893415 Zoonotic & Neglected Diseases 10 ZND plate MMV893417 Zoonotic & Neglected Diseases 10 ZND plate MMV897662 Zoonotic & Neglected Diseases 10 ZND plate MMV977436 Zoonotic & Neglected Diseases 10 25 2.3 Bacteria and host cells 2.3.1 MAB cells and culture media Mycobacterium abscessus subspecies abscessus ATCC 19977T strain was transformed with pTEC27 expressing tomato red fluorescent protein (RFP) and had selective resistance to hygromycin.78 The solid media contained DifcoTM Middlebrook 7H10 agar supplemented with 0.5 % glycerol, 0.5 % bovine serum albumin, 2.0 % dextrose, and 0.8 % sodium chloride (10 % ADS), and 500 µg/mL hygromycin B (Roche Diagnostics). ADS was sterilized by filtration using a Stericup® Filter Unit (Millipore Sigma). The liquid media contained DifcoTM Middlebrook 7H9 broth supplemented with 10 % ADS, 0.05 % Tween® 80 (Fisher Chemical), and 500 µg/mL hygromycin. An incomplete solid or liquid media was one without antibiotics. MAB stock was retrieved from -80 °C freezer and streaked on solid media plates, which were grown in a 37 °C incubator for 3-5 days. A single colony was transferred to liquid media and bacteria were grown at 37 °C with shaking for 2-3 days or until they reached a mid-log phase, optical density (OD), 600 nm of 0.3 - 0.7 (Thermo ScientificTM Genesys 20). Preculture was used to prepare bacterial stocks for use in the future. Bacterial culture was diluted with sterile glycerol (final 15 % glycerol) and stored in -80 °C freezer as glycerol stocks. 2.3.2 THP-1 cells and culture media THP-1 (ATCC TIB-202TM) are monocyte cells which originated from the peripheral blood tissue of a donor with acute monocytic leukemia.79 Cells were maintained in Gibco RPMI 1640 growth media supplemented with 10 % fetal bovine serum (GibcoTM), 2 % glutamine (GibcoTM), and 1 % non-essential amino acids (GibcoTM). THP-1 cells were diluted with incomplete RPMI media (no antibiotics) every 2-3 days to maintain a density between 0.25 - 1 million cells/mL. Cells were incubated at 37 °C and 5 % carbon dioxide (CO2) using tissue culture flasks at volumes 26 between 20 - 50 mL. A new batch of cells was grown from a liquid nitrogen stock every two months to prevent cells from altering their phenotype or replication rate. 2.3.3 RAW 264.7 cells and culture media RAW 264.7 (ATCC TIB-71TM) are macrophage cells which were harvested from ascites tissue of a male mouse with Abelson murine leukemia virus-induced tumor.80 RAW 264.7 cells were kindly supplied to our laboratory from Dr. Erwin Schurr’s research group at McGill University. We used RAW 264.7 cells as an additional cancer cell line to test for the cytotoxicity of our active compounds. Dulbecco’s Modified Eagles Medium (incomplete DMEM) was used to maintain RAW 264.7 cells and cells were kept in a 37 °C and 5 % CO2 incubator. RAW 264.7 cells adhere to the surface of the tissue culture flask as they grow. Cells were passaged every 2-3 days or when the cells had reached ~70 % confluency. This step was performed by detaching RAW 264.7 cells from the surface of the flask using a sterile scraping tool. The suspended cells were mixed well, counted, and then diluted with fresh incomplete DMEM at a ratio of 1:3 to 1:6. 2.3.4 HEK293T cells and culture media 293T (ATCC CRL-3216TM) are human embryonic kidney (HEK293T) cells which resemble epithelial cells.81 HEK293T cells were kindly supplied to our laboratory from Dr. Khaled Abdelrahman’s research group at UBC. We needed to determine the toxicity of our active compounds against HEK293T cells as these cells are among others that are commonly used for cytotoxicity assays. Dulbecco’s Modified Eagles Medium (DMEM) was used to maintain HEK293T cells and cells were kept in a 37 °C and 5 % CO2 incubator. HEK293T cells adhere to the surface of the tissue culture flask as they grow. Cells were passaged every 2-3 days or when the cells had reached ~70 % confluency. This step was performed using GibcoTM trypsin-ethylenediaminetetraacetic acid (0.25 %), an enzyme which detaches proteins that have adhered to the flask. Detached cells were mixed well, counted, and then diluted at a ratio of 1:3 to 1:8. 27 2.4 Single concentration activity determination of libraries against MAB in broth MAB pre-culture was grown in 7H9 media supplemented with 10 % ADS and 0.05 % Tween 80 for 2-3 days or until the bacteria reached a mid-log phase. In 96-well plates, TCM and ME were diluted to a final concentration of 100 µg/mL. PMC and GHP were diluted to 20 µg/mL and 10 µM, respectively. Plates containing diluted natural extracts and pure compounds libraries were seeded with 5 x 105 bacterial cells/mL (OD600, 0.1 [1 x 108 CFU/mL]. The negative and positive controls were 1 % DMSO and 4 µM bedaquiline (BDQ), respectively. Plates were sealed with parafilm, transferred to a secondary container with a beaker of water to prevent plates from drying out, and incubated for five days in 37 °C incubator. A plate reader was used to measure OD600 units (VarioSkanTM, ThermoFisher), which were used to calculate the percentage of inhibition. Compounds that displayed ≥90 % of inhibition against MAB were reported as active compounds. Further, a statistical Z’-factor was applied to determine the reliability of our assay. Z’-factor indicates how distinct the positive and negative controls are from each other and this measurement helps to exclude false positive and negative results.82 Z’-factor score of 1, 0.5-1, 0-0.5 indicates an ideal, excellent, and marginal assay, respectively.82 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (−100) 𝑥 Signalsample – SignalDMSOSignalDMSO – SignalBDQ 𝑍′ − 𝐹𝑎𝑐𝑡𝑜𝑟 = 1 − 3(Standard deviationBDQ + Standard deviationDMSO)|MeanBDQ – MeanDMSO| 2.5 THP-1 infection model THP-1 cells were centrifuged at 3220 x g (25 °C) for 10 minutes, and then washed once with 7H9 media supplemented with 0.075 % Tween 80. Human serum (10 %) was used to opsonize bacteria (30 minutes in 37 °C rolling incubator) for recognition by THP-1 cells. Meanwhile, THP-1 cells were counted under a microscope using a hemocytometer to determine the density of cells. 28 The volume of cells was calculated to seed 1 x 105 cells per well in 96-well plates. This volume was spun down at 750 rpm, 25 °C for 10 minutes and then resuspended to 1 x 106 cells/mL. THP-1 monocytes were differentiated to macrophages by phorbol 12-myristate-13-acetate (PMA) at a final concentration of 40 ng/mL. Opsonized bacteria were de-clumped to a single cell suspension using a 25G 1.5'' blunt needle (SAI Infusion Technologies). THP-1 cells and opsonized bacteria were mixed to establish a multiplicity of infection (MOI = 5) whereby, one host cell was infected by five bacterial cells. Then, an infection was established by incubation of cells with bacteria for three hours in a 37 °C rolling incubator. After incubation, infected macrophages were centrifuged at 113 x g (25 °C) for 10 minutes and then washed three times with incomplete RPMI media. In 96-well plates, TCM was diluted to a final concentration of 100 µg/mL. PMC and GHP were diluted to 20 µg/mL and 10 µM, respectively. Infected cells were added to diluted compounds in complete RPMI containing amikacin at 70 µM to kill extracellular bacteria. The negative and positive controls were 1 % DMSO and 4 µM BDQ, respectively. Plates were sealed with parafilm, transferred to a secondary container, and incubated for 72 hours in a 37 °C and 5 % CO2 incubator. Afterwards, the wells were washed with incomplete RPMI, stained with Hoescht 33342 nucleic acid stain at 1.4 µg/mL (InvitrogenTM) for 30 minutes, and fixed with 4 % paraformaldehyde/formaldehyde (Sigma-Aldrich) for 40 minutes. The staining and fixing media were aspirated, and then phosphate-buffered saline (PBS) solution was added to plates. Plates were scanned using a high throughput platform (Thermo Fisher CellInsight CX5) to measure the number of bacteria per macrophage cells. The percentage of inhibition and Z’-factor scores were calculated using the formulas outlined in section 2.4. Compounds that displayed above 30 % of inhibition against MAB and above 70 % THP-1 cell survival were reported as active compounds. 29 2.6 Minimum inhibitory concentration determination assay 2.6.1 Minimum inhibitory concentration of active compounds in broth The MIC of active compounds in broth was calculated using the broth microdilution method (Figure 2).78 We determined the concentration at which the active compounds inhibited the growth of ≥90 % of the bacteria (MIC90). In a single column on a 96-well plate, active compounds were diluted to double the highest concentration that was intended to be tested. The rest of the columns did not contain active compounds but had 7H9 media with 2 % DMSO. The column with active compounds had double the volume of 7H9 media contained in the rest of the columns. Half the volume of diluted active compounds was aspirated and then added to the rest of the columns in a two-fold dilution in every subsequent column. MAB preculture that was grown to mid-log phase was used to dilute the inoculum where each well was seeded with 5 x 105 CFU/mL (OD600 0.1 [1 x 108 CFU/mL). An equal volume of the diluted inoculum was added to plates containing active compounds. Plates were sealed with parafilm, transferred to a secondary container with a beaker of water, and incubated at 37 °C for five days. VarioSkanTM plate reader was used to measure OD600 units to calculate the percentage of inhibition. Percentage of inhibition and Z’-factor were calculated using the formulas outlined in section 2.4. The negative and positive controls were 1 % DMSO and 4 µM BDQ, respectively. MIC90 values were calculated using a nonlinear regression model (log (agonist) versus response – Find EC90) on GraphPad Prism application. 2.6.2 Minimum inhibitory concentration of active compounds intracellularly The MIC of active compounds intracellularly was calculated using the broth microdilution method (Figure 2).78 We determined the concentration at which the active compounds inhibited the growth of ≥90 % of the bacteria (MIC90). In a single column on a 96-well plate, active compounds were diluted to double the highest concentration that was intended to be tested. The 30 rest of the columns did not contain active compounds but had complete RPMI media with 2 % DMSO. The column with active compounds had double the volume of complete RPMI media contained in the rest of the columns. Half the volume of diluted active compounds was aspirated and then added to the rest of the columns in a two-fold dilution in every subsequent column. MAB preculture that was grown to mid-log phase and then centrifuged to wash the pellet in preparation for a 30-minute bacterial opsonization with 10 % human serum. THP-1 cells were counted to calculate the volume needed to seed 1 x 105 cells/well. After washing the cells, 40 ng/mL PMA was added. THP-1 cells and opsonized MAB were incubated at 37°C for three hours. The multiplicity of infection was five MAB cells per one THP-1 cell (MOI = 5). After the 3-hour incubation period the infected THP-1 cells were added to plates containing the diluted active compounds. Plates were incubated at 37 °C for 72 hours and then, infected macrophages were washed with incomplete RPMI media, stained for 30 minutes with Hoescht 33342, and fixed for 40 minutes with 4 % paraformaldehyde/formaldehyde. The staining and fixing media was aspirated, and then PBS was added to plates. The negative and positive controls were 1 % DMSO and 4 µM BDQ, respectively. Plates were scanned using CX5® to measure the number of bacteria per macrophage cells. The percentage of inhibition and Z’-factor scores were calculated using the formulas in section 2.4. MIC90 values were calculated using a nonlinear regression model (log (agonist) versus response – Find EC90) on GraphPad Prism application. 31 Figure 2. Broth microdilution method. Double the highest intended concentration of active compounds was added to column 2 in a 96-well plate. This column contained twice the volume of assay media (7H9 or complete RPMI) contained in columns 3-11. A two-fold serial dilution was performed by aspirating half the volume from column 2 to 11, consecutively. Column 1 and 12 contained 1 % DMSO (D) and 4 µM BDQ (B) as the negative and positive controls, respectively. 32 2.7 Cytotoxicity assay using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) We tested the toxicity of our active compounds against THP-1, RAW 264.7, and HEK293T cells assay using the MTT assay. In 96-well plates, 10, 000 cells per well (separate plates for each cell line) were seeded and plates were kept in a 37 °C and 5 % CO2 incubator. THP-1 cells were differentiated using PMA at 40 ng/mL while both RAW 264.7 and HEK293T cells were left to adhere to the bottom of the wells. After 24 hours, a ten-point series of two-fold dilutions of active compounds was prepared using broth microdilution method as outlined under section 2.6.1. Active compounds were added to differentiated and adherent cells, which were incubated for 72 hours at 37 °C and 5 % CO2. Two and a half hours before the end of incubation period, 5 mg/mL yellow MTT solution was added to wells. Viable cells can chemically reduce yellow MTT solution to purple formazan.83 At the end of the 72-hour incubation time, an MTT extraction solution (50 % dimethylformamide, sodium dodecyl sulfate, 2.5 % acetic acid and 2.5 % 1M HCl acid) was added to each well and plates were incubated overnight. The negative and positive controls were 1 % DMSO and 4 µM BDQ, respectively. Measurements on a VarioSkanTM plate reader were taken at 570 nm and 690 nm. The difference in optical density between the higher and lower wavelengths was used to calculate the percentage of viable cells. The cytotoxicity concentration at 50 % cell death (CC50) and MIC at 50 % MAB growth inhibition were calculated using a nonlinear regression model (log (inhibitor) versus normalized response – Variable slope) on GraphPad Prism application. Each active compound’s selectivity index was calculated as CC50/MIC50. 2.8 Antibacterial activity determination of the active compounds Representatives of mycobacteria, Gram-positive, and Gram-negative bacteria were selected to determine the antibacterial activity of active compounds in broth (Table 6). We used M. smegmatis as a representative RGM, B. subtilis and S. aureus as representatives for Gram-positive 33 bacteria, and E. coli and S. Tm as representatives for Gram-negative bacteria. M. smegmatis was streaked on 7H10 incomplete solid media and a single colony was picked and grown in incomplete 7H9 liquid media over two nights. B. subtilis, S. aureus, E. coli, and S. Tm were streaked on LB agar plates and single colonies were picked and grown in incomplete LB broth overnight. S. Tm was grown in a complete LB broth with 50 µg/mL ampicillin. All species were grown to mid-log phase and their precultures were diluted in incomplete media in 96-well plates containing active compounds to seed 5 x 104 bacterial cells/well. Active compounds were prepared using the broth microdilution method as outlined under the minimum inhibitory concentration determination assay section above. Plates containing bacterial species and active compounds were sealed and kept in 30 °C incubator overnight except for the plate containing M. smegmatis which was kept over two nights at 37 °C. VarioSkanTM plate reader was used to measure bacterial OD600, and percentage of bacterial growth inhibition was calculated using the formula listed under the single concentration activity determination assay section above. The negative and positive controls were 1% DMSO and 100 µg/mL gentamicin, respectively for all the tested bacterial species except for M. smegmatis whereby the positive control was 4 µM BDQ. Table 6. Bacterial species that were used to determine antibacterial activity of active compounds. Class Species OD600 0.1 equivalent to CFU/mL Mycobacteria Mycobacterium smegmatis MC2 155 (M. smegmatis) 1 x 108 CFU/mL Gram-positive Bacillus subtilis (B. subtilis) 0.17 x 108 CFU/mL Staphylococcus aureus ATCCTM 25923 (S. aureus) 0.21 x 108 CFU/mL Gram-negative Escherichia coli ATCCTM 25922 (E. coli) 0.8 x 108 CFU/mL Salmonella enterica serovar Typhimurium (S. Tm) 0.17 x 108 CFU/mL 34 Chapter Three 3. Results 3.1 Single concentration activity determination in broth 3.1.1 Seven compounds were active against MAB in broth We aimed to assess our natural extracts and pure compounds libraries for activity against MAB in broth using a single concentration activity determination assay. Libraries from TCM and ME were tested at 100 µg/mL, PMC at 20 µg/mL, and GHP box at 10 µM. We incubated MAB cells with the libraries in 96-well plates at 37 °C for five days. Then, we measured OD600 to calculate the percentage of MAB growth inhibition. Our assay proved to be excellent with an average Z’-factor score = 0.7 calculated using 1 % DMSO and 4 µM BDQ as negative and positive controls, respectively (Figure 3a). Compounds that exhibited ≥90 % MAB growth inhibition in broth were considered active. We identified seven active compounds (1 - 7) in broth: compounds 1 - 3, 4 – 6, and 7 from the ME, PMC, and GHP box libraries, respectively (Figure 3b). Compounds 1 - 7 advanced to hit validation studies including MIC determination, cytotoxicity tests, and specificity of antimicrobial activity. 35 Figure 3. Phenotypic screening of natural extracts and pure compounds libraries against MAB in broth. a Screening assay validation. Each screened plate contained at least four replicates of the negative (1 % DMSO) and positive (4 µM BDQ) controls, respectively. The average Z’-factor was calculated using percentages of growth inhibition of all the controls used in screening libraries against MAB in broth. b Percentages of growth inhibition of libraries using single concentration activity determination assay against MAB in broth. The screen was performed once for each library. TCM and marine extracts libraries were screened in duplicates. Two active extracts were identified from the ME and four active compounds from the pure marine compounds and GHP box libraries. 36 In general, percentages of growth inhibition of libraries showed an uneven distribution but a similar trend (Table 7). The higher the percentage of growth inhibition, the fewer the number of extracts and pure compounds detected. For example, 0.2 % of the TCM library reached > 75 % MAB growth inhibition in broth while 89 % of this library exhibited < 25 % inhibition. For the ME and PMC libraries 0.5 % and 2.7 %, respectively indicated > 75 % growth inhibition while 82 % and 86 %, respectively had < 25 % growth inhibition against MAB in broth. Also, 12 % of the GHP library showed > 75 % MAB growth inhibition in broth while 48 % of this library exhibited < 25 % inhibition. Table 7. Distribution of the percentages of growth inhibition from TCM, ME, PMC, and GHP against MAB growth in broth MAB Growth inhibition TCM (%) ME (%) PMC (%) GHP (%) 90 – 100 % 0 0.5 2.79 1.67 75 – 90 % 0.27 0 0 10.42 50 – 75 % 0.81 3 2.39 19.17 25 – 50 % 9.97 14.75 8.37 20.42 0 – 25 % 88.9 81.75 86.45 48.33 3.1.1.1 TCM Library From 371 TCM extracts, using the single concentration activity determination assay, we did not identify active compounds against MAB in broth (Figure 3b). 3.1.1.2 ME Library We reported two active extracts, RJA76309 and RJA03-264 against MAB in broth from testing 400 ME library at 100 µg/mL using the single concentration activity determination assay (Figure 3b). Bioassay guided fractionation of RJA76309 and RJA03-264 was performed, and fractions were generated by our collaborators, Andersen Lab. We tested the inhibitory activity of fractions against MAB and active ones underwent further fractionation until active pure compounds were isolated. Our collaborators isolated three active pure compounds and determined 37 their chemical structures; haliclonadiamine (1) (368 g/mol), papuamine (2) (368 g/mol), and haliclonacyclamine (3) (468.8 g/mol). Compounds 1 and 2 had structurally similar pentacyclic compounds, except for the location of one hydrogen bond (Figure 4). Compound 3 was a cyclic compound, heavier in molecular weight compared to the other two compounds. 3.1.1.3 PMC Library We found three additional active compounds against MAB in broth from testing 251 PMC at 20 µg/mL using single concentration activity determination assay (Figure 3b). These compounds were ascididemin (4) (283.3 g/mol), manzamine A (5) (548.8 g/mol), and 1,2,3,4-tetrahydro-2-N-methyl-8-hydroxymanzamine A (6) (581.8 g/mol). Compounds 5 and 6 were β-carboline polycyclic compounds, and compound 6 contained hydroxyl and methyl groups (Figure 4). 3.1.1.4 GHP Box Our study found one active compound, MMV1829412 against MAB in broth from testing 240 GHP compounds at 10 µM using single concentration activity determination assay (Figure 3b). MMV1829412 was the designated compound ID from MMV, and the compound name was 4,6-dimethyl-N-[(1S)-1-phenylethyl]-1H-indole-2-carboxamide (7) (292.4 g/mol). Compound 7 came from a plate containing active compounds against zoonotic and neglected diseases. The activity of this compound was originally indicated against Mtb, according to the library details provided by MMV. 38 Figure 4. Chemical structures of the seven active compounds in broth. Haliclonadiamine (1). Papuamine (2). Haliclonacyclamine (3). Ascididemin (4). Manzamine A (5). 1,2,3,4-tetrahydro-2-N-methyl-8-hydroxymanzamine A (6). 4,6-dimethyl-N-[(1S)-1-phenylethyl]-1H-indole-2-carboxamide (7). 39 3.1.2 Compounds 1 - 7 displayed a dose dependent antimycobacterial activity against MAB in broth We used the broth microdilution assay to determine the MIC90 of compounds 1 – 7 against MAB growth. The highest tested concentrations of compounds 1 – 3, 4 - 6, and 7 were 100 µg/mL, 20 µg/mL, and 7.3 µg/mL, respectively. Compounds were diluted two-fold in a ten-point series and incubated with MAB in 96-well plates for five days at 37 °C. We measured OD600 units to calculate the percentages of MAB growth inhibition in broth and then determined the MIC90. We expected our active compounds to display MIC90 values that were lower than the compounds’ highest tested concentrations. Compounds 1 - 7 displayed a dose dependent antimycobacterial activity against MAB in broth and had MIC90 values ranging from 3.6 - 25 µg/mL (Table 8, Figure 5). Compounds 2 and 7 from ME and GHP libraries indicated the highest (25 µg/mL) and lowest (3.6 µg/mL) MIC90 values, respectively. The rest of the active compounds from ME (1 and 3) and PMC (4 - 6) libraries had comparable MIC90 values that fell between 8.7 – 14 µg/mL. 3.1.2.1 Cytotoxicity profiles of active compounds indicated varying selectivity Compounds 1 – 7 were tested for cytotoxicity against THP-1, RAW 264.7, and HEK293T cells using the MTT assay. The highest tested concentrations of compounds 1 – 3, 4 - 6, and 7 were 100 µg/mL, 20 µg/mL, and 7.3 µg/mL, respectively. Compounds were diluted using the broth microdilution method and then added to pre-differentiated/adherent cells in 96-well plates for a 72-hour incubation period at 37 °C and 5 % CO2. A yellow MTT solution was added to plates followed by an extraction buffer for an overnight incubation. OD570 and OD690 measurements were used to calculate the percentages of cell survival. We did not test compounds 4, 5, and 6 for cytotoxicity against THP-1 and RAW 264.7 cells and we did not test compound 7 against RAW 264 cells. We had a limited amount of these compounds and thus we prioritized testing them against HEK293T cells, one of the standard models for cytotoxicity assays. We determined the 40 compounds’ selectivity index (SI) by diving the active compounds’ concentrations at 50 % cell death and 50 % MAB growth inhibition (CC50 /MIC50). Active compounds showed varying selectivity where some compounds were less or more selective to MAB than to HEK293T cells (Figure 6, Table 8). Compounds 1 – 4 were less selective to the bacteria than to host cells with low SI between 0.1 – 0.5. Compounds 5 – 7 were more selective to the bacteria than to host cell with SI between 3.4 - >9.6. Table 8. Antimycobacterial and cytotoxicity activities from compounds 1 - 7 against THP-1, RAW 264.7, and HEK293T cells. nd* Not determined. SI = CC50 / MIC50 using HEK293T cells. 95% Confidence Interval values were determined using GraphPad Prism Variable Slope log(dose) versus response curve. Active compounds MIC90 (µg/mL) MIC50 (µg/mL) CC50 (µg/mL) SI THP-1 RAW 264.7 HEK293T 1. Haliclonadiamine 8.7 [7.5, 10.2] 2.7 [2.5, 2.9] 2.6 0.6 0.4 0.1 2. Papuamine 25 [21, 32.5] 7.9 [7.1, 8.8] 2.6 0.5 0.3 0.04 3. Haliclonacyclamine 8.9 [7.8, 10.8] 2.9 [2.7, 3.1] 3.6 0.4 0.9 0.3 4. Ascididemin 14 [10.4, 30.8] 7.0 [5.5, 8.9] nd* nd* 3.8 0.5 5. Manzamine A 9.5 [nd*] 4.9 [3.0, 6.4] nd* nd* 17 3.4 6. 1,2,3,4-tetrahydro-2-N-methyl-8-hydroxymanzamine A 12.5 [8.1, 14.1] 4.6 [3.9, 5.3] nd* nd* 16.7 3.6 7. 4,6-dimethyl-N-[(1S)-1-1-phenylethyl]-1H-indole-2-carboxamide 3.6 [1.0, 7.1] 0.3 [0.1, 0.6] nd* >2.9 >2.9 >9.6 41 Figure 5. Dose response curves and MIC90 determination of compounds 1 - 7 against MAB in broth. The assay was performed twice with two technical replicates each time (Z’-factor = 0.8). Dotted line point to 90 % growth inhibition and the corresponding MIC90 value. 42 Figure 6. Dose-dependent cytotoxic concentrations of active compounds against HEK293T cells. The cytotoxicity assay using MTT was performed once with two technical replicates per active compound. The horizontal dotted line shows cytotoxicity concentration of active compounds at 50 % HEK293T cell death (CC50) and MIC at 50 % MAB growth inhibition. 43 3.1.2.2 Compounds 5, 6, and 7 exhibited antibacterial selectivity against mycobacteria and Gram-positive bacteria in broth Compounds 1 - 7 were further tested against five representative bacterial species in broth. The species included were mycobacteria (M. smegmatis), Gram-positive bacteria (B. subtilis and S. aureus), and Gram-negative bacteria (E. coli and S. Tm). We aimed to determine the antibacterial selectivity of compounds 1 - 7 using the broth microdilution method. The highest tested concentrations of compounds 1 & 3, 4 - 6, and 7 were 100 µg/mL, 20 µg/mL, and 7.3 µg/mL, respectively. We received a limited amount of compound 2 from our collaborators and therefore we could not determine its antibacterial activity against our tested bacterial species. Species were incubated with the diluted compounds at 37 °C for 1-2 nights and MIC90 values were calculated from bacterial growth OD600 units. Compounds 5 - 7 indicated antibacterial selectivity against mycobacteria and Gram-positive bacteria (Table 9). Compound 5 inhibited growth against B. subtilis and S. aureus at MIC90 8.7 µg/mL and 11.3 µg/mL, respectively. These values against Gram-positive bacteria were relatively comparable to the MIC90 of compound 5 against MAB (9.5 µg/mL). Compound 6 was active against M. smegmatis and B. subtilis at MIC90 17.8 µg/mL and 13.7 µg/mL, respectively. These values were comparable to the MIC90 of compound 6 against MAB (12.5 µg/mL). Compound 7 showed antibacterial activity against MAB in broth (MIC90 3.6 µg/mL) but lacked activity against B. subtilis, S. aureus, E. coli, and S. Tm. Since we received a limited supply of compounds in the GHP box, we could not determine the antibacterial activity of compound 7 against M. smegmatis. Compounds 1, 3, and 4 were active against most of the tested representative bacterial species with mostly lower MIC90 values (0.5 – 11.4 µg/mL) than those found against MAB (8.7 – 25 µg/mL). 44 Table 9. Antibacterial activities of active compounds against mycobacteria, Gram-positive, and Gram-negative bacterial species. Active compounds MIC90 (µg/mL) Mycobacteria Gram-positive Gram-negative M. abscessus M. smegmatis B. subtilis S. aureus E. coli S. Tm 1. Haliclonadiamine 8.7 2.3 0.5 3.6 3.9 11.4 2. Papuamine 25 nd* nd* nd* nd* nd* 3. Haliclonacyclamine 8.9 3.4 1.6 8.6 10.8 40 4. Ascididemin 14 1.6 3.6 0.7 3.7 10 5. Manzamine A 9.5 61.7 8.7 11.3 74.4 >100 6. 1,2,3,4-tetrahydro-2-N-methyl-8-hydroxymanzamine A 12.5 17.8 13.7 32.1 73.7 >100 7. 4,6-dimethyl-N-[(1S)-1-1-phenylethyl]-1H-indole-2-carboxamide 3.6 nd* >4 48.3 47.3 >4 nd* Not determined. 3.2 Single concentration activity determination using the THP-1 infection model 3.2.1 TCM Fractions 53, 56, and 73 were active against MAB intracellularly We aimed to identify intracellularly active compounds from natural extracts and pure compounds libraries using our THP-1 infection model. We tested TCM at 100 µg/mL, PMC at 20 µg/mL, and GHP box at 10 µM. In 96-well plates, the libraries were incubated with MAB-infected THP-1 cells for 72 hours at 37 °C and 5 % CO2 to identify active extracts and pure compounds. Our assay had an average Z’-factor score = 0.3 (marginal assay), calculated using 1 % DMSO and 4 µM BDQ as negative and positive controls, respectively (Figure 7a). Untreated wells (1 % DMSO) displayed great variability in percentages of MAB growth inhibition intracellularly, but most wells displayed less than 30 % inhibition. TCM extracts were considered active intracellularly by attaining above 70 % THP-1 cell survival and above 30 % MAB growth inhibition. This threshold was set after calculating the average THP-1 cell survival and intracellular-MAB growth inhibition of the libraries. We identified 21 active TCM extracts, and 45 bioassay guided fractionation of these extracts yielded three intracellularly active TCM fractions. Overall, percentages of TCM, PMC, and GHP box that displayed above 70 % THP-1 cell survival were 80 %, 54 %, and 71 %, respectively, (Table 10). However, only the TCM library had active extracts displaying above 70 % THP-1 cell survival and above 30 % MAB inhibition intracellularly. Table 10. Distribution of percentages of THP-1 cell survival from TCM, PMC, and GHP against MAB growth intracellularly. THP-1 cell survival TCM (%) PMC (%) GHP (%) 70 – 100 % 80.86 54.06 70.98 50 – 70 % 3.77 5.69 6.25 30 – 50 % 4.58 6.097 4.02 0 -30 % 10.78 34.15 18.75 3.2.1.1 TCM Library We identified 21 intracellularly active TCM extracts tested at 100 µg/mL against MAB from 371 TCM extracts using our THP-1 infection model (Figure 7b). Most of these extracts displayed 80 - 100 % THP-1 cell survival and 30 - 50 % MAB growth inhibition intracellularly (Table 11) except for extract TCM 132, which had 77 % THP-1 cell survival and 87 % MAB growth inhibition intracellularly. These results were reported to our collaborators at Angal Biotechnology company who fractionated the active extracts and provided us with 80 active fractions for further testing against MAB growth. 46 Figure 7. Phenotypic screening of natural extracts and pure compounds libraries against MAB intracellularly. a Screening assay validation. Each screened plate contained at least four replicates of the negative (1 % DMSO) and positive (4 µM BDQ) controls, respectively. The average Z’-factor was calculated using percentages of growth inhibition of all the controls used in screening libraries against MAB intracellularly. b Percentages of growth inhibition and THP-1 cell survival of the libraries using single concentration activity determination assay against MAB intracellularly. The screen was performed once for each library. TCM library was screened in duplicates and yielded 21 intracellularly active TCM extracts 47 Table 11. Percentages of THP-1 cell survival and MAB growth inhibition of 21 intracellularly active TCM extracts. TCM Extract % THP-1 cell survival % MAB inhibition 57 90.69 44.94 60 100 33.97 77 97.67 46.9 78 100 39.74 79 95.83 41.34 83 99.7 45.37 84 99.36 55.42 89 100 37.11 93 99.74 38.27 95 90.54 39.64 100 86.05 44.27 105 96.89 48.91 132 77.30 87.04 188 88.12 41.42 257 76.80 45.75 288 96.32 49.52 298 92.57 45.6 303 92.25 38.01 329 80.65 40.50 332 95.81 37.54 335 83.86 44.32 In a THP-1 infection assay using 80 TCM fractions we did not identify intracellularly active fractions against MAB (Figure 8a). However, ten TCM fractions displayed > 80 % MAB growth inhibition intracellularly but with < 40 % THP-1 cell survival. We decided to lower the highest tested concentration from 100 µg/mL to 12.5 µg/mL and identified three intracellularly active fractions: TCM Fractions 53, 56, and 73 (Figure 8b, c, and d). Concentration-response assays revealed these active fractions worked in a dose-dependent manner. A decrease in the concentration of the active fractions led to a decrease in their inhibitory activity against MAB growth intracellularly coupled with an increase in THP-1 cell survival. 48 TCM Fraction 53, at 50 µg/mL displayed 100 % MAB growth inhibition intracellularly but only 5 % THP-1 cell survival (Figure 8b). However, this fraction at 25 µg/mL had a 77 % MAB growth inhibition and 78.5 % THP-1 cell survival. At 12.5 µg/mL, TCM Fraction 53 had a 94 % THP-1 cell survival and 23 % MAB growth inhibition. TCM Fraction 56, at 50 µg/mL had a 98 % percentage of THP-1 survival and 39 % MAB growth inhibition (Figure 8c). At 25 µg/mL or lower, TCM Fraction 56 reached 100 % THP-1 cell survival but with less than three percentage of MAB growth inhibition. TCM Fraction 73, at 50 µg/mL showed an 88 % THP-1 cell survival and 33 % MAB growth inhibition (Figure 8d). At 25 µg/mL or lower, TCM Fraction 73 displayed 96 % THP-1 cell survival but had below 12 % MAB growth inhibition. TCM fractions 53, 56, and 73 have been reported to our collaborators who will perform further fractionation to isolate the active compound against MAB intracellularly. 3.2.1.2 ME Library We did not test the ME against MAB using the THP-1 infection model. 3.2.1.3 PMC Library Our assay reported no intracellularly active compounds from the 251 PMC library. More than half of this library (54 %) attained above 70 % THP-1 cell survival but, of these compounds none displayed above 30 % MAB growth inhibition intracellularly. 3.2.1.4 GHP Box Our screen did not yield active compounds from 240 GHP box compounds using our THP-1 infection model. More than two-thirds of this library displayed above 70 % THP-1 cell survival but, of these compounds none displayed above 30 % MAB growth inhibition intracellularly. 49 Figure 8. Phenotypic screening of 80 TCM fractions against MAB intracellularly. a Percentages of growth inhibition and THP-1 cell survival of the 80 TCM fractions using single concentration activity determination assay against MAB intracellularly. The screen was performed once. b c d Dose dependent intracellular inhibitory activity against MAB and THP-1 cell survival of three active TCM fractions. The assay was run twice with two technical replicates each time (Z’-factor = 0.4). 50 Chapter Four 4.1 Discussion MAB drug discovery effort has been ongoing but the global increase in the incidence and prevalence of MAB-PD necessitates more research. The current MAB treatment regimen is long and often unsuccessful due to the pathogen’s intrinsic and acquired resistance to antibiotics. To accelerate the drug discovery effort, we performed a phenotypic screen using natural products and pure compounds libraries to identify active compounds against MAB in broth and intracellularly. Our work was in collaboration with Angal Biotechnology (Suzhou) Ltd, Dr. Raymond Andersen research group, and MMV team who supplied us with TCM, ME and PMC, and GHP box libraries, respectively. 4.1.1 Compounds 1 – 7 were active and displayed a dose dependent activity against MAB in broth Compounds 1 - 7 were active against MAB in broth, using a single concentration activity determination assay from ME at 100 µg/mL (compounds 1 – 3), PMC at 20 µg/mL (compound 4 – 6), and GHP box at 7.3 µg/mL (compound 7). Inhibitory activities against MAB in broth have not yet been reported for these compounds. Also, we determined MIC90 of compounds 1 – 7 against MAB in broth using the broth microdilution method. These compounds had MIC90 ranging from 3.6 – 25 µg/mL and displayed dose dependent antimycobacterial activities against MAB in broth. Dose dependency was a desirable outcome for these compounds to be considered as potential therapeutics. The use of NPs was advantageous to our work due to the diversity of compounds and their chemical structures, especially in the TCM, ME and PMC libraries. Our active compounds originated mostly from a marine sponge, Haliclona sp. (Order Haploscerida)84 except compounds 51 4 and 7. Compound 4 was derived from a marine ascidian, Didemnum sp.85 and compound 7 came from indole alkaloids.86 However, we cannot rule out the possibility that the active compounds can originate from residing microbial flora within the marine sponge population. Upon their initial isolation in the late 1980s, compounds 1 and 2 were found to have inhibitory activities against Candida albicans (yeast), B. subtilis, and S. aureus.87 In our assay, compound 1 had a lower MIC90 (8.7 µg/mL) than that of compound 2 (25 µg/mL). However, the chemical structures of these compounds are mostly similar except for the position of one hydrogen molecule. MAB’s extreme drug resistance could require higher inhibitory concentrations than those needed against other less resistant NTM. For instance, compound 2 was found, by a different group, to have inhibitory activity at a relatively low MIC (6.25 µg/mL) against a SGM, Mycobacterium marinum.88 Also, compound 1 and 2 MIC90 values against MAB in broth were lower than or comparable to those published by the Clinical and Laboratory Standards Institute (CLSI) for antibiotics against susceptible MAB isolates. For instance, MIC values against MAB in broth indicated by CLSI for clarithromycin, amikacin, imipenem, and linezolid were 8, 64, 32, and 32 µg/mL.89 Compound 3 has been a marine alkaloid of research interest since the 1990s when it was isolated and found to have inhibitory activity against B. subtilis, yeast, and Trichophyton mentagrophytes.90,91 We reported anti-MAB activity of this compound at MIC 8.9 µg/mL. Previous work had reported inhibitory activities of compound 3 against M. smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) at relatively low MIC, 2.5 and 1 µg/mL, respectively.91,92 However, compound 3 MIC90 was similar to that of clarithromycin and lower than amikacin’s, imipenem’s, and linezolid’s, according to CLSI data for antibiotics against susceptible MAB clinical strains. 52 Compound 4 was isolated for the first time in 1988 and was found to have an inhibitory activity ex-vivo against L1210 murine leukemia cells.93 In the present study, compound 4 inhibited MAB growth in broth at MIC90 14 µg/mL. This value is lower than that indicated by CLSI for amikacin, imipenem, and linezolid against susceptible MAB isolates in broth. When compound 5 was first isolated in 1986, it was found to be active against P388, murine leukemia cells.94 We calculated MIC90 9.5 µg/mL from compound 5 against MAB in broth. Compound 6 was isolated in 1994 as one of the derivatives of compound 5 and also had an antitumor activity against P388 cells.95 Our assay determined MIC90 12.5 µg/mL from compound 6 against MAB in broth. Compounds 5 and 6 had lower MIC90 values compared with the CLSI’s value for amikacin, imipenem, and linezolid against susceptible MAB isolates in broth. Indole alkaloids span a wide range of origins including plants and marine organisms such as sponges, tunicates, seaweeds, and shellfishes.96 Derived from a genus Penicillium, oxindole is a natural product that was used to synthesize indole for the first time in 1866.96 Since then numerous indole-based compounds have been generated and in particular, compound 7 had been reported to show inhibitory activity against Mtb97,98 and MAB99,100. In our screen, compound 7 had the lowest MIC90 (3.6 µg/mL) against MAB in broth. Our results were comparable to others who tested the inhibitory activity of this compound’s derivatives and reported a wide range of MIC (0.06 – 32 µg/mL).99,101 Also, compound 7 had a lower MIC90 value compared to CLSI’s values for clarithromycin, amikacin, imipenem, linezolid, and moxifloxacin against susceptible MAB clinical strains.89 Researchers suggest that MmpL3 could be the target protein of compound 7 since genetic mutations associated with this target have caused drug resistance.99,102 The antimycobacterial activity of our active compounds could be improved through generating compound derivatives and testing them against MAB. Structure-activity relationship 53 studies could be performed in collaboration with medicinal chemists, aiming to identify active compounds with decreased MIC values against MAB but maintaining dose dependency. 4.1.1.1 Cytotoxicity profiles of active compounds indicated varying selectivity We performed an MTT assay using compounds 1 - 7 and reported cytotoxicity profiles of active compounds with varying selectivity to HEK293T cells. The highest tested concentrations of compound 1 - 3, 4 - 6, and 7 were 100, 20, and 7.3 µg/mL, respectively. Compounds 1 - 4 had lower CC50 than MIC50, which indicated lower selectivity of these compounds to MAB than HEK293T cells (SI: 0.1 - 0.5). Compounds 5 - 7 had higher CC50 than MIC50, which indicated higher selectivity of these compounds to MAB than HEK293T cells (SI: 3.4 - >9.6). Compounds 1 and 2 had CC50 of 0.4 - 2.6 and 0.3 - 2.6 µg/mL, respectively against HEK293T, RAW264.7, and THP-1 cells. These values were comparable to those reported by others. For example, compounds 1 and 2 were previously found to inhibit growth at CC50 0.3 - 1.6 µg/mL and 0.3 - 0.5 µg/mL, respectively in breast, liver, prostate, blood, lymph, and colon cancer cells.103 Also, CC50 range of compound 3 (0.9 - 3.6 µg/mL) against HEK293T, RAW264.7, and THP-1 cells was similar to values published by others (1.8 - 6.0 µg/mL) against skin, blood, colon, and fibroblast cancer cells.104 The CC50 of compound 4 against HEK293T (CC50 3.8 µg/mL) was slightly higher than values reported by others (0.002 - 0.25 µg/mL) for this compound against brain, bladder, colon, breast, lung, and prostate cancer cells.105 Although the mechanism of action is unknown, the target of our compounds, for instance 1 - 4 could be specific to cancer cells. Moreover, in our study, compounds 5 and 6 were more selective to MAB than HEK293T cells with CC50 of 17 and 16.7 µg/mL, respectively. Also, when testing for antimalarial activity, other researchers found that compound 5 had higher selectivity to the parasite than to monkey kidney fibroblast cells (Vero).106,107 However, in another study, compounds 5 and 6 were reported to inhibit 54 the growth of leukemia cells with CC50 of 0.07108 and 0.8 µg/mL95, respectively. In addition, compound 5 was prior shown to reduce the growth of papilloma (KB) and colorectal (LoVo) cancer cells with CC50 of 0.05 and 0.15 µg/mL, respectively.109 Our data indicated that compound 7 was more selective to MAB than HEK293T cells with CC50 of >2.9 µg/mL. Other indole-2-carboxamide compounds have been previously found to be more selective to mycobacterial than host cells including both normal97,110 and cancer cells111. Our results on the cytotoxicity profiles of compounds 1 - 7 serve as preliminary data and these active compounds need to be tested further against a broad range of host cells. Also, in vivo studies using animal models need to be applied to determine the toxicity of these compounds to organisms. 4.1.1.2 Compounds 5, 6, and 7 exhibited antibacterial selectivity against mycobacteria and Gram-positive bacteria in broth We performed a broth microdilution assay to determine the antibacterial activity of compounds 1 - 7 against representative mycobacteria (M. smegmatis), Gram-positive bacteria (B. subtilis and S. aureus) and Gram-negative bacteria (E. coli and S. Tm) in broth. These bacteria were chosen since they are well-established models for microbiology studies. Compounds 5, 6, and 7 showed antibacterial selectivity against mycobacteria and Gram-positive bacteria. Determination of antibacterial selectivity was essential to our study because we aimed to identify active compounds with high antibacterial specificity against MAB, which could suggest their affinity toward a conserved mycobacterial target. Compounds 5 and 6 inhibited B. subtilis growth with MIC90 8.7 and 13.7 µg/mL, respectively. The mechanism of action of these compounds against Gram-positive bacteria haven’t been established. Gram positive bacteria are characterized by the presence of an inner cytoplasmic 55 membrane and a peptidoglycan-based cell wall.112,113 Large molecules can pass through the cell wall of Gram-positive bacteria due to a loose structural arrangement of peptidoglycan and its embedded components.114 This reason could have facilitated the growth inhibition of compounds 5 and 6 against B. subtilis regardless of these compounds’ high molecular weight, 548.8 and 581.8 g/mol, respectively. Further, we did not observe antibacterial activity from these compounds against E. coli and S. Tm. These results were consistent with those published by others115,116 and a likely cause of inactivity could have been membrane impermeability brought by lipopolysaccharides and phospholipids that effectively block antibacterial molecules from crossing the bacterial cell wall.114,117 Also, we reported growth inhibition of compound 5 against S. aureus with MIC90 11.3 µg/mL, a finding that aligned with data presented in previous studies.107,118,119 However, compound 6 displayed a weaker inhibitory activity against S. aureus than that of compound 5. This finding could be due to slight differences in the chemical structure of these two compounds. Compound 7 exhibited selective growth inhibition against MAB only. Since compound 7 has been hypothesized to inhibit MmpL3, we did not expect this compound to inhibit growth against bacteria lacking MmpL3. MmpL3 protein is so far known to be present only in mycobacteria and corynebacterial species.120,121 Compounds 1, 3, and 4 were non-selective in inhibiting growth against most of the representative bacterial species. The mechanisms of inhibition of these compounds have not been confirmed. In the past, a Haliclona sp. sponge sample containing algae species (dinoflagellates) was collected and researchers isolated compound 3, which was detected to have originated from the sponge, perhaps as an antimicrobial defense mechanism.122 It has previously been proposed 56 that compound 4 acted by cleaving DNA.123,124 Possibly, DNA damage drove non-selective inhibitory activity against the tested species in the present work. In our study, antibacterial activity from compounds 5, 6, and 7 was selective against the representative mycobacteria and Gram-positive bacteria. However, the growth inhibition of compounds 1, 3, and 4 was non-selective against the tested representative bacteria. Further studies are required to decipher the mechanism of action using active compounds, especially the ones that exhibited antibacterial selectivity. 4.1.2 TCM Fractions 53, 56, and 73 were active against MAB intracellularly Phenotypic screening of 371 TCM at 100 µg/mL identified 21 intracellularly active extracts using our THP-1 infection model. Bioassay guided fractionation of the active extracts in collaboration with Angal Biotech Ltd yielded 80 TCM fractions and further screening found three intracellularly active TCM fractions (53, 56, and 73). In these initial screening stages, we have observed most of the TCM library samples to maintain > 70 % THP-1 cell survival possibly due to role of these products as herbal medicine for humans. For example, TCM library had the highest percentage of samples (80 %) that had > 70 % THP-1 cell survival. However, one of the challenges pertaining to the use of herbal medicine is inadequate scientific knowledge to augment the administration of herbal medicine and ensure safety of the used products among people.72,74,125 We observed ten TCM fractions that reached 60 % THP-1 cell loss when tested at 100 µg/mL but at reduced concentrations (50, 25, 12.5 µg/mL), cell survival was achieved. From our work, we can attest to the importance of monitoring the consumption of herbal medications to maintain drug safety. Our collaborative work in identifying active TCM compounds against intracellular MAB growth is contributing efforts toward utilizing herbal medicine with sufficient knowledge of efficacy and safety. 57 4.2 Conclusion Our work involved a phenotypic screening of natural extracts and pure compounds libraries against MAB growth in broth and intracellularly. We obtained TCM extracts, marine products, and GHP box from our collaborators Angal Biotechnology (Suzhou) Ltd., Dr. Raymond Andersen Laboratory, and MMV team, respectively. We identified seven active pure compounds (1 – 7) against MAB in broth and the antimycobacterial activity of these compounds against MAB in broth had not been published before. Compounds 1 - 7 inhibited MAB growth in a dose-dependent activity with MIC90 values ranging from 3.6 – 25 µg/mL. These values were comparable to those reported by the CLSI (≤ 64 µg/mL) for some commonly prescribed antibiotics against susceptible MAB isolates. We determined the cytotoxicity profiles of compounds 1 - 7 and overall, these compounds showed varying selectivity to MAB than HEK293T cells. Further cytotoxicity studies need to be done using a broad range of host cells and importantly, testing the toxicity of our active compounds to organisms using animal models. The antimicrobial activity of compounds 1 - 7 showed that compounds 1 - 4 inhibited growth of the representative Gram-positive and Gram-negative bacterial species non-selectively. However, compounds 5 - 7 indicated selective antibacterial inhibition against mycobacteria and Gram-positive bacteria. This selective antibacterial activity could be associated with Gram-negative cell wall impermeability (compound 5 and 6) and specificity to a mycobacterial target (compound 7). Using our THP-1 infection model, we identified three active TCM fractions against intracellular MAB. TCM Fractions 53, 56, and 73 were reported to our collaborators for further fractionation to isolate the pure active compound(s). 58 4.3 Significance The incidence and prevalence of MAB-PD have been on the rise. MAB is mostly associated with PD due to this pathogen’s opportunistic nature and intrinsic resistance to most antibiotics. Individuals living with underlying lung diseases such as CF and bronchiectasis suffer the most from MAB-PD. Even worse, MAB treatment is long, but the outcome has not been impressive. Currently, there is no FDA-approved drug for the treatment of MAB-PD. Effort to populate the MAB drug pipeline has so far been channeled toward testing compounds that show activity against other pathogens, mainly Mtb. This initiative is an important starting point, but further work needs to be done to develop effective therapies for MAB treatment. Our work aimed to bridge the gap in MAB drug discovery field by testing natural products, which are known to possess rich, unique, and diverse chemicals for the identification of hits against MAB. Our research has identified active compounds against MAB in broth and intracellularly. This step is crucial in drug discovery since chemical modifications and further tests pertaining to the identified hits could advance them into lead compounds in pre-clinical studies. Collaboration with other researchers would eventually upgrade lead compounds to clinical trials. The few clinical studies that took place in the past one and a half decades have not led to approval of new anti-MAB drugs. Thus, this outcome highlights the need to approach drug discovery effort with robust and consistent initiatives. 4.4 Limitations In our phenotypic screening assay, we incubated MAB in broth for five days before taking OD600 measurements. It is possible that we could have obtained different results if we analyzed 59 data taken earlier during the incubation period because of the likelihood of denaturation of our natural extracts and pure compounds. Moreover, MIC90 determination of active compounds could have been conducted using a more robust assay such as the resazurin assay. In this colorimetric assay, a blue-colored resazurin is chemically reduced by viable cells, a reaction which leads to a color change to pink.126 The resazurin assay could have allowed for the analysis of visual results from plates in addition to the optical density measurements. Further, to effectively compare the MIC90 results from our active compounds with the published standard values for antibiotics, the cytotoxicity profiles of commonly used MAB antibiotics should have been determined. 4.5 Future directions Our identified active compounds in broth could benefit from future structure-activity relationship studies in collaboration with medicinal chemists. The aim would be to generate derivatives of the active compounds that would improve inhibitory activity against MAB, induce less toxicity against host cells, and increase antimycobacterial specificity. For example, compounds 1 - 3 would be excellent compounds to begin investigating further because their antimycobacterial activities have been underreported. Moreover, derivatives of our active compounds with improved activities could be tested against MAB clinical strains. Target identification is another relatively essential step in drug discovery work, especially for MAB because of the many unknown details about how this mycobacterium establishes an infection within the host organism. Mechanism of action studies could begin with target identification using our active compounds. Target identification can be performed through generating resistant bacteria from spontaneous mutations by increasing the concentration of active compounds above 2x the MIC against MAB. After extracting the genomic DNA of resistant strains, 60 whole genome sequencing could be conducted to identify single nucleotide polymorphisms as well as indels. Gene analysis would then determine genes or targets associated with resistance and study their function(s) in bacterial growth and survival. 61 References 1. Johnson, M. M. & Odell, J. A. Nontuberculous mycobacterial pulmonary infections. J Thorac Dis 6, 210–220 (2014). 2. Kwon, Y.-S. & Koh, W.-J. Diagnosis and Treatment of Nontuberculous Mycobacterial Lung Disease. J Korean Med Sci 31, 649–659 (2016). 3. Ratnatunga, C. N. et al. The Rise of Non-Tuberculosis Mycobacterial Lung Disease. Front Immunol 11, 303 (2020). 4. Honda, J. R., Virdi, R. & Chan, E. D. Global Environmental Nontuberculous Mycobacteria and Their Contemporaneous Man-Made and Natural Niches. Front. Microbiol. 9, (2018). 5. Bento, C. M., Gomes, M. S. & Silva, T. Looking beyond Typical Treatments for Atypical Mycobacteria. Antibiotics 9, 18 (2020). 6. Gopalaswamy, R., Shanmugam, S., Mondal, R. & Subbian, S. Of tuberculosis and non-tuberculous mycobacterial infections – a comparative analysis of epidemiology, diagnosis and treatment. J Biomed Sci 27, 74 (2020). 7. Cruz-Aguilar, M., Castillo-Rodal, A. I., Arredondo-Hernández, R. & López-Vidal, Y. Non-tuberculous mycobacteria immunopathogenesis: Closer than they appear. a prime of innate immunity trade-off and NTM ways into virulence. Scandinavian Journal of Immunology 94, e13035 (2021). 8. Dahl, V. N. et al. Global trends of pulmonary infections with nontuberculous mycobacteria: a systematic review. International Journal of Infectious Diseases 125, 120–131 (2022). 9. Przybylski, G., Bukowski, J., Kowalska, W., Pilaczyńska-Cemel, M. & Krawiecka, D. Trends from the Last Decade with Nontuberculous Mycobacteria Lung Disease (NTM-LD): Clinicians’ Perspectives in Regional Center of Pulmonology in Bydgoszcz, Poland. Pathogens 12, 988 (2023). 10. Ringshausen, F. C. et al. Predictive modeling of nontuberculous mycobacterial pulmonary disease epidemiology using German health claims data. International Journal of Infectious Diseases 104, 398–406 (2021). 11. Johansen, M. D., Herrmann, J.-L. & Kremer, L. Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus. Nat Rev Microbiol 18, 392–407 (2020). 62 12. Franco-Paredes, C. et al. Cutaneous Mycobacterial Infections. Clinical Microbiology Reviews 32, e00069-18 (2018). 13. Lee, M.-R. et al. Mycobacterium abscessus Complex Infections in Humans. Emerg Infect Dis 21, 1638–1646 (2015). 14. Ryan, K. & Byrd, T. F. Mycobacterium abscessus: Shapeshifter of the Mycobacterial World. Frontiers in Microbiology 9, (2018). 15. Quang, N. T. & Jang, J. Current Molecular Therapeutic Agents and Drug Candidates for Mycobacterium abscessus. Frontiers in Pharmacology 12, (2021). 16. Victoria, L., Gupta, A., Gómez, J. L. & Robledo, J. Mycobacterium abscessus complex: A Review of Recent Developments in an Emerging Pathogen. Frontiers in Cellular and Infection Microbiology 11, (2021). 17. Marshall, J. E., Mercaldo, R. A., Lipner, E. M. & Prevots, D. Rebecca. Incidence of nontuberculous mycobacteria infections among persons with cystic fibrosis in the United States (2010–2019). BMC Infectious Diseases 23, 489 (2023). 18. Wetzstein, N. et al. Molecular Epidemiology of Mycobacterium abscessus Isolates Recovered from German Cystic Fibrosis Patients. Microbiol Spectr 10, e01714-22. 19. Yoshida, M. et al. Molecular Epidemiological Characteristics of Mycobacterium abscessus Complex Derived from Non-Cystic Fibrosis Patients in Japan and Taiwan. Microbiology Spectrum 10, e00571-22 (2022). 20. Abdelaal, H. F. M., Chan, E. D., Young, L., Baldwin, S. L. & Coler, R. N. Mycobacterium abscessus: It’s Complex. Microorganisms 10, 1454 (2022). 21. Degiacomi, G., Riabova, O., Makarov, V. & Pasca, M. R. Mycobacterium abscessus, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients. Int. J. Mol. Sci. 21 (2019). 22. Laencina, L. et al. Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus. Proceedings of the National Academy of Sciences 115, E1002–E1011 (2018). 23. Dubois, V. et al. Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages. PLOS Pathogens 15, e1008069 (2019). 63 24. Dedrick, R. M. et al. The problem of Mycobacterium abscessus complex: multi-drug resistance, bacteriophage susceptibility and potential healthcare transmission. Clinical Microbiology and Infection 29, 1335.e9-1335.e16 (2023). 25. Redondo, N. et al. Genomic Analysis of Mycobacterium abscessus Complex Isolates Collected in Ireland between 2006 and 2017. Journal of Clinical Microbiology (2020) doi:10.1128/jcm.00295-20. 26. Doyle, R. M. et al. Cross-transmission Is Not the Source of New Mycobacterium abscessus Infections in a Multicenter Cohort of Cystic Fibrosis Patients. Clinical Infectious Diseases 70, 1855–1864 (2020). 27. Fujiwara, K. et al. Potential Cross-Transmission of Mycobacterium abscessus among Non-Cystic Fibrosis Patients at a Tertiary Hospital in Japan. Microbiol Spectr 10, e00097-22. 28. Parmar, S. & Tocheva, E. I. The cell envelope of Mycobacterium abscessus and its role in pathogenesis. PLOS Pathogens 19, e1011318 (2023). 29. Kam, J. Y. et al. Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish. Nat Commun 13, 952 (2022). 30. Daher, W. et al. Glycopeptidolipid glycosylation controls surface properties and pathogenicity in Mycobacterium abscessus. Cell Chemical Biology 29, 910-924.e7 (2022). 31. Schorey, J. S. & Sweet, L. The mycobacterial glycopeptidolipids: structure, function, and their role in pathogenesis. Glycobiology 18, 832–841 (2008). 32. Rhoades, E. R. et al. Mycobacterium abscessus Glycopeptidolipids Mask Underlying Cell Wall Phosphatidyl-myo-Inositol Mannosides Blocking Induction of Human Macrophage TNF-α by Preventing Interaction with TLR21. The Journal of Immunology 183, 1997–2007 (2009). 33. Roux, A.-L. et al. The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages. Open Biol 6, 160185 (2016). 34. Sancho-Vaello, E., Albesa-Jové, D., Rodrigo-Unzueta, A. & Guerin, M. E. Structural basis of phosphatidyl-myo-inositol mannosides biosynthesis in mycobacteria. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1862, 1355–1367 (2017). 64 35. Boldrin, F. et al. The Phosphatidyl-myo-Inositol Dimannoside Acyltransferase PatA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo. Journal of Bacteriology 203, 10.1128/jb.00439-20 (2021). 36. Sakamoto, K. et al. Mycobacterial Trehalose Dimycolate Reprograms Macrophage Global Gene Expression and Activates Matrix Metalloproteinases. Infection and Immunity 81, 764–776 (2013). 37. Actor, J. K. Trehalose Dimycolate (Cord Factor) as a Contributing Factor to Tuberculosis Pathogenesis. in Tuberculosis Host-Pathogen Interactions (eds. Cirillo, J. D. & Kong, Y.) 43–61 (Springer International Publishing, Cham, 2019). doi:10.1007/978-3-030-25381-3_3. 38. Llorens-Fons, M. et al. Trehalose Polyphleates, External Cell Wall Lipids in Mycobacterium abscessus, Are Associated with the Formation of Clumps with Cording Morphology, Which Have Been Associated with Virulence. Front. Microbiol. 8, (2017). 39. Richter, A., Shapira, T. & Av-Gay, Y. THP-1 and Dictyostelium Infection Models for Screening and Characterization of Anti-Mycobacterium abscessus Hit Compounds. Antimicrob Agents Chemother 64, e01601-19 (2019). 40. Hanh, B. T. B. et al. Etamycin as a Novel Mycobacterium abscessus Inhibitor. Int J Mol Sci 21, 6908 (2020). 41. Daley, C. L. et al. Treatment of Nontuberculous Mycobacterial Pulmonary Disease: An Official ATS/ERS/ESCMID/IDSA Clinical Practice Guideline. Eur Respir J 56, 2000535 (2020). 42. Fröberg, G. et al. Towards clinical breakpoints for non-tuberculous mycobacteria – Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution. Clinical Microbiology and Infection 29, 758–764 (2023). 43. Kwak, N. et al. Mycobacterium abscessus pulmonary disease: individual patient data meta-analysis. European Respiratory Journal 54, (2019). 44. Sullivan, J. R. et al. Natural Products Lysobactin and Sorangicin A Show In Vitro Activity against Mycobacterium abscessus Complex. Microbiology Spectrum 0, e02672-22 (2022). 45. Wu, M.-L., Aziz, D. B., Dartois, V. & Dick, T. NTM drug discovery: status, gaps and the way forward. Drug Discovery Today 23, 1502–1519 (2018). 65 46. Richard, M. et al. Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus. Antimicrobial Agents and Chemotherapy 63, 10.1128/aac.01316-18 (2018). 47. Luthra, S., Rominski, A. & Sander, P. The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance. Front. Microbiol. 9, (2018). 48. Bleicher, K. H., Böhm, H.-J., Müller, K. & Alanine, A. I. Hit and lead generation: beyond high-throughput screening. Nat Rev Drug Discov 2, 369–378 (2003). 49. Hughes, J., Rees, S., Kalindjian, S. & Philpott, K. Principles of early drug discovery. Br J Pharmacol 162, 1239–1249 (2011). 50. Nguyen, P. C. et al. Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis. Sci Rep 7, 11751 (2017). 51. Aragaw, W. W. et al. In Vitro Resistance against DNA Gyrase Inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus. Microbiol Spectr 10, e01321-21. 52. Study Details | Tigecycline for Treatment of Rapidly Growing Mycobacteria | ClinicalTrials.gov. https://clinicaltrials.gov/study/NCT00600600. 53. Study Details | Inhaled Nitric Oxide for Patients With MABSC | ClinicalTrials.gov. https://clinicaltrials.gov/study/NCT03208764?intr=Nitric%20Oxide&aggFilters=phase:2&cond=NTM&rank=1. 54. Study Details | Liposomal Amikacin for Inhalation (LAI) in the Treatment of Mycobacterium abscessus Lung Disease | ClinicalTrials.gov. https://clinicaltrials.gov/study/NCT03038178?cond=NTM%20pulmonary%20m.%20abscessus&intr=Liposomal%20amikacin%20for%20inhalation%20%5C(LAI%5C)&rank=1. 55. Li, Z., Chen, K., Rose, P. & Zhu, Y. Z. Natural products in drug discovery and development: Synthesis and medicinal perspective of leonurine. Front. Chem. 10, (2022). 56. Sahile, H. A. et al. Screening of diverse marine invertebrate extracts identified Lissoclinotoxin F, Discodermin B, and other anti-Mycobacterium tuberculosis active compounds. J Antibiot 75, 213–225 (2022). 57. García-Davis, S. et al. Antimycobacterial Activity of Laurinterol and Aplysin from Laurencia johnstonii. Mar Drugs 18, 287 (2020). 66 58. Dias, D. A., Urban, S. & Roessner, U. A Historical Overview of Natural Products in Drug Discovery. Metabolites 2, 303–336 (2012). 59. Porras, G. et al. Ethnobotany and the Role of Plant Natural Products in Antibiotic Drug Discovery. Chem. Rev. 121, 3495–3560 (2021). 60. PubChem. Artemisinin. https://pubchem.ncbi.nlm.nih.gov/compound/68827. 61. Atanasov, A. G., Zotchev, S. B., Dirsch, V. M. & Supuran, C. T. Natural products in drug discovery: advances and opportunities. Nat Rev Drug Discov 20, 200–216 (2021). 62. Zhu, J., Liu, Y., Liu, Z., Wang, H. & Zhang, H. Bioactive Nitrogenous Secondary Metabolites from the Marine Sponge Genus Haliclona. Mar Drugs 17, 682 (2019). 63. Bae, S.-J. et al. Protective effects of a traditional herbal extract from Stellaria dichotoma var. lanceolata against Mycobacterium abscessus infections. PLoS One 13, e0207696 (2018). 64. Persaud, R. et al. Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1. Cell Chemical Biology 29, 870-882.e11 (2022). 65. Sirichoat, A. et al. Assessment of antimycobacterial activities of pure compounds extracted from Thai medicinal plants against clarithromycin-resistant Mycobacterium abscessus. PeerJ 9, e12391 (2021). 66. Maiolini, M. et al. The War against Tuberculosis: A Review of Natural Compounds and Their Derivatives. Molecules 25, 3011 (2020). 67. Addison, W., Frederickson, M., G. Coyne, A. & Abell, C. Potential therapeutic targets from Mycobacterium abscessus ( Mab ): recently reported efforts towards the discovery of novel antibacterial agents to treat Mab infections. RSC Medicinal Chemistry 13, 392–404 (2022). 68. Subhash, N. & Sundaramurthy, V. Advances in host-based screening for compounds with intracellular anti-mycobacterial activity. Cellular Microbiology 23, e13337 (2021). 69. Grzelak, E. M. et al. Strategies in anti-Mycobacterium tuberculosis drug discovery based on phenotypic screening. J Antibiot 72, 719–728 (2019). 70. Burke, C. et al. Development of a novel secondary phenotypic screen to identify hits within the mycobacterial protein synthesis pipeline. FASEB Bioadv 2, 600–612 (2020). 71. Homeyer, N. et al. A platform for target prediction of phenotypic screening hit molecules. Journal of Molecular Graphics and Modelling 95, 107485 (2020). 67 72. Zhu, Y.-P. & Woerdenbag, H. J. Traditional Chinese herbal medicine. Pharm World Sci 17, 103–112 (1995). 73. Xu, Q. et al. The quest for modernisation of traditional Chinese medicine. BMC Complement Altern Med 13, 132 (2013). 74. Zhou, X., Li, C.-G., Chang, D. & Bensoussan, A. Current Status and Major Challenges to the Safety and Efficacy Presented by Chinese Herbal Medicine. Medicines 6, 14 (2019). 75. Zhou, X. et al. Synergistic Effects of Chinese Herbal Medicine: A Comprehensive Review of Methodology and Current Research. Front. Pharmacol. 7, (2016). 76. Williams, D. E. & Andersen, R. J. Biologically Active Marine Natural Products and Their Protein Targets Discovered Using a Chemical Genetics Approach. Nat Prod Rep 37, 617–633 (2020). 77. About the Global Health Priority Box. Medicines for Malaria Venture https://www.mmv.org/mmv-open/global-health-priority-box/about-global-health-priority-box. 78. Richter, A., Strauch, A., Chao, J., Ko, M. & Av-Gay, Y. Screening of Preselected Libraries Targeting Mycobacterium abscessus for Drug Discovery. Antimicrob Agents Chemother 62, e00828-18 (2018). 79. THP-1 - TIB-202 | ATCC. https://www.atcc.org/products/tib-202. 80. RAW 264.7 - TIB-71 | ATCC. https://www.atcc.org/products/tib-71. 81. 293T - CRL-3216 | ATCC. https://www.atcc.org/products/crl-3216. 82. Zhang, J.-H., Chung, T. D. Y. & Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. SLAS Discovery 4, 67–73 (1999). 83. Zheng, X. & Av-Gay, Y. System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis. JoVE 55273 (2017) doi:10.3791/55273. 84. Althagbi, H. I., Alarif, W. M., Al-Footy, K. O. & Abdel-Lateff, A. Marine-Derived Macrocyclic Alkaloids (MDMAs): Chemical and Biological Diversity. Marine Drugs 18, 368 (2020). 85. Palanisamy, S. K., Rajendran, N. M. & Marino, A. Natural Products Diversity of Marine Ascidians (Tunicates; Ascidiacea) and Successful Drugs in Clinical Development. Nat Prod Bioprospect 7, 1–111 (2017). 68 86. Chehardoli, G. & Bahmani, A. Synthetic strategies, SAR studies, and computer modeling of indole 2 and 3-carboxamides as the strong enzyme inhibitors: a review. Mol Divers 25, 535–550 (2021). 87. Fahy, E. et al. Haliclonadiamine, an antimicrobial alkaloid from the sponge Haliclona SP. Tetrahedron Letters 29, 3427–3428 (1988). 88. Barker, L. P. et al. A Mycobacterium marinum zone of inhibition assay as a method for screening potential antimycobacterial compounds from marine extracts. Planta Med 73, 559–563 (2007). 89. Kim, Y. J. et al. Trend of nontuberculous mycobacteria species and minimal inhibitory concentration in a referral laboratory in Korea from 2013 to 2019. Journal of Infection and Public Health 17, 212–216 (2024). 90. Clark, R. J. et al. The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp. Tetrahedron 54, 8811–8826 (1998). 91. Charan, R. D., Garson, M. J., Brereton, I. M., Willis, A. C. & Hooper, J. N. A. Haliclonacyclamines A and B, cytotoxic alkaloids from the tropical marine sponge Haliclona sp. Tetrahedron 52, 9111–9120 (1996). 92. Arai, M., Ishida, S., Setiawan, A. & Kobayashi, M. Haliclonacyclamines, Tetracyclic Alkylpiperidine Alkaloids, as Anti-dormant Mycobacterial Substances from a Marine Sponge of Haliclona sp. Chem. Pharm. Bull. 57, 1136–1138 (2009). 93. Kobayash, J. et al. Ascididemin, a novel pentacyclic aromatic alkaloid with potent antileukemic activity from the okinawan tunicate didemnum sp. Tetrahedron Letters 29, 1177–1180 (1988). 94. Sakai, Ryuichi., Higa, Tatsuo., Jefford, C. W. & Bernardinelli, Gerald. Manzamine A, a novel antitumor alkaloid from a sponge. J. Am. Chem. Soc. 108, 6404–6405 (1986). 95. Crews, P. et al. 1,2,3,4-tetrahydro-8-hydroxymanzamines, alkaloids from two different haplosclerid sponges. Tetrahedron 50, 13567–13574 (1994). 96. Bajad, N. G., Singh, S. K., Singh, S. K., Singh, T. D. & Singh, M. Indole: A promising scaffold for the discovery and development of potential anti-tubercular agents. Current Research in Pharmacology and Drug Discovery 3, 100119 (2022). 97. Lun, S. et al. Indoleamides are active against drug-resistant Mycobacterium tuberculosis. Nat Commun 4, 2907 (2013). 69 98. Onajole, O. K. et al. Preliminary Structure–Activity Relationships and Biological Evaluation of Novel Antitubercular Indolecarboxamide Derivatives Against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains. J. Med. Chem. 56, 4093–4103 (2013). 99. Kozikowski, A. P. et al. Targeting Mycolic Acid Transport by Indole-2-carboxamides for the Treatment of Mycobacterium abscessus Infections. J. Med. Chem. 60, 5876–5888 (2017). 100. Pandya, A. N. et al. Indole-2-Carboxamides Are Active against Mycobacterium abscessus in a Mouse Model of Acute Infection. Antimicrobial Agents and Chemotherapy 63, 10.1128/aac.02245-18 (2019). 101. Franz, N. D. et al. Design, Synthesis and Evaluation of Indole-2-carboxamides with Pan Anti-mycobacterial Activity. Bioorg Med Chem 25, 3746–3755 (2017). 102. Bhattarai, P. et al. Structural Determinants of Indole-2-carboxamides: Identification of Lead Acetamides with Pan Antimycobacterial Activity. J. Med. Chem. 66, 170–187 (2023). 103. Yamazaki, H. et al. Papuamine and haliclonadiamine, obtained from an Indonesian sponge Haliclona sp., inhibited cell proliferation of human cancer cell lines. Natural Product Research 27, 1012–1015 (2013). 104. Torres, Y. R. et al. Antibacterial activity against resistant bacteria and cytotoxicity of four alkaloid toxins isolated from the marine sponge Arenosclera brasiliensis. Toxicon 40, 885–891 (2002). 105. Delfourne, E. et al. Synthesis and In Vitro Antitumor Activity of Novel Ring D Analogues of the Marine Pyridoacridine Ascididemin: Structure−Activity Relationship. J. Med. Chem. 45, 3765–3771 (2002). 106. Shilabin, A. G., Kasanah, N., Tekwani, B. L. & Hamann, M. T. Kinetic Studies and Bioactivity of Potential Manzamine Prodrugs. J. Nat. Prod. 71, 1218–1221 (2008). 107. Peng, J. et al. Structure Activity Relationship and Mechanism of Action Studies of Manzamine Analogues for the Control of Neuroinflammation and Cerebral Infections. J Med Chem 53, 61–76 (2010). 108. Sakai, Ryuichi., Higa, Tatsuo., Jefford, C. W. & Bernardinelli, Gerald. Manzamine A, a novel antitumor alkaloid from a sponge. J. Am. Chem. Soc. 108, 6404–6405 (1986). 109. Ichiba, T., Corgiat, J. M., Scheuer, P. J. & Kelly-Borges, M. 8-Hydroxymanzamine A, a β-Carboline Alkaloid from a Sponge, Pachypellina sp. J. Nat. Prod. 57, 168–170 (1994). 70 110. Stec, J. et al. Indole-2-carboxamide-based MmpL3 Inhibitors Show Exceptional Antitubercular Activity in an Animal Model of Tuberculosis Infection. J. Med. Chem. 59, 6232–6247 (2016). 111. Bhakhar, K. A. et al. Indole-2-carboxamides as New Anti-Mycobacterial Agents: Design, Synthesis, Biological Evaluation and Molecular Modeling against mmpL3. ChemistrySelect 7, e202201813 (2022). 112. Rohde, M. The Gram-Positive Bacterial Cell Wall. Microbiology Spectrum 7, 10.1128/microbiolspec.gpp3-0044–2018 (2019). 113. Silhavy, T. J., Kahne, D. & Walker, S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). 114. Lambert, P. a. Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. Journal of Applied Microbiology 92, 46S-54S (2002). 115. Ibrahim, M. A. et al. 2-N-Methyl modifications and SAR studies of manzamine A. Bioorganic & Medicinal Chemistry 16, 6702–6706 (2008). 116. Samoylenko, V. et al. Bioactive (+)-Manzamine A and (+)-8-Hydroxymanzamine A Tertiary Bases and Salts from Acanthostrongylophora ingens and their Preparations. Nat Prod Commun 4, 185–192 (2009). 117. Arzanlou, M., Chai, W. C. & Venter, H. Intrinsic, adaptive and acquired antimicrobial resistance in Gram-negative bacteria. Essays in Biochemistry 61, 49–59 (2017). 118. Edrada, R. A. et al. Four New Bioactive Manzamine-Type Alkaloids from the Philippine Marine Sponge Xestospongia ashmorica. J. Nat. Prod. 59, 1056–1060 (1996). 119. Hu, J.-F., Hamann, M. T., Hill, R. & Kelly, M. THE MANZAMINE ALKALOIDS. Alkaloids Chem Biol 60, 207–285 (2003). 120. Williams, J. T. & Abramovitch, R. B. Molecular Mechanisms of MmpL3 Function and Inhibition. Microbial Drug Resistance 29, 190–212 (2023). 121. Varela, C. et al. MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria. Chem Biol 19, 498–506 (2012). 122. Garson, M. J., Flowers, A. E., Webb, R. I., Charan, R. D. & McCaffrey, E. J. A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation. Cell Tissue Res 293, 365–373 (1998). 71 123. Matsumoto, S. S., Biggs, J., Copp, B. R., Holden, J. A. & Barrows, L. R. Mechanism of Ascididemin-Induced Cytotoxicity. Chem. Res. Toxicol. 16, 113–122 (2003). 124. Watters, D. J. Ascidian Toxins with Potential for Drug Development. Marine Drugs 16, 162 (2018). 125. Wang, J. et al. Quality and safety of Chinese herbal medicines guided by a systems biology perspective. Journal of Ethnopharmacology 126, 31–41 (2009). 126. Petiti, J., Revel, L. & Divieto, C. Standard Operating Procedure to Optimize Resazurin-Based Viability Assays. Biosensors 14, 156 (2024).
Thesis/Dissertation
eng
Microbiology and Immunology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Av-Gay, Yossef
Identification of active compounds against Mycobacterium abscessus
Text