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UBC Theses and Dissertations

Screening, characterization and inhibition of viral cysteine proteinases Huitema , Carly


3C proteinases (3Cpros) are a family of essential cysteine proteinases found in various viruses of medical and agricultural significance. Three lines of research related to the characterization of 3Cpros were pursued. In the first, biological selections and screens were developed to evaluate proteinase activity in a high-throughput fashion. A selection system based on the cleavage of an engineered transcriptional regulator, XylR, by hepatitis A virus (HAV) 3Cpro failed. However, this strategy facilitated the development of a screen based on the cleavage of fused green fluorescent protein variants, CyPet and YPet. The screen was used to demonstrate that HAV 3Cpro prefers Ile, Val or Leu at the P₄ position of the cleavage sequence, and Gly, Ser or Ala at the P₁’ position. In the second project, the 3Cpro from Israeli acute paralysis virus (IAPV), a dicistrovirus, was investigated. IAPV has been associated with the recent colony collapse disorder afflicting commercial hives. A portion of the replicase including 3Cpro was heterologously produced. The resulting autoprocessed fragments were analyzed using mass spectrometry to identify the 3Cpro cleavage sequence PIVIE/AQT. This cleavage sequence likely occurs between the 3A/3B proteins of the polypeptide and is the first within the replicase to be described in this family of viruses. Thirdly, inhibitors of HAV 3Cpro and SARS 3CLpro were developed. The keto- glutamine analogue (HIP2-171-2) competitively inhibited SARS 3CLpro with a Kic = 0.17 ± 0.03 μM and is among the most potent peptide inhibitors developed against this proteinase. The azapeptide epoxide (APE) KAE-3-91 irreversibly inhibited SARS 3CLpro with a kinact/Ki of 1900 ± 400 M−¹s−¹, which is similar in magnitude to that of the first generation APE’s produced to inhibit caspases. Finally, both SARS 3CLpro and HAV 3Cpro were screened against a library of inhibitory halopyridinyl esters. Each of three halopyridinyl esters inhibited 3Cpro with apparent Kic’s of 120-240 nM. However, further study revealed that the inhibitors were slowly hydrolyzed by both proteinases. Overall, the described screens and inhibitors should facilitate the further characterization of 3C and related proteinases as well as the development of novel antivirals.

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