Our long term objectives are twofold: 1) to use a novel chemical tool set to study a critical host-pathogen inter- face and its role in virus life cycles;2) We also seek to validate a focused set of targets that can serve as the basis for future chemical screens to identify drug candidates, as well as an annotate all available pathogen genomes that are susceptible to chemical inhibition of these targets. Phosphoinositide (PI) 4-kinase activity is essential for generating specific phosphoinositides such as PI-4,5-bisphosphate (PI(4,5)P2, or "PIP2"). We discovered a novel PIP2 binding motif within the hepatitis C virus (HCV) NS5A protein. This motif, termed a Basic Amino Acid PIP2 Pincer (or "BAAPP domain"), mediates specific interaction with PIP2, and point mutations in the BAAPP domain abrogate PIP2 binding and HCV RNA replication. Similar BAAPP domains are found in pathogens as diverse as rhinovirus and P. falciparum. Finally, small molecules recently developed to inhibit PIP2 production without host toxicity by targeting specific PI 4-kinase (PI4K) family members-the PI4KIII alpha (PI4KIII?) and beta (PI4KIII?) isoforms--potently inhibit HCV replication in vitro (e.g. inhibitor PT423 exhibits IC50 365 nM;CC50 >10microM), and display comparable activity against several BAAPP domain-containing pathogens tested to date. We now seek to: 1) Further probe and exploit the role of PI4KIII in the HCV life cycle by: a) determining the effect of PT423 treatment on HCV replication complex integrity and enzymatic activity using biochemical fractionation and membrane-associated replicase assays;b) evaluating the potential for inhibitors of PI4KIII to synergize with other classes of anti-HCV agents targeting replicase complex assembly and function;c) providing proof-of-concept that PI4KIII inhibitors can inhibit multidrug resistant viruses by determining the susceptibility to PT423 of HCV variants resistant to various classes of HCV antivirals;2) Further validate PI4KIII as a target for future drug screening efforts by: a) performing cellular pull-down assays, in the presence or absence of HCV, with PT423-linked beads coupled with mass spectrometry to identify candidate off-target proteins and how that spectrum might change in the face of viral infection;b) further validating the mechanism of action of our model PI4KIII inhibitors by studying PT423 levels required for HCV inhibition in the presence of either siRNA against PI4KIII?, or PI4KIII? mutants with engineered candidate mutations conferring resistance to PT423;3) Determine the critical BAAPP domain features that define susceptibility to chemical inhibitors of PI4KIII by: a) determining the crystal structure of full length NS5A, with and withou PIP2;b) performing a structure/function analysis of BAAPP domain peptides to define their key determinants of PIP2 binding using quartz crystal microbalance, fluorescence polarization, and the nuclear magnetic resonance structures of the peptides;c) developing an algorithm to automatically identify the above determined key features of a PIP2 binding BAAPP domain within databases of all available pathogen genomes;d) providing proof-of-concept that identified pathogens are susceptible to inhibition with PI4KIII inhibitors.
We seek to use a novel chemical toolset to both study the role of a subset of PI 4-kinases in viral life cycles and validate these kinases as targets that ca serve as the basis for future chemical screens to identify drug candidates. As such, this will be a powerful example of how chemical tools can lead to exciting new treatments for a wide array of infectious diseases, and provide a critical solution to the emergence of resistance to other types of anti-infectives.
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