structure and function of viral polymerases€¦ · structure and function of viral polymerases ....
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Structure and Function of Viral Polymerases
Human Immunodeficiency Virus (HIV)Reverse Transcriptase (RT)
Hepatitis C Virus (HCV)RNA-dependent RNA Polymerase
Herpes VirusesDNA-dependent DNA Polymerase
Major Research Interests
Viral Polymerases are Important Targets in Drug Discovery/Development Efforts
HIV Reverse Transcriptase (RT): nucleoside and non-nucleoside analogues are approved for clinical use.
HCV RNA-dependent RNA Polymerase: nucleoside and non-nucleoside analogues are currently evaluated in clinical trials.
Herpes virus associated DNA-dependent DNA Polymerases: nucleoside analogues, such as acyclovir and the pyrophosphate mimic foscarnet are approved and used in the clinic.
Limitations with Existing Drugs
Available drugs do not work against each of the eight different Herpes viruses:HSV-1 and HSV-2, VZV, HCMV, EBV, HHV-6, HHV-7 and HHV-8.
Infection with these viruses can cause a wide range of human disease, including neurological disorders, retinitis, chickenpox, shingles, cancer….
Immunocompromised individuals, including transplant patients, are the most vulnerable.
Adverse effects, poor bioavailability, and/or the emergence of drug resistance can compromise therapy with existing anti-herpetic drugs.
There is an urgent need for safe and potent anti-herpetic drugs that can also be used pre-emptively in transplant patients.
Problems in the Discovery and Development of anti-herpetic Polymerase Inhibitors
Herpes virus associated DNA polymerases are difficult to express/purify.
Success in the development of assays that facilitate the screening and characterization for polymerase inhibitors has therefore been limited.
Cell-based drug susceptibility assays are time-consuming, (often) inaccurate, or not even available for each of the eight different viruses.
Objective
Develop a surrogate system that facilitate the discovery and development of polymerase active site inhibitors
Similarities among Viral DNA polymerases and their Orthologs in Phages The polymerase of bacteriophage RB69 (gp43) can be expressed and purified at high yields
Yang et al, Biochemistry 2002
Approach: Design of Phage/Virus Chimeras
Predicted/desired properties:
1. The chimeric enzymes can be expressed in bacteria (E.coli)
2. Chimeras are sensitive to antiviral drugs
Swap the natural substrate binding sites:Introduce helix N and helix P of herpesvirus polymerases against a backbone of gp43
RB69 gp43 is not sensitive to PFA; The Chimera ABC is!
sp
0Min
us M
g2+
0.4
1.2
3.7
PFAµM
gp43
11 33 100
0 0.4
1.2
3.7
PFAµM
ABC
11 33 100
0 0.4
1.2
3.7
PFAµM
ABC/R784A
11 33 100
0 0.4
1.2
3.7
PFAµM
ABC/Q807A
11 33 100
gp43 ABC ABC/R784A ABC/Q807A
IC50, µM > 100 2.9 27 29
Tchesnokov et al. , J Biol Chem 2009
Major Deliverables
Constructs for chimeric enzymes that represent each of the eight herpes virus associated DNA polymerases.
Potential Applications
Use of purified chimeras in screening efforts.
Use of purified enzymes in the characterization of investigational compounds with respect to potency and selectivity.
Use of chimeras to identify antiviral compounds in bacteria.
Collaborators
Guy Boivin, MD, MSC, FRCPC
Holder of the Canada Research Chair on emerging viruses and antiviral resistance
Centre de recherche en infectiologie du CHULUniversité Laval
Develop cell-based drug susceptibility assays to validate the new platform.
Contribute to Discovery and Development of Antivirals
TargetValidation
AssayDevelopment
Drug DiscoveryDevelopment Clinic
Resistance
Impact of CQDM funding on our Research
CQDM
Academia Industry Academia