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

Structure-Based Alignment of Herpes Virus and Phage associated Polymerases

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

dTTP

Preliminary Data: Design of HCMV/RB69 Chimeras

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

Egor Tchesnokov Greg Beilhartz Brian Scarth Maryam Ehteshami Suzanne McCormick Megan Powdrill Colins Vasquez Mia Biondi Jean Bernatchez Bruno Marchand

McGill UniversityDepartment of Microbiology and Immunology

CRS, CFI, NCRTPFRSQ, CANFAR, NIH, Tibotec, Merck, Gilead, ViroChem, GSK

Funding