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Discovery Novel Allosteric Fragment Inhibitors of HIV-1 Reverse

Transcriptase for HIV Prevention

A/Prof Gilda Tachedjian Retroviral Biology and Antivirals Laboratory

Centre for Biomedical ResearchBurnet Institute

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Discover Novel Allosteric Inhibitors of HIV-1 RT

Polymerase active site

NNRTI binding pocket

RNase H active site

HIV RT inhibitors approved or being developed for oral and topical PrEP same drug classes used for therapy

Potential for drug resistance in the contextof PrEP use in a real life setting

13 RT inhibitors used in the clinic they onlybelong to two classes: NRTIs and NNRTIs

Conformational flexibility of RT – function

Additional druggable allosteric sites in RT

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• “Fragments” are chemicals MW < 250 Da• More efficient at probing the chemical space – sample greater

chemical diversity by screening a smaller library• Bind with weak affinity • Strategically elaborated into larger high affinity inhibitors• Validated approach – US FDA approved drug Vemurafinib

Fragment Based Drug Discovery (FBDD)

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Three Novel Fragments Inhibit NNRTI Resistant HIV-1 RT

Inhibition of HIV-1 RT DDDP

4A2 inhibits RNase H

IC50 178 µM

Screen

13%

1.1%

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V89 and 4A2 Competes with Template/Primer and Cell culture data

4A2 inhibits HIV-1

EC50 18 ± 4 µM

4A2 Competes with T/P

IC50 93 ± 1 µM

V89 Competes with dNTP

Ki 220 ± 74 µM

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Identified fragments with novel scaffolds and modes of action compared to HIV RT inhibitors used clinically

Structure activity relationship (SAR) and X-ray crystallography studies are in progress to: - identify optimised fragments (more potent than original hits) - identify binding sites on HIV-1 RT - elaborate fragments into potent inhibitors by structure-based drug design

Summary

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Tachedjian Lab

Univ of PittsburghNicolas Sluis-Cremer

Acknowledgments

Rutgers University Eddy ArnoldJoe Bauman

Monash Institute of Pharmaceutical Sciences (MIPS)David ChalmersMartin ScanlonSteve Headey

Jennifer LaCath LathamDavid TyssenAdam Johnson

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•Detects weak binders•Can screen mixtures of compounds (x 5)•Pulse saturates entire protein (receptor) with magnetization transferring to protein bound ligand•Once ligand dissociates, saturated ligands can be detected•Resonances of small fragments not directly affected by pulse

Magnetic Field

RT

fragmentDissociate

Magnetisation transfer to fragment

Detect bound fragments

Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR)

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• Library of 630 fragments (ave MW 208) - Astex “Rule of Three” to make sure fragment like i.e. mass≤300 Da, ≤3 H-bond acceptors, ≤3H-bond donors, a clogP of ≤3, rotatable bonds ≤3 and a polar surface <60A2

Maybridge Ro3 library

Fragment Library

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Fragments are generally less potent against MoMLV and do not inhibit Klenow DNA pol