1111 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 Research Burnet Institute

2222 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 context of PrEP use in a real life setting 13 RT inhibitors used in the clinic they only belong to two classes: NRTIs and NNRTIs Conformational flexibility of RT – function Additional druggable allosteric sites in RT

3333 “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)

4444 Three Novel Fragments Inhibit NNRTI Resistant HIV-1 RT Inhibition of HIV-1 RT DDDP 4A2 inhibits RNase H IC µM Screen 13% 1.1%

5555 V89 and 4A2 Competes with Template/Primer and Cell culture data 4A2 inhibits HIV-1 EC ± 4 µM 4A2 Competes with T/P IC ± 1 µM V89 Competes with dNTP Ki 220 ± 74 µM

6666 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

7777 Tachedjian Lab Univ of Pittsburgh Nicolas Sluis-Cremer Acknowledgments Rutgers University Eddy Arnold Joe Bauman Monash Institute of Pharmaceutical Sciences (MIPS) David Chalmers Martin Scanlon Steve Headey Jennifer La Cath Latham David Tyssen Adam Johnson

8888 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 fragment Dissociate Magnetisation transfer to fragment Detect bound fragments Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR)

9999 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 <60A 2 Maybridge Ro3 library Fragment Library

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