Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket \u2013 targeting P-pocket by fragment screening

Abhimanyu K Singh,Dominique Schols,Kalyan Das,Weijie Gu,Hoai Nguyen,Steven De Jonghe,Sergio E Martinez,Piet Herdewijn

doi:10.1038/s41467-021-27409-y

Abhimanyu K Singh, Dominique Schols + Show 6 more

Open Access

https://doi.org/10.1038/s41467-021-27409-y

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Abstract

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3’-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.

Highlights

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA
Our study revealed a highly conserved transient P-pocket that is created in the process of sliding of RT over a dsDNA substrate and established an experimental platform for structure-based drug design using single-particle cryo-electron microscopy (cryo-EM)
The I63C RT cross-linked with a 27/21-mer dsDNA was crystallized as a polymerase active complex in which, the primer 3′-end occupied the priming site (P site) (P complex; Fig. 1b) and an incoming d4T-TP was bound at the N site,[16] the extra nucleotide in the 28/21-mer DNA is on the template overhang, which is primarily disordered in the structures

Summary

Introduction

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and templateprimer that are resilient to drug-resistance mutations. Fostemsavir, a gp[120] inhibitor, was FDA-approved in 2020 primarily for treating multi-drug resistant HIV infection[3]. NcRTIs compete with dNTPs for binding at the N site and can be categorized as metaldependent inhibitors such as α-CNP8–10 and metal-independent inhibitors such as INDOPY-111,12. Despite these developments, the discovery of new druggable sites of RT that are highly conserved is important for developing new classes of drugs with resilience to existing drug-resistance mutations

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