Detecting Allosteric Sites of HIV-1 Reverse Transcriptase by X-ray Crystallographic Fragment Screening

Abstract

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography. A total of 775 fragments were grouped into 143 cocktails, which were soaked into crystals of RT in complex with the non-nucleoside drug rilpivirine (TMC278). Seven new sites were discovered, including the Incoming Nucleotide Binding, Knuckles, NNRTI Adjacent, and 399 sites, located in the polymerase region of RT, and the 428, RNase H Primer Grip Adjacent, and 507 sites, located in the RNase H region. Three of these sites—Knuckles, NNRTI Adjacent, and Incoming Nucleotide Binding—are inhibitory and provide opportunities for discovery of new anti-AIDS drugs.

Studies on Cycloheptathiophene‐3‐carboxamide Derivatives as Allosteric HIV‐1 Ribonuclease H Inhibitors

“…Q500 has been reported as one of the key residues responsible for RNA:DNA hybrid binding, [35] and it was recently proposed as a possible druggable binding site for allosteric RNase H inhibitors. [36][37][38] Theoretically, compounds interacting with residue Q500 (Figure 3), or residues in its vicinity that are important for RNA:DNA substrate positioning, could potentially interfere with duplex accommodation in the active site of RNase H. [36] On the basis of these results, we hypothesized that the binding of cHTC derivatives to the allosteric site of RNase H could be affected by competition with the RNA:DNA duplex, especially for compounds that were weaker inhibitors. To test this hypothesis, the RNase H activity of compounds 31 and 33 was re-assayed by modifying the order of addition of the RNA:DNA duplex substrate.…”

Advantages of crystallographic fragment screening: Functional and mechanistic insights from a powerful platform for efficient drug discovery

“…Prior to conducting a fragment screening campaign against the influenza A endonuclease (PA N ), it was necessary to identify a new crystal form because the two published crystal forms were not useful for structure-based drug design. One crystal form was very difficult to reproduce while other published structure of PA N had an active site occluded by a crystallographically symmetric copy of the protein (Dias et al, 2009; Yuan et al, 2009; Bauman et al, 2013b). Additionally, binding sites should not be occupied by chemical components from the crystallization condition.…”

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain

“…Subsequent optimization using a combination of pre-seeding and reducing the well volume from 500 l to 50 l improved crystal production to approximately 3 suitable crystals per drop. High throughput fragment screening of a chemically diverse library of 971 fragments, consisting of cocktails containing 4 -8 compounds each, was conducted using a previously described protocol (37).…”