Crystallographic Fragment‐Based Drug Discovery: Use of a Brominated Fragment Library Targeting HIV Protease

Abstract

A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV-1 protease in order to probe surface sites in soaking experiments. Often fragments are weak binders with partial occupancy, resulting in weak, difficult-to-fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3-bromo-2,6-dimethoxybenzoic acid (Br6) in the flap site, and 1-bromo-2-naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br-fragments provides further insight into the nature of these surface pockets.

A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413

“…HIV 50 values were determined by using PHA-PBM as target cells, and the inhibition of p24 Gag protein production by each drug was used as the endpoint. The numbers in parentheses represent the fold changes of EC 50 s for each isolate compared to the EC 50 s for HIV-1 ERS104pre .…”

“…Recently, the impact of halogen bonding on favorable proteinligand interactions has received increased attention, with several successful applications (49,50). In particular, fluorine and chlorine substitutions not only enhance binding affinity and specificity but also facilitate membrane permeability and metabolic stability (51,52).…”

“…On day 7 of culture, the supernatants were harvested, and the amounts of p24 (capsid [CA]) Gag protein were determined by using a fully automated chemiluminescence enzyme immunoassay system (Lumipulse F; Fujirebio Inc., Tokyo, Japan) (27,28). Drug concentrations that suppressed the production of p24 Gag protein by 50% (50% effective concentrations [EC 50 s]) were determined by comparison with the p24 production level in a drugfree control cell culture. All assays were performed in duplicate.…”

“…All assays were performed in duplicate. Each compound's EC 50 shown in the current report is the average value for the data obtained from two to four independently conducted experiments. PHA-PBM were derived from a single donor for each independent experiment.…”

“…MT-4 cells (10 5 /ml) were exposed to HIV-1 NL4-3 (500 TCID 50 ) and cultured in the presence of various HIV-1 PIs, initially at their EC 50 s. Viral replication was monitored by determination of the amount of p24 Gag produced by MT-4 cells. The culture supernatants were harvested on day 7 and were used to infect fresh MT-4 cells for the next round of culture in the presence of increasing concentrations of each drug.…”