A 5-μM docking hit has been optimized to an extraordinarily potent (55 pM) non-nucleoside inhibitor of HIV reverse transcriptase. Use of free energy perturbation (FEP) calculations to predict relative free energies of binding aided the optimizations by identifying optimal substitution patterns for phenyl rings and a linker. The most potent resultant catechol diethers feature terminal uracil and cyanovinylphenyl groups. A halogen bond with Pro95 likely contributes to the extreme potency of compound 42. In addition, several examples are provided illustrating failures of attempted grafting of a substructure from a very active compound onto a seemingly related scaffold to improve its activity.

Efficient optimization of an inactive 2-anilinyl-5-benzyloxadiazole core has been guided by free energy perturbation (FEP) calculations to provide potent non-nucleoside inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (NNRTIs). An FEP "chlorine scan" was performed to identify the most promising sites for substitution of aryl hydrogens. This yielded NNRTIs 8 and 10 with activities (EC 50 ) of 820 and 310 nM for protection of human T-cells from infection by wild-type HIV-1. FEP calculations for additional substituent modifications and change of the core heterocycle readily led to oxazoles 28 and 29, which were confirmed as highly potent anti-HIV agents with activities in the 10-20 nM range. The designed compounds were also monitored for possession of desirable pharmacological properties by use of additional computational tools. Overall, the trends predicted by the FEP calculations were well borne out by the assay results. FEPguided lead optimization is confirmed as a valuable tool for molecular design including drug discovery; chlorine scans are particularly attractive since they are both straightforward to perform and highly informative.

HIV/AIDS is a global pandemic for which new treatment strategies are desperately needed. We have designed a novel small molecule with the potential to interfere with HIV survival through two mechanisms: (1) by recruiting antibodies to gp120-expressing virus particles and infected human cells, thus enhancing their uptake and destruction by the human immune, and (2) by binding the viral glycoprotein gp120, inhibiting its interaction with the human protein CD4, and preventing virus entry. Here we demonstrate that ARM-H is capable of simultaneously binding gp120, a component of the Env surface viral glycoprotein (found on the surface of both HIV and virus-infected cells) and anti-2,4-dinitrophenyl antibodies (already present in the human bloodstream). The ternary complex formed between antibody, ARM-H, and gp120 is immunologically active and leads to the complement-mediated destruction of Env-expressing cells. Furthermore, ARM-H prevents virus entry into human T-cells, and should therefore be capable of inhibiting virus replication through two mutually reinforcing mechanisms (inhibition of virus entry and antibody-mediated killing). These studies demonstrate the viable anti-HIV activity of antibody-recruiting small molecules, and have the potential to initiate novel paradigms in HIV treatment.

We evaluated the replication efficiency of the HIV reverse transcriptase (RT) mutants K103N, G190A, and G190S, which confer resistance to the non-nucleoside RT inhibitor efavirenz, using growth competition assays in cell culture. In the absence of efavirenz, the fitness hierarchy was G190S < G190A < K103N < wild-type. The fitness reduction of G190S relative to K103N was less evident at high efavirenz concentrations, although K103N still replicated more efficiently. Efficiency of RNase H cleavage and RNA-dependent DNA synthesis from tRNA(Lys, 3) correlated with relative fitness, in biochemical studies of mutant RTs. Presteady state and steady state polymerization assays using DNA primers detected no abnormalities. This work is consistent with previous studies demonstrating that initiation of viral DNA synthesis is reduced in mutants with slowed RNase H cleavage, and suggests that both abnormalities contribute to the replication defect of these mutants. It also suggests that high concentrations of efavirenz are unlikely to favor the selection of G190S clinically.

Virtual screening of the Maybridge library of ca. 70,000 compounds was performed using a similarity filter, docking, and MM-GB/SA post-processing to seek potential non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Though known NNRTIs were retrieved well, purchase and assaying of representative, top-scoring compounds from the library failed to yield any active anti-HIV agents. However, the highest-ranked library compound, oxadiazole 1, was pursued as a potential "near-miss" with the BOMB program to seek constructive modifications. Subsequent synthesis and assaying of several polychloro-analogs did yield anti-HIV agents with EC 50 values as low as 310 nM. The study demonstrates that it is possible to learn from a formally unsuccessful virtual-screening exercise and, with the aid of computational analyses, to evolve efficiently a false positive into a true active. In addition, the need for adequate structure validation was confirmed by the apparent misrepresentation of a purchased compound elsewhere as the present oxadiazole core compound, 16.