Efficient discovery of potent anti-HIV agents targeting the Tyr181Cys variant of HIV reverse transcriptase

Abstract

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) that interfere with the replication of human immunodeficiency virus (HIV) are being pursued with guidance from molecular modeling including free-energy perturbation (FEP) calculations for protein-inhibitor binding affinities. The previously reported pyrimidinylphenylamine 1 and its chloro analogue 2 are potent anti-HIV agents; they inhibit replication of wild-type HIV-1 in infected human T-cells with EC(50) values of 2 and 10 nM, respectively. However, they show no activity against viral strains containing the Tyr181Cys (Y181C) mutation in HIV-RT. Modeling indicates that the problem is likely associated with extensive interaction between the dimethylallyloxy substituent and Tyr181. As an alternative, a phenoxy group is computed to be oriented in a manner diminishing the contact with Tyr181. However, this replacement leads to a roughly 1000-fold loss of activity for 3 (2.5 μM). The present report details the efficient, computationally driven evolution of 3 to novel NNRTIs with sub-10 nM potency toward both wild-type HIV-1 and Y181C-containing variants. The critical contributors were FEP substituent scans for the phenoxy and pyrimidine rings and recognition of potential benefits of addition of a cyanovinyl group to the phenoxy ring.

Figure 1

Two orientations of NNRTI 2 bound to WT HIV-RT, as created using BOMB and optimized using MCPRO. The full model contains ca. 175 protein residues and the inhibitor. Carbon atoms of the NNRTI are colored gold. The image on the right is rotated 90° from the one on the left.

Figure 2

The same views as in Figure 1, but with NNRTI 3 bound to HIV-RT. Modeling suggested substitutions at the 3- and 5-positions in the phenoxy ring with possible passage of a substituent between Tyr181 and Tyr188 or towards Pro95.

Figure 3

(Left) Alternative conformer of 3 bound to HIV-RT. (Right) Comparison of the two ligand conformations. In the top one, the faces of the phenyl rings are roughly perpendicular, while the geometry is more like a clamshell in the alternative conformer.

Figure 4

Computed differences in free energies of binding, ΔΔG_b (kcal/mol), for chlorophenyl analogs. The hysteresis of 0.50 kcal/mol for the cycle provides a measure of the precision of the calculations.

Figure 5

Computed differences in free energies of binding, ΔΔG_b (kcal/mol), with HIV-RT and observed anti-HIV activities for phenoxy and thiophenoxy analogs. The FEP calculations were run in both directions yielding the indicated results in kcal/mol.

Figure 6

Optimized structures for 19 bound to wild-type (left) HIV-RT and the Tyr181Cys mutant (right). Highlighted residues are labeled in Figure 1.

Figure 7

Computed structures illustrating alternative placement of the cyanovinyl group for analogs of 15 bound to wild-type HIV-RT: projecting (A) between Tyr181 and Tyr188; (B) towards Pro95 and Pro97; (C) between Tyr188 and Phe227; and (D) below Pro95.

Figure 8

Depiction from the 2zd1 crystal structure of rilpivirine bound to WT HIV-RT illustrating the positioning of the cyanovinyl group between Tyr188 and Phe227. Carbon atoms of rilpivirine are in yellow.

Figure 9

Computed differences in free energies of binding, ΔΔG_b (kcal/mol), with HIV-RT for conversion of pyrimidines to 1,3,5-triazines. The computed statistical uncertainties in both cases are ± 0.33 kcal/mol.

Scheme 1

General procedure for synthesis of heteroarylaminodiphenyl ethers

Scheme 2

General procedures for synthesis of diphenyl ethers and thioethers

Scheme 3

Synthesis of cyanovinyl-substituted phenols

Scheme 4

Synthesis of cyclopropyltriazine analogs