Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M

Abstract

The potent new antiviral inhibitor TMC-114 (UIC-94017) of HIV-1 protease (PR) has been studied with three PR variants containing single mutations D30N, I50V, and L90M, which provide resistance to the major clinical inhibitors. The inhibition constants (K(i)) of TMC-114 for mutants PR(D30N), PR(I50V), and PR(L90M) were 30-, 9-, and 0.14-fold, respectively, relative to wild-type PR. The molecular basis for the inhibition was analyzed using high-resolution (1.22-1.45 A) crystal structures of PR mutant complexes with TMC-114. In PR(D30N), the inhibitor has a water-mediated interaction with the side chain of Asn30 rather than the direct interaction observed in PR, which is consistent with the relative inhibition. Similarly, in PR(I50V) the inhibitor loses favorable hydrophobic interactions with the side chain of Val50. TMC-114 has additional van der Waals contacts in PR(L90M) structure compared to the PR structure, leading to a tighter binding of the inhibitor. The observed changes in PR structure and activity are discussed in relation to the potential for development of resistant mutants on exposure to TMC-114.

Figure 1

(a) Molecular diagram of the TMC114 inhibitor. (b) PR dimer structure (green ribbons). The sites of mutation are indicated using ball-and-stick representation for Asp30, Ile50, and Leu90. Catalytic aspartate residues are shown in a stick representation. Only one subunit is labeled.

Figure 2

2F_o-F_c electron density map for residues Pro79-Thr80-Pro81 of PR_I50V complex. The contour level is 2.6σ.

Figure 3

Alternate side-chain positions of PR_I50V structure shown in 2F_o-F_c map. The residues drawn are Ile84 (62/38% occupancies) (a) and Arg57 (66/34% occupancies) (b). The electron density map is contoured at a level of 1.6σ (a) and 1.1σ (b). The major conformations are colored by atom type, and the minor conformations are in pink.

Figure 4

2F_o-F_c electron density map for the sodium cation region in PR_L90M structure. The map is contoured at 2.0σ level. The sodium atom has a distorted square bipyramidal coordination of water and carbonyl oxygen atoms. The short contacts in the Na⁺ coordination sphere and hydrogen bonds are indicated by dashed lines, with the interatomic distances in Å. The side-chain of Tyr59 is omitted for clarity.

Figure 5

The hydrogen bond network between the inhibitor and the protease in PR_L90M structure. Residues 30, 30′, 50 and 50′ have two alternate conformations including main-chain atoms, the minor conformations are colored in pink.

Figure 6

2F_o-F_c electron density maps for residues Asn30 (a) and Asn30′ (b) of PR_D30N. The maps clearly show the two alternate conformations of Asn30′ (56/44% occupancies) and water-mediated contacts to the aniline moiety of TMC114. Contour level is 1.6σ. Interactions of bis-THF moiety are not shown.

Figure 7

Interactions of the aniline group of TMC114 with residue Asp30 in PR and PR_I50V structures. PR is colored by the atom type, and PR_I50V is shown as green sticks. The alternate conformation of the Asp30 side-chain in PR, and the second orientation of the inhibitor for both structures are omitted for clarity. The interactions of the other inhibitor orientation with residue Asp30′ of the other protease subunit are similar in all the structures.

Figure 8

C-H…O interactions between the sulfonamide moiety of TMC114 and the protease atoms. PR is colored by the atom type, and PR_I50V is shown as green sticks. The alternate conformation of Ile50′ in PR_WT and Val50′ in PR_I50V, and the second orientation of the inhibitor for both structures are omitted for clarity. The alternate conformations of residue 50′ that do not make C-H…O interactions with TMC114 are omitted. (a) and (b) show the contacts with the residues in different subunits of PR. For the other structures these contacts are similar to PR.

Figure 9

C-H…π interactions between the side-chains of Ile50 or Val50 (a), and Ile50′ or Val50′ (b) with the aromatic ring of TMC114’s aniline moiety in PR_D30N (blue), PR_I50V (red ball-and-stick representation) and PR_L90M (green). Alternate conformations of residue 50 that do not form close contacts to the aromatic ring are omitted for clarity.

Figure 10

Interactions of mutated residues 90 and 90′ with catalytic residue 25 and 25′ shown in superimposed mutant and PR_WT structures. Interatomic distances are indicated by broken lines with the separation in Å. PR_L90M is shown in magenta bonds, PR in atom-type colored fashion. The side-chain conformations of Met90 and Met90′ that make shortened contacts to the main-chain atoms of aspartates are shown.