Lack of synergy for inhibitors targeting a multi-drug-resistant HIV-1 protease

Abstract

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to approximately 2.2 A resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.

Fig. 1.

(a) A chemical schematic diagram of indinavir, with the protease substrate subsites labeled. (b) A schematic representation of the synthesis pathway for indinavir analogs.

Fig. 2.

Ribbon diagrams of two views of the drug-resistant variant of HIV-1 protease dimer (in cyan and yellow) bound to indinavir (in magenta). The three modifications L63P, V82T, I84V are displayed and labeled in blue and green for each monomer, respectively. Figures were made with MIDAS (Ferrin et al. 1988).

Fig. 3.

Stereo views of the variant HIV-1 protease inhibitor complexes showing all atoms within at least 3.5 Å of their respectively bound inhibitors. The side chains adjacent to the protease active site are labeled, with the sites of mutation labeled in red. Eighty percent of the van der Waals surface is shown around each inhibitor. Figures were made with MIDAS (Ferrin et al. 1988). (a) Indinavir complex (in gray). (b) XN1336–51 complex (in yellow). An arrow indicates the addition of an s-methyl group in the benzylic position relative to indinavir. (c) 807–29–4 complex (in cyan). An arrow indicates the methylenedioxyphenyl group, which has replaced the pyridine ring of indinavir. (d) XN1336–52 complex (in magenta). Arrows indicate both of the modifications relative to indinavir described in b and c.(e) All four complexes superimposed. An arrow indicates where in the protease the 807–29–4 complex varies from the rest of the set.

Fig. 4.

Inhibition of 3X-protease by the four inhibitors in the presence of a substrate peptide, AcSQNYPVV-NH2. Percent activity is measured by the amount of remaining uncleaved peptide after incubation with protease and varying amounts of inhibitors. The IC₅₀ is the concentration at which 50% of the activity is remaining.

Fig. 5.

Stereo views overlapping each of the three indinavir analogs on indinavir (in gray). Also shown are the catalytic aspartic acids at residue 25 in each monomer of the protease. Figures were made with MIDAS (Ferrin et al. 1988). (a) XN1336–51 (in yellow). The arrow indicates the direction of the shift relative to indinavir (in gray). (b) 807–29–4 (in cyan). The arrow indicates the tilt of the inhibitor relative to indinavir (in gray). (c) XN1336–52 (magenta). The arrow indicates the direction of the shift relative to indinavir (in gray). (d) All four inhibitors superimposed.

Fig. 5.

Stereo views overlapping each of the three indinavir analogs on indinavir (in gray). Also shown are the catalytic aspartic acids at residue 25 in each monomer of the protease. Figures were made with MIDAS (Ferrin et al. 1988). (a) XN1336–51 (in yellow). The arrow indicates the direction of the shift relative to indinavir (in gray). (b) 807–29–4 (in cyan). The arrow indicates the tilt of the inhibitor relative to indinavir (in gray). (c) XN1336–52 (magenta). The arrow indicates the direction of the shift relative to indinavir (in gray). (d) All four inhibitors superimposed.

Fig. 6.

Double difference distance plots for the various inhibitor complexes of HIV-1 protease. The value plotted is D_ij = d_ij (first complex) − d_ij (second complex), where d_ij indicates the distance between the α carbons i and j in a particular complex. Contours in the plots show whether the respective distances in the two complexes being compared are closer or further apart. Black indicates a difference of <−0.6 Å, red a difference of between −0.59 and −0.3 Å, blue a difference of between 0.3 and 0.59 Å, and yellow a difference of >0.6 Å. (a) Drug-resistant variant HIV-1 protease complexes bound with indinavir and XN1336–51. (b) Drug-resistant variant HIV-1 protease complexes bound with indinavir and 807–29–4. (c) Drug-resistant variant HIV-1 protease complexes bound with indinavir and XN1336–52. (d) Drug-resistant variant HIV-1 protease complexes bound with XN1336–51 and XN1336–52.