Synthesis, Stability, Antiviral Activity, and Protease-Bound Structures of Substrate-Mimicking Constrained Macrocyclic Inhibitors of HIV-1 Protease

Abstract

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Potencies of Human Immunodeficiency Virus Protease Inhibitors In Vitro againstPlasmodium falciparumand In Vivo against Murine Malaria

“…For the coformulation of lopinavir and ritonavir, the EC 50 with respect to the ritonavir component was the same as that obtained for ritonavir alone, whereas the EC 50 of lopinavir, with respect to ritonavir, was approximately fourfold higher. Two other structurally quite distinct HIV-1 protease inhibitors, BR124 and BR314, which show potent antiretroviral activity in vitro (56), were also found to inhibit the growth of P. falciparum in vitro at comparable concentrations (Table 1).…”

Computational inhibitor design against malaria plasmepsins

“…Subsequently, the impact of cyclization was investigated in four compounds (18 -21 in Table 1) [16]. The general idea of cyclization is to preorganize the ligand in a bioactive conformation thereby reducing the entropy loss upon binding [89]. Further benefits of cyclization are protection of the amide bonds from proteolytic cleavage and improved cell permeability [90].…”

An insight into the opening path to semi-open conformation of HIV-1 protease by molecular dynamics simulation

“…The X-ray diffraction structure of HIV-1 protease, PDB ID: 1D4L, reported by Tyndall [3], was used as the template of this study. In order to properly compare the simulation results with the experimental data, the mutant residues in 1D4L were restored to the original residues by the model building method using the standard geometry with the residue operation command of CHARMM.…”

“…It has a highly conserved pair of catalytic triads to fold into an active form for facilitating the hydrolysis of peptide bonds [2] at a specific active site (Asp25, Thr26, Gly27) [3]. The catalytic triads are located at the periphery of the substrate-binding pocket and opposite the flap region, which is believed to open and close for enabling the entry and binding of the substrates [2].…”