Human Immunodeficiency Virus Type 1 Protease Inhibitors: Evaluation of Resistance Engendered by Amino Acid Substitutions in the Enzyme's Substrate Binding Site

Abstract

The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.

Resistance of human immunodeficiency virus type 1 to protease inhibitors: selection of resistance mutations in the presence and absence of the drug

“…To date, several studies have examined the acquisition of HIV-1 resistance to protease inhibitors and have determined the changes required in the protease to reduce sensitivity to a variety of these compounds (Condra et al, 1995;Danziani et al, 1993;el-Farrash et al, 1994;Ho et al, 1994;Kaplan et al, 1994;Markowitz et al, 1995;Otto et al, 1993 ;Patick et al, 1995 ;Sardana et al, 1994). In vivo and in vitro, HIV-1 resistance to several distinct protease inhibitors is often induced by common amino acid substitutions that can confer various levels of cross-resistance (Condra et al, 1995).…”

A Potent Human Immunodeficiency Virus Type 1 Protease Inhibitor, UIC-94003 (TMC-126), and Selection of a Novel (A28S) Mutation in the Protease Active Site

“…A number of studies indicate that cross-resistance is a major obstacle inherent to most PIs (4,16,30). This observation should not be a surprise because all the inhibitors were designed to bind wild-type enzyme.…”

Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

“…Modeling studies by Gustchina et al revealed that differences in the affinity of HIV-1 and HIV-2 proteases to two aspartic PI appeared to be due in part to I32V (27). Sardana and colleagues mutated the four amino acid positions in HIV-1 protease to the corresponding HIV-2 amino acids and performed enzyme activity and inhibition studies using purified proteases in the presence of three inhibitors: L-689,502, L-731,723, and RO 31-8959 (later licensed as SQV) (29). Although their results supported the findings of Gustchina et al, the authors noted that substitutions at residues 47, 76, and 82 had relatively minor effects on protease inhibition by these compounds.…”

“…In HIV-1, a substitution at any one of these residues confers multi-PI resistance (26), and common drug resistance mutations at positions 32, 47, and 82 encode the corresponding wild-type (WT) amino acids from HIV-2. Early structural and biochemical studies showed that these residues contribute to substrate selectivity (27,28), as well as inhibitor sensitivity (29), although most of these observations were made using investigational PI which were never licensed. More recently, crystallographic studies have linked three of the four residues (32, 47, and 82) to differential amprenavir (APV) and DRV sensitivities (30)(31)(32)(33).…”