Viability of a Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variant: Structural Insights for Better Antiviral Therapy

Abstract

Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.

Structural Basis for Coevolution of a Human Immunodeficiency Virus Type 1 Nucleocapsid-p1 Cleavage Site with a V82A Drug-Resistant Mutation in Viral Protease

“…Although the peptides used for crystallization span a slightly different region of the cleavage site (P5 to P5Ј and P4 to P6Ј), these differences are likely to have minimal or no effect on the bound conformation of the substrate. From our previous experience with to determine substrate complexes with HIV protease (38,39,49), the P5 and P5Ј residues usually have no contact with the protease and are often disordered in the electron density. Thus, the presence or absence of P5 or P6Ј is unlikely to impact how the substrate is packed in the active site.…”

“…Crystals were grown by the hanging drop, vapor diffusion method as previously described (38,39,49). Stock solutions (25 mM) of substrate peptides used for cocrystallization were dissolved in refolding buffer rather than in dimethyl sulfoxide.…”

“…Our previous studies have shown that V82A is a prime site for drug resistance to occur, as the valine at residue 82 is not critical for substrate recognition but does extensively contact many of the commonly used inhibitors (39). However, the apparent coevolution of the NC-p1 cleavage site (AP2V) with the V82A mutation in the protease (1,10,20,55) implies that Val82 plays an important role in the protease's recognition of the NC-p1 substrate, a role that is lost when the V82A mutation occurs.…”

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

“…This concept has been successfully used in designing robust protease inhibitors and in predicting the sites of drug-resistant mutations. [12][13][14] Additionally, recent studies examined substrates from patient samples and found that the sequences of the substrates coevolved with major drug-resistant mutations in the protease. 15,16 These findings suggest that for resistant protease variants, there is an alteration of substrate specificity that is selected for in the coevolving substrate sites.…”

“…Studies of HIV-1 protease in complex with substrates and inhibitors indicate that the drug-resistant mutants of this enzyme have altered molecular recognition abilities; they still recognize the enzyme's substrates but binding to inhibitors is disrupted. [9][10][11][12] The mechanisms involved in substrate recognition and binding of these drug-resistant mutants have yet to be fully elucidated. To gain insight into these processes, an improved understanding of which residues dictate substrate binding specificity and how these sites evolve with the emergence of drug resistance is required.…”