A Major Role for a Set of Non-Active Site Mutations in the Development of HIV-1 Protease Drug Resistance

Abstract

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.

Comparing the Accumulation of Active- and Nonactive-Site Mutations in the HIV-1 Protease

“…Dissociation Constants. The K i values for the wild-type enzyme with all of the inhibitors tested were subnanomolar to low nanomolar in agreement with other studies (Table 4) (5,(27)(28)(29). The V6 variant showed a large decrease in susceptibility to ritonavir.…”

Analysis of HIV-1 CRF_01 A/E Protease Inhibitor Resistance: Structural Determinants for Maintaining Sensitivity and Developing Resistance to Atazanavir

“…It was originally believed that non-active site mutations were acquired to regain the loss of catalytic activity due to active site mutations. Recent studies from our laboratory and others have shown that many non-active site mutations in HIV-1 subtype-B proteases will contribute to decreasing the binding affinity of inhibitors, while maintaining catalytic efficiency (10,(22)(23)(24)26,27). In this study we provide insights into the effects of natural polymorphisms and the contributions to resistance made by therapy-selected active site and non-active site mutations found in the CRF_01 AE protease.…”

“…The Ile84 residue is located in the center of the active site and we previously suggested that the I84V mutation may function to destabilize the interactions of the core of the inhibitor and its interactions with the flaps (23). Previous studies have also shown the importance of mutations in the flaps in providing a high level of resistance (10,22,23,26), and that the addition of non-active site mutations may decrease binding affinity by destabilizing the flaps in the bound conformation decreasing binding affinity (23). Recent studies by Yanchunas and colleagues, utilizing thermodynamic and modeling techniques, have suggested the importance of ATV P2 and P2′ groups interactions with Ile50 in maintaining binding affinity (33).…”

Inference of epistatic effects leading to entrenchment and drug resistance in HIV-1 protease

“…Chang and Torbett (2011) demonstrate that stability is compromised by the acquisition of primary mutations and this loss of stability can be rescued by known compensatory mutations, sometimes in excess of the reference stability. Muzammil et al (2003) and Louis et al (2011) have shown that patterns of up to ten or more resistance mutations do not necessarily suffer from reduced stability relative to the wildtype, and that nonactive site mutations can lead to resistance in certain sequence contexts. In figure 3A, the change in statistical Potts energies, DE ¼ E À E ref is plotted versus the change in thermal stability, where E and E ref are the statistical energies of the mutated and reference sequences corresponding to each pair of stability measurements.…”