Defective Hydrophobic Sliding Mechanism and Active Site Expansion in HIV-1 Protease Drug Resistant Variant Gly48Thr/Leu89Met: Mechanisms for the Loss of Saquinavir Binding Potency

Abstract

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis–Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/L89M-SQV has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.

Recent Progress in the Development of HIV-1 Protease Inhibitors for the Treatment of HIV/AIDS

“…56 The flap region, composed of residues 39–57, is a main regulator for substrate and ligand binding. 60 HIV protease exists in multiple conformations, including the free “flap-open” conformation and the ligand-bound “flap-closed” conformation (Figure 4). Mutations in this region tend to change the extent to which the flaps may be open or closed and thereby change the shape of and access to the binding pocket, consequently reducing the ability of the inhibitor to bind in the active site.…”

“…This reduced the van der Waals interactions between the PI and enzyme, reducing binding affinity. 60 …”

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

“…These mutations produce small changes in the size and shape of the amino acid side chains that interact with inhibitors. A recent example of this mechanism is a double mutant, G48T/L89M, that displays resistance to SQV via an expanded active site cavity from a defective hydrophobic sliding mechanism [31];…”

Improving Viral Protease Inhibitors to Counter Drug Resistance

“…Altered dynamics differentially impact substrate processing versus inhibitor binding, as the inhibitor needs to stay bound at the active site for efficient inhibition, while the substrates need to be processed and released for efficient turnover. Such changes in dynamics have been revealed in molecular dynamics simulations as well as experimental NMR and electron paramagnetic resonance (EPR) dynamics of HIV-1 protease drug resistant variants [79–82]. This resistance mechanism through changes in conformational dynamics propagating to the active site [72] may be common to distal mutations in other proteases.…”