Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance

Abstract

In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended >10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.

Structural Adaptation of Darunavir Analogues against Primary Mutations in HIV-1 Protease

“…Accordingly, we previously designed, synthesized and evaluated a panel of 10 DRV analogs with varying P1' and P2' moieties, and demonstrated that substrate envelope-guided design can achieve improved inhibition and potency against resistant variants 34 . These inhibitors, named UMass1-10, have subsequently served us as tools to probe subsite interdependency 35 , water structure 36 , and structural changes in solution by NMR 37 . Among these analogs, UMass1 and UMass6 share the same P2' moiety with DRV but have larger hydrophobic groups at the P1' position [ Figure 2a] leveraging the unexploited space in the S1' pocket of the substrate envelope.…”

Exploring molecular mechanism of allosteric inhibitor to relieve drug resistance of multiple mutations in HIV‐1 protease by enhanced conformational sampling

“…Except for the experimental studies, different simulation methods were also employed to partially probe drug resistance induced by the mutated PR . It is worth noting that Schiffer's group used crystallography and simulations to study bindings of inhibitors to PR, and their results clarified drug‐resistant mechanism of mutations in PR and binding modes of inhibitors to PR . Although more traditional approaches, namely design and development of inhibitors targeting the active site, are adopted by many research groups to overcome drug resistance of PR, by now it is still a big challenge to solve this issue.…”

An inhibitor-interaction intermediate of HIV-1 protease, revealed by Isothermal Titration Calorimetry and NMR spectroscopy

“…5c) and DRV-bound forms (Fig. 5d) [60]. Flap+ (I54) spectra also exhibited very similar resonance patterns to those of Flap+ (I54V) in both forms ( Fig.…”

“…The clones were confirmed by DNA sequencing. We expressed 15 N isotope labeled proteins and purified using the protocols published previously [60]. Proteins were folded with 10 mM acetate at pH 6.0, buffer exchanged to a 20 mM sodium phosphate at pH 5.8, and concentrated approximately to 5 or 50 µM (assuming a dimer).…”

“…[60]. Thus, the simplest scenario for the two forms in state (I) of our model may be closed-(I a )and open-(I b ) flap forms: when the flaps are more closed, inhibitor binding will need a PR conformational change, which may involve significant enthalpy changes.…”