Two-dimensional infrared spectra reveal relaxation of the nonnucleoside inhibitor TMC278 complexed with HIV-1 reverse transcriptase

Abstract

The two nitrile groups at the wings of the nonnucleoside HIV-1 reverse transcriptase (RT) inhibitor TMC278 are both identified in high-sensitivity 2D IR spectroscopy experiments of the HIV-1 RT/ TMC278 complex. The vibrational spectra indicate that the two arms of the inhibitor sense quite different environments within the hydrophobic pocket. The vibrational relaxation of the two arms are almost equal at 3 ps from model studies. The 2D IR spectra expose a significant distribution of nitrile frequencies that diffuse at equilibrium on ultrafast time scales ranging from hundreds of femtoseconds to tens of picoseconds. The slow spectral diffusion of the cyanovinyl arm of the inhibitor is attributed to its interaction with the backbone and side chains in the hydrophobic tunnel. The results show that the inhibitor cyano modes lose memory of their structural configurations relative to the hydrophobic pocket within tens of picoseconds. The cross-peaks between the two arms of the drug are tentatively attributed to relaxation of the nitrile state with both arms excited.2D IR spectroscopy ͉ ultrafast protein response ͉ vibrational relaxation R everse transcriptase (RT) enables the conversion of a singlestranded RNA into a double-stranded DNA in a process that is essential for viral replication. The nonnucleoside inhibitors of HIV-1 RT (NNRTIs) bind in a hydrophobic pocket between two ␤-sheets, one containing the polymerase active site and the other containing the primer grip, a structural element that is involved in positioning the 3Ј primer terminus for catalysis (1). Resistance to NNRTIs can arise from mutations in the residues in and around the binding pocket (2). An NNRTI that has strategic flexibility can overcome the effects of drug-resistance mutations by adapting its conformation and position to accommodate mutation-induced changes in the binding pocket (3). The diarylpyrimidine (DAPY) NNRTI TMC278 (4) is a flexible molecule and inhibits a broad spectrum of NNRTI-resistant mutants. Questions then arise as to freedom of motion of the inhibitor and the time scales of such structural reorganizations. To obtain answers there is a need for time-sensitive probes of structure within the pocket that can respond to motions of the drug and the nearby residues. One set of probes that do not perturb the structure and that span the short time regime are the vibrations of the chemical bonds of the inhibitor. They sense the fluctuations of the surrounding structure through the time correlation functions of their vibrational frequencies. These correlation functions can be measured with the methods of 2D IR spectroscopy (5), and the present work is an application of 2D IR spectroscopy aimed at exposing dynamical aspects of the enzyme inhibitor complex at the chemical bond-length scale. There have been many recent experimental and theoretical advances showing that these methods can expose novel aspects of ultrafast processes in biological assemblies (6-18). The present article on the vibrational spectra and dynamics of an HIV-1 RT i...

TMC278, a Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor (NNRTI), Active against Wild-Type and NNRTI-Resistant HIV-1

“…Crystal structures of the binding site between TMC278 and the HIV-1 RT complex, as well as freedom-of-motion analyses, revealed that TMC278 binds to the HIV-1 RT and adapts to changes in the NNRTI-binding pocket. TMC278 thereby compensates for the presence of drug resistance mutations, which further explains the mechanism of the increased genetic barrier to resistance to this compound (15,20 50 values ranging from 0.07 to 1.01 nM and 2.88 to 8.45 nM, respectively. TMC278 was on average approximately 10-to 15-fold less active against the HIV-1 group O isolates than against the HIV-1 group M primary isolates.…”

Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase

“…Two-dimensional IR spectroscopy probes the FFCF, and thereby the protein motions that are sensed by the transition-state analogue (the IR chromophore), only for a few tens of picoseconds at most. Although these experiments cannot identify the time scales for spectral diffusion for motions that occur beyond this time range, they do characterize the distribution of frequencies determined by the distribution of structures resulting from these slower motions (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)44). Motions at all time scales contribute to the initial value of the FFCF, but only those motions that occur on the femtosecond to picosecond time scale contribute to the FFCF decay probed in the 2D IR measurements.…”

“…A close experimental approximation to this ideal is to study the dynamics of an enzyme in a complex with a transition-state-analog inhibitor. There have been several infrared spectroscopic studies of enzyme-ligand interaction dynamics with substrate-analog complexes that mimic the ground state (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). These studies use infrared echo and 2D IR spectroscopies to measure the frequencyfrequency time correlation function (FFCF) that reveals the time .…”

Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy

“…Two 2D IR studies have been performed on biological systems using CN as the probe. One study involved HIV reverse transcriptase using a bound substrate containing a CN group (27). The other studied nitrile-labeled HP35 in its native state in water (28).…”