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16th International HIV Drug Resistance Workshop12-16 June 2007, Barbados |
STRATEGIC FLEXIBILITY OF THE NONNUCLEOSIDE RT INHIBITOR TMC278 EXPLAINS ITS POTENCY AGAINST DRUG-RESISTANT MUTANTS
Antivir Ther. 2007; 12:S33 (abstract no. 31)
K Das1, JD Bauman1, M Baweja1, AD Clark Jr1, PL Boyer2, AJ Shatkin1, PJ Lewi3, SH Hughes2 and E Arnold1
1Center for Advanced Biotechnology and Medicine, and Rutgers University, Piscataway, NJ, USA; 2NCI-Frederick, Frederick, MD, USA; 3Katholieke Universiteit, Leuven, Belgium
BACKGROUND: TMC278 is highly effective in treating HIV-1 infection at relatively low doses (~25–75 mg/day) in clinical trials. TMC278 is a potent inhibitor of HIV-1 strains that are resistant to approved NNRTI drugs (Janssen et al., J Med Chem. 2005 Mar 24;48(6):1901-9). Strategic flexibility (Das et al., J Med Chem. 2004 May 6;47(10):2550-60) helps TMC278 and other DAPY analogues overcome the effects of drug-resistance mutations. In structural studies, the drug flexibility interferes with the formation of well-diffracting crystals of the RT/TMC278 complex.
METHODS: Systematic protein engineering was performed to improve diffraction from crystals of the HIV-1 RT/TMC278 complex. Primary design concepts were: 1) create RT mutations based on HIV-1 strains for which crystal structures had been determined; 2) remove lysine and glutamic acid patches to reduce surface entropy; 3) alter amino acid residues to make new lattice contacts and/or remove some of the contacts in earlier crystal forms; and 4) remove disordered residues. Iterative rounds of mutagenesis and crystallization trials were performed using engineered RT constructs in complex with TMC278 and other NNRTIs.
RESULTS: Crystals of the engineered RT/TMC278 complex diffracted X-rays to 1.8 Å resolution. Crystal structures of wild-type, and the L100I+K103N and K103N+Y181C double mutant RTs in complexes with TMC278 were determined at 1.8, 2.1, and 2.9 Å resolution, respectively. The strategically positioned cyanovinyl group of TMC278 is important in retaining the potency of the drug against drug-resistance mutants. Loss of stabilizing aromatic ring interactions resulting from Y181C mutation is largely compensated by a new set of interactions developed between the cyanovinyl group of TMC278 and Y183. The structure of I100L+K103N mutant RT/TMC278 complex demonstrated that TMC278 undergoes significant conformational (wiggling) and positional (jiggling) changes when binds to the mutant RT.
CONCLUSIONS: High-resolution structures of RT are critical for reliable description of inhibitor–protein interactions, better understanding of the effects of resistance mutations, and systematic structure-based drug design. The structures demonstrate the important role of strategic flexibility of TMC278 in evading the effects of drug resistance mutations. Incorporation of strategic flexibility into ligands appears to be an important consideration in designing drugs against rapidly evolving targets.
2007-06-12
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