Background:  TMC278, a product of a multidisciplinary effort—involving medicinal chemists, virologists, crystallographers, molecular modelers, toxicologists, analytical chemists, and pharmacologists—is currently under clinical investigation. TMC278 is a potent inhibitor of available non-nucleoside reverse transcriptase inhibitor (NNRTI) -resistant HIV-1 strains, including L100I/K103N and K103N/Y181C double mutants, which are resistant to all approved NNRTI. The concept of structural flexibility in overcoming the effects of drug-resistance mutations evolved from systematic structural studies throughout the drug-development process. However, experiments spanning a 5-year period were unsuccessful in yielding a crystal structure of the HIV-1 reverse transcriptase (RT)/TMC278 complex .

Methods:  A systematic protein engineering study was performed to:  create RT mutations based on other HIV-1 strains; remove lysine and glutamic acid patches to reduce surface entropy; alter amino acid residues that were shown to make lattice contacts in earlier crystal forms; attempt to enhance contacts seen in previous crystal forms; and remove disordered residues. Several iterative rounds of mutagenesis and crystallization trials with TMC278 and other NNRTI were carried out.

Results:  A systematic protein engineering study has resulted in crystals of HIV-1 RT/NNRTI complexes diffracting to 1.8-Å resolution. The crystal structures of wild type, L100I/K103N and K103N/Y181C double-mutant RT in complex with TMC278 illustrate the role of conformational flexibility (wiggling) and repositioning (jiggling) in overcoming the effects of drug-resistance mutations. High-resolution data sets, better than 2.0-Å resolution, are now produced routinely for numerous NNRTI. This is in contrast to earlier structures of HIV-1 RT commonly determined at ~2.5- to 3.0-Å resolution.

Conclusions:  The swiftness of crystallization of engineered RT that brought success in obtaining high-resolution structures of RT provides an excellent opportunity for accurate understanding of inhibitor-protein interactions, and the effects of resistance mutations. It also offers the opportunity to peruse systematic structure-based design of new RT inhibitors, and structure-based screening for new lead compounds directed to existing and possible new targets of RT. The technique of iterative crystal engineering can also be used in obtaining and improving crystals of important yet difficult proteins.