12   A Femtomolar HIV-1 Protease Inhibitor with Subnanomolar Activity against Multidrug Resistant HIV-1 Strains.

J. Erickson*¹, S. Gulnik¹, L. Suvorov¹, E. Gustchina¹, D. Xie¹, G. Wang¹, M. A. Eissenstat¹, M.-P. De Bethune², H. Jonckheere², S. De Meyer², H. Azijn², R. Pauwels², P. Wigerinck², D. Surleraux², W. Verschueren², A. Tahri², A. Ghosh³, K. Yoshimura⁴, and H. Mitsuya⁴.
¹Tibotec, Rockville, MD;²Tibotec, Mechelen, Belgium;³Univ. of Illinois at Chicago; and⁴NCI, NIH, Bethesda, MD.

Background:The widespread clinical use of HIV-1 reverse transcriptase and protease inhibitors (PIs) has resulted in the emergence of resistant strains of HIV-1 that severely compromise the usefulness of such drugs. Cross-resistance within a mechanistic class of inhibitors is the rule, rather than the exception, and can render an entire class of inhibitors ineffective once high level resistance to one of them has developed.

Methods:We have pursued a structure- and fitness-based strategy, in combination with multidrug resistance profiling, to discover PIs that possess the following properties: 1) high antiretroviral potency; 2) activity against HIV-1 variants that are highly cross-resistant to current PIs; and 3) ability to suppress the evolution of drug resistant variants along commonly observed genetic pathways.

Results:We have identified a new series of PIs, exemplified by TMC126, that inhibit wild type and multi-drug resistant clinical isolates of HIV-1 with IC₅₀values down to 0.1 x 10^-9M. The K_dof TMC126 against wild type protease was measured to be 0.38 x 10^-15M using a thermodynamic linkage assay. Enzyme fitness values were measured using a panel of cross-resistant proteases and predicted barriers to the emergence of common active site mutations in the presence of TMC126.

Conclusions:Comparative selection experiments with TMC126 showed that drug resistant strains emerged more slowly and evolved using different genetic pathways than commonly observed for current PIs, consistent with the enzyme fitness data. Crystallographic analysis revealed a structural basis for the resistant-repellent properties of TMC126 and related inhibitors. Several compounds in this series are being evaluated for suitability as drug candidates.