Design of HIV Protease Inhibitors Targeting Protein Backbone: An Effective Strategy for Combating Drug Resistance

Arun K. Ghosh received his Ph.D. from the University of Pittsburgh and pursued postdoctoral research with Professor E. J. Corey at Harvard University. He was a Professor of Chemistry at the University of Illinois at Chicago from 1994 to 2005. From 2005 to present, he is a Professor in the departments of chemistry and medicinal chemistry at Purdue University.

Bruno D. Chapsal obtained his M.S. in chemistry from CPE Lyon, France, and received his Ph.D. from Stony Brook University, NY, under the direction of Professor Iwao Ojima. He is currently carrying out postdoctoral research in Professor Ghosh’s laboratories.

Irene T. Weber received her Ph.D. from the University of Oxford, England, under the supervision of Professor Louise Johnson. She pursued postdoctoral research with Professor Thomas Steitz at Yale University. She was Professor of Microbiology and Immunology at Thomas Jefferson University in Philadelphia from 1991 to 2000. From 2001 to the present she is Professor of Biology and Chemistry at Georgia State University in Atlanta.

Hiroaki Mitsuya received his M.D. and Ph.D. from the Kumamoto University School of Medicine, Japan. He was a Visiting Scientist at the National Cancer Institute from 1982 to 1990. From 2001 to present, he is Principal Investigator & Chief, Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, Bethesda, MD. From 1997 to present, he is also Professor of Medicine and Chairman, Department of Internal Medicine, Kumamoto University School of Medicine, Japan.

The discovery of human immunodeficiency virus (HIV) protease inhibitors (PIs) and their utilization in highly active antiretroviral therapy (HAART) have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

The present line of research focuses on the presumption that an inhibitor that can maximize interactions in the HIV-1 protease active site, particularly with the enzyme backbone atoms, will likely retain these interactions with mutant enzymes. Our structure-based design of HIV PIs specifically targeting the protein backbone has led to exceedingly potent inhibitors with superb resistance profiles.

We initially introduced new structural templates, particulary nonpeptidic conformationally constrained P₂ ligands that would efficiently mimic peptide binding in the S₂ subsite of the protease and provide enhanced bioavailability to the inhibitor. Cyclic ether derived ligands appeared as privileged structural features and allowed us to obtain a series of potent PIs. Following our structure-based design approach, we developed a high-affinity 3(R),3a(R),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) ligand that maximizes hydrogen bonding and hyrophobic interactions in the protease S₂ subsite. Combination of this ligand with a range of different isosteres led to a series of exceedingly potent inhibitors.

Darunavir, initially TMC-114, which combines the bis-THF ligand with a sulfonamide isostere, directly resulted from this line of research. This inhibitor displayed unprecedented enzyme inhibitory potency (K_i = 16 pM) and antiviral activity (IC₉₀ = 4.1 nM). Most importantly, it consistently retained is potency against highly drug-resistant HIV strains. Darunavirs IC₅₀ remained in the low nanomolar range against highly mutated HIV strains that displayed resistance to most available PIs.

Our detailed crystal structure analyses of darunavir-bound protease complexes clearly demonstrated extensive hydrogen bonding between the inhibitor and the protease backbone. Most strikingly, these analyses provided ample evidence of the unique contribution of the bis-THF as a P₂-ligand. With numerous hydrogen bonds, bis-THF was shown to closely and tightly bind to the backbone atoms of the S₂ subsite of the protease. Such tight interactions were consistently observed with mutant proteases and might therefore account for the unusually high resistance profile of darunavir. Optimization attempts of the backbone binding in other subsites of the enzyme, through rational modifications of the isostere or tailor made P₂ ligands, led to equally impressive inhibitors with excellent resistance profiles.

The concept of targeting the protein backbone in current structure-based drug design may offer a reliable strategy for combating drug resistance.