Molecular Recognition of Cyclic Urea HIV-1 Protease Inhibitors

Paul J Ala,Richard J Deloskey,Edward E Huston,Prabhakar K Jadhav,Patrick Y.S Lam,Charles J Eyermann,C Nicholas Hodge,Margaret C Schadt,Frank A Lewandowski,Patricia C Weber,Denise D Mccabe,Jodie L Duke,Chong-Hwan Chang

doi:10.1074/jbc.273.20.12325

Abstract

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.

Highlights

An essential step in the life cycle of the human immunodeficiency virus (HIV)1 is the proteolytic cleavage of the viral polyprotein gene products of gag and gag-pol into active structural and replicative proteins [1, 2]
In an attempt to delay the onset of drug resistance, the FDA approved the use of combination therapy, i.e. a mixture of protease and reverse transcriptase antiretroviral agents
In a continuing effort to identify new interactions that might increase the potency of our inhibitors, and other members of the cyclic family, we have performed a structural analysis of HIV-1 protease in complex with a series of CUs, which have IC90 values ranging from 5.1 to 4700 nM

Summary

EXPERIMENTAL PROCEDURES

Inhibitors and Ki Measurements—The inhibitors XK216, XK263, DMP323, DMP450, XV638, and SD146 were synthesized as reported (14, 19 –22), and their Ki values were measured as described previously [23]. HIV-1 Protease Preparation and Purification—The protease was mutated at a single position (Cys Ala), expressed in Escherichia coli BL21 (DE-3) [24], purified from inclusion bodies, and refolded using a hydrophobic interaction column [25]. Crystallization and Data Collection—Frozen aliquots (ϳ120 ml) of the protease (32 ␮g/ml) in 50 mM sodium acetate buffer (pH 5.5), 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 5% ethylene glycol, and 350 mM NaCl were thawed and immediately mixed with one of the six inhibitors at a concentration equal to a 1000 –5000-fold molar excess over its Ki value. The protein was concentrated to 150 ␮g/ml, using an Amicon-stirred cell equipped with a YM3 membrane, and exchanged

Molecular Recognition of Cyclic Urea Inhibitors

RESULTS

Distance between atoms

DISCUSSION