ritonavir and kynostatin-272

Water molecules are commonly observed in crystal structures of protein-ligand complexes where they mediate protein-ligand binding. It is of considerable theoretical and practical importance to determine quantitatively the individual free energy contributions of these interfacial water molecules to protein-ligand binding and to elucidate factors that influence them. The double-decoupling free energy molecular dynamics simulation method has been used to calculate the binding free energy contribution for each of the four interfacial water molecules observed in the crystal structure of HIV-1 protease complexed with KNI-272, a potent inhibitor. While two of these water molecules contribute significantly to the binding free energy, the other two have close to zero contribution. It was further observed that the protonation states of two catalytic aspartate residues, Asp25 and Asp125, strongly influence the free energy contribution of a conserved water molecule Wat301 and that different inhibitors significantly influence the free energy contribution of Wat301. Our results have important implications on our understanding of the role of interfacial water molecules in protein-ligand binding and to structure-based drug design aimed at incorporating these interfacial water molecules into ligands.

Topics: Binding Sites; Computer Simulation; Crystallization; HIV Protease; HIV Protease Inhibitors; Kinetics; Models, Chemical; Models, Molecular; Oligopeptides; Protein Binding; Protein Conformation; Ritonavir; Thermodynamics; Water

The activity of three human immunodeficiency virus (HIV) protease inhibitors was investigated in human primary monocytes/macrophages (M/M) chronically infected by HIV-1. Saquinavir, KNI-272, and ritonavir inhibited the replication of HIV-1 in vitro, with EC50s of approximately 0.5-3.3 microM. However, only partial inhibition was achievable, even at the highest concentrations tested. Also, the activity of these drugs in chronically infected M/M was approximately 7- to 26-fold lower than in acutely infected M/M and approximately 2- to 10-fold lower than in chronically infected H9 lymphocytes. When protease inhibitors were removed from cultures of chronically infected M/M, production of virus rapidly returned to the levels found in untreated M/M. Therefore, relatively high concentrations of protease inhibitors are required to suppress HIV-1 production in chronically infected macrophages, and such cells may be a vulnerable point for the escape of virus in patients taking these drugs.

Topics: Anti-HIV Agents; Cell Line; Cells, Cultured; HIV Core Protein p24; HIV Protease Inhibitors; HIV-1; Humans; Macrophages; Monocytes; Oligopeptides; Reverse Transcriptase Inhibitors; Ritonavir; Saquinavir; Zidovudine

We asked whether human immunodeficiency virus type 1 (HIV-1) protease plays a major role in the early stages of infection (i.e. from viral entry to reverse transcription) by using various protease inhibitors (saquinavir, ritonavir, and KNI-272). When assessed in the two-day multinuclear activation of a galactosidase indicator (MAGI) assay, involving a single cycle of HIV-1 replication, all protease inhibitors failed to block infection of HeLa-CD4-LTR-beta-gal cells by HIV-1, while reverse transcriptase (RT) inhibitors (AZT and ddI) completely blocked the infection. Moreover, when HIV-1 proviral DNA synthesis was examined by polymerase chain reaction in HeLa-CD4-LTR-beta-gal cells exposed to HIV-1 and cultured in the presence of protease inhibitors, a significant amount of proviral DNA was detected, while no proviral DNA synthesis was detected when the cells were cultured in the presence of RT inhibitors. Protease inhibitors also failed to block chloramphenicol acetyltransferase (CAT) expression in HLCD4-CAT cells exposed to HIV-1, while RT inhibitors completely suppressed CAT expression. These results strongly suggest, contrary to a previous report by Nagy et al. (1994), that HIV-1 protease does not play a major role in the early stages of infection.

Topics: Chloramphenicol O-Acetyltransferase; HeLa Cells; HIV Infections; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Oligopeptides; Ritonavir; Saquinavir

Eleven different recombinant, drug-resistant HIV-1 protease (HIV PR) mutants--R8Q, V32I, M46I, V82A, V82F, V82I, I84V, V32I/I84V, M46I/V82F, M46I/I84V, and V32I/K45I/F53L/A71V/I84V/L89M--were generated on the basis of results of in vitro selection experiments using the inhibitors A-77003, A-84538, and KNI-272. Kinetic parameters of mutant and wild-type (WT) enzymes were measured along with inhibition constants (Ki) toward the inhibitors A-77003, A-84538, KNI-272, L-735,524, and Ro31-8959. The catalytic efficiency, kcat/Km, for the mutants decreased relative to WT by a factor of 1.2-14.8 and was mainly due to the elevation of Km. The effects of specific mutations on Ki values were unique with respect to both inhibitor and mutant enzyme. A new property, termed vitality, defined as the ratio (Kikcat/Km)mutant/(Kikcat/Km)WT was introduced to compare the selective advantage of different mutants in the presence of a given inhibitor. High vitality values were generally observed with mutations that emerged during in vitro selection studies. The kinetic model along with the panel of mutants described here should be useful for evaluating and predicting patterns of resistance for HIV PR inhibitors and may aid in the selection of inhibitor combinations to combat drug resistance.

Topics: Amino Acid Sequence; Binding Sites; Carbamates; Cloning, Molecular; Drug Resistance, Microbial; HIV Protease; HIV Protease Inhibitors; HIV-1; Indinavir; Isoquinolines; Kinetics; Methylurea Compounds; Mutagenesis, Site-Directed; Oligopeptides; Point Mutation; Pyridines; Quinolines; Recombinant Proteins; Saquinavir; Structure-Activity Relationship; Thiazoles; Valine