Major enhancement of the affinity of an enzyme for a transition-state analog by a single hydroxyl group.

The compound 1,6-dihydropurine ribonucleoside, prepared by reduction of nebularine in the presence of ultraviolet light, is bound by adenosine deaminase approximately 10(8)-fold less tightly than 6-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog. This difference in affinities, which is associated with the presence of a single hydroxyl group in the second compound, suggests the degree to which one or a few hydrogen bonds may stabilize the transition state in an enzyme reaction of this type.

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.

The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.

An orally active inhibitor of renin.

A potent renin inhibitor, U-71038 (Boc-Pro-Phe-N-MeHis-Leu psi[CHOHCH2]Val-Ile-Amp), was tested for oral effectiveness. Enzyme kinetic studies indicated that U-71038 was a competitive inhibitor of hog renin with an inhibitor constant (Ki) value of 12 nM. Intravenous as well as oral administration of U-71038 to anesthetized, ganglion-blocked rats infused with hog renin elicited dose-related hypotensive responses. Intravenous administration of U-71038 to conscious, sodium-depleted monkeys caused dose-related decreases of blood pressure and plasma renin activity without affecting heart rate. Similarly, the oral administration of U-71038 at 50 mg/kg to conscious, sodium-depleted monkeys elicited a pronounced hypotension and decrease in plasma renin activity that persisted for 5 hours. The hypotensive responses elicited by intravenous and oral administration of U-71038 to hog renin-infused rats and sodium-depleted monkeys were shown to be due entirely to inhibition of the renin-angiotensin system. A comparison of the results obtained after the intravenous administration of U-71038 with the results obtained after the oral administration of U-71038 implied that at least 10% of the orally administered U-71038 must have been absorbed to cause the observed effects in hog renin-infused rats and sodium-depleted monkeys. The studies demonstrated that an inhibitor of renin with a long duration of action and with oral effectiveness is a feasible entity.

Design and synthesis of potent and specific renin inhibitors containing difluorostatine, difluorostatone, and related analogues.

Peptides that contain difluorostatine and difluorostatone residues have been shown to be potent inhibitors of the aspartyl protease renin. The readily hydrated fluoro ketone is proposed to mimic the tetrahedral intermediate that forms during the enzyme-catalyzed hydrolysis of a peptidic bond. It is suggested that the sp3-hybridized ketal acts as a transition-state analogue renin inhibitor. The fluoro ketone is shown to be a much more effective inhibitor than the corresponding nonfluorinated ketone, which acts as a pseudosubstrate. More lipophilic side chains at the P1 site can enhance the inhibitory potency of the difluorostatine analogue, but this cannot be demonstrated in the difluorostatone series. Additionally, high renin specificity has been shown for a difluorostatone-containing peptide.

Design and synthesis of a potent and specific renin inhibitor with a prolonged duration of action in vivo.

A structure-activity analysis of peptides containing backbone C alpha-methyl and N alpha-methyl modifications led to the discovery of potent renin inhibitors with high metabolic stability. In vitro, Boc-Pro-Phe-N alpha-MeHis-Leu psi-[CHOHCH2]Val-Ile-Amp (XII) is a potent inhibitor of human plasma renin with IC50 of 0.26 nM. It is a much weaker inhibitor of other aspartic proteases such as porcine pepsin or bovine cathepsin D (IC50 = 6 microM). It was shown not to be degraded by a rat liver homogenate preparation. In vivo, it inhibited plasma renin activity and lowered blood pressure of furosemide-treated cynomolgus monkeys. At a dose of 5 mg/kg iv, the pronounced hypotensive response persisted for greater than 3 h postinfusion.

Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy.

The structure-activity studies leading to the potent and clinically efficacious HIV protease inhibitor ritonavir are described. Beginning with the moderately potent and orally bioavailable inhibitor A-80987, systematic investigation of peripheral (P3 and P2') heterocyclic groups designed to decrease the rate of hepatic metabolism provided analogues with improved pharmacokinetic properties after oral dosing in rats. Replacement of pyridyl groups with thiazoles provided increased chemical stability toward oxidation while maintaining sufficient aqueous solubility for oral absorption. Optimization of hydrophobic interactions with the HIV protease active site produced ritonavir, with excellent in vitro potency (EC50 = 0.02 microM) and high and sustained plasma concentrations after oral administration in four species. Details of the discovery and preclinical development of ritonavir are described.

Novel triterpene sulfates from Fusarium compactum using a rhinovirus 3C protease inhibitor screen.

Two novel triterpene sulfates have been isolated from Fusarium compactum by bioactivity-directed fractionation using an assay which measures the inhibition of proteolytic activity of rhinovirus 3C protease on a fluorogenic peptide substrate. The compounds were purified by countercurrent and reverse phase chromatographies. NMR, MS, UV and IR studies revealed two triterpene sulfates, uncommon metabolites of terrestrial fungi.

Contribution of a single hydroxyl group to transition-state discrimination by adenosine deaminase: evidence for an "entropy trap" mechanism.

Adenosine deaminase was found to bind 6-hydroxy-1,6-dihydropurine ribonucleoside (II), formed by reversible addition of water to purine ribonucleoside (I) in a reaction analogous to formation of a tetrahedral intermediate in substrate deamination, with an apparent Ki value of 3 x 10(-13) M at 20 degrees C. 1,6-Dihydropurine ribonucleoside (IV), synthesized by photolysis of purine ribonucleoside in the presence of NaBH4, exhibited a Ki value of 5.4 x 10-6 M. After correction for differences between the relative free energies of solvation of II and IV, the 6-hydroxyl group of II was estimated to contribute more than 16 kcal to the free energy of binding, approaching the enthalapy of formation of a single hydrogen bond to charged group in the vapor phase. The relatively weak binding of IV and of substrate water suggests that entropic effects, arising from the cooperative action of binding determinants contained within these separate molecules, contribute more than 10 kcal/mol to the free energy of binding of II in which these binding determinants are contained within a single molecule. In free solution, the entropy of reversible hydration of I was evaluated by measuring the temperature dependence of equilibria of protonation of I and of pseudobase formation from I-methylpurinium ribonucleoside as -35 eu, comparable with the entropy of activation for the uncatalyzed hydrolysis of adenosine. In the active site of adenosine deaminase, this thermodynamic obstacle is evidently climbed spontaneously as a result of attractive interactions between the active site and the critical hydroxyl group at the 6-position.(ABSTRACT TRUNCATED AT 250 WORDS)

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.

Kinetics of the inhibition of human renin by an inhibitor containing a hydroxyethylene dipeptide isostere.

We have studied the inhibition of both human and hog renins by compound 1 [Boc-Pro-Phe-N alpha-MeHis-Leu psi(CHOHCH2)Val-Ile-(aminomethyl)pyridine] using kinetics. The inhibition of human renin was shown to be time dependent and followed a minimal two-step mechanism. A loosely bound EI complex was formed rapidly with a dissociation constant, KI, of 12 nM. A second EI complex was slowly formed and was found to be 64-fold more strongly bound with an overall KI of 0.19 nM. The inhibition of human renin was shown to be competitive by both initial and final steady-state velocities. Compound 1 was also shown to be a competitive inhibitor of hog renin with a KI of 12 nM, but no evidence for time-dependent inhibition was detected. The differences in overall KI and inhibition kinetics may be a consequence of the similarities in structure between 1 and human angiotensinogen.

Mechanism and fidelity of HIV reverse transcriptase.

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.

Cardiovascular effects of a renin inhibitor in relation to posture in nonhuman primates.

Orthostatic cardiovascular reflexes were evaluated in conscious cynomolgus monkeys during interruption of the renin-angiotensin system with the renin inhibitor: RIP (Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys-OH). RIP was synthesized via solid phase techniques and purified to homogeneity. In vitro studies indicated that it exhibited classical competitive inhibition of renin with a KI of 2.3 microM. In vivo, RIP at 2 mg/kg per min inhibited renin and angiotensin I pressor responses indicating that it was not a specific renin inhibitor at this dose. However, in spite of the nonspecificity, RIP did not affect the supine blood pressure of sodium-replete monkeys, but did evoke hypotension in supine sodium depleted monkeys. RIP did not elicit significant orthostatic hypotension in either sodium-replete or sodium depleted monkeys. The cardiovascular effects of RIP described in this study appear to be due to inhibition of the renin-angiotensin system.

GS4071 is a slow-binding inhibitor of influenza neuraminidase from both A and B strains.

The kinetics of inhibition of purified influenza neuraminidases from A/Tokyo/3/67 and B/Memphis/3/89 influenza viruses by (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene- 1-carboxylic acid (GS4071) were investigated. Progress curve experiments established that GS4071 is a time dependent inhibitor of both A and B strains of influenza neuraminidase. The apparent association and dissociation rate constants, as well as the overall Ki values, were only modestly different for the two neuraminidase strains. The time dependent inhibition phenomenon, often referred to as slow-binding inhibition, appears to be a consequence of the very slow rate of dissociation of the compound from influenza neuraminidase.

Novel alpha- and beta-amino acid inhibitors of influenza virus neuraminidase.

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 alpha- and beta-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K(i) values in the 50 microM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the alpha-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the beta-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the alpha- or beta-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.

Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides.

The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA. The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.