Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).
J Am Chem Soc 2001 Sep 12
PMID:Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site. 1153 77

In the search for novel anti-human immunodeficiency virus type 1 (anti-HIV-1) agents from natural sources, 49 MeOH extracts of Korean plants were screened for their inhibitory effects against RNA-dependent DNA polymerase (RT) and ribonuclease H (RNase H) activities of HIV-1 reverse transcriptase and HIV-1 protease, and anti-HIV-1 activity. Regarding the HIV-1 reverse transcriptase, Agrimonia pilosa (whole plant), Cornus kousa (stem and leaf), Limonium tetragonum (root) and Mallotus japonicus (stem) showed significant inhibitory activity on RT activity with 50% inhibitory activity (IC(50)) of 8.9, 6.3, 7.5 and 11.9 microg/mL, respectively, whereas Agrimonia pilosa was also active against RNase H activity (IC(50) = 98.4 microg/mL). Four plants, namely Agrimonia pilosa (whole plant), Atractylodes japonica (root), Clematis heracleifolia (whole plant) and Syneilesis palmata (whole plant), were appreciably active (<35%) against recombinant HIV-1 protease at a concentration of 100 microg/mL. Crinum asiaticum var. japonicum (root) showed significant anti-HIV-1 activity (ED(50) = 12.5 microg/mL) with a favourable SI value of 16.
Phytother Res 2001 Sep
PMID:Inhibitory effects of Korean plants on HIV-1 activities. 1153 75

Implementation of derivatized carbohydrates as C(2)-symmetric HIV-1 protease inhibitors has previously been reported. With the objective of improving the anti-HIV activity of such compounds, we synthesized a series of fluoro substituted P1/P1' analogues. These compounds were evaluated for antiviral activity toward both wild type and mutant virus. The potency of the analogues in blocking HIV-1 protease was moderate, with K(i) values ranging from 1 to 7 nM. Nonetheless, compared to the parent nonfluorous inhibitors, a majority of the compounds exhibited improved antiviral activity, for example the 3-fluorobenzyl derivative 9b, which had a K(i) value of 7.13 nM and displayed one of the most powerful antiviral activities in the cellular assay of the series. Our results strongly suggest that fluoro substitution can substantially improve antiviral activity. The X-ray crystal structures of two of the fluoro substituted inhibitors (9a and 9f) cocrystallized with HIV-1 protease are discussed.
J Med Chem 2001 Sep 13
PMID:Design and synthesis of potent C(2)-symmetric diol-based HIV-1 protease inhibitors: effects of fluoro substitution. 1154 77

To inhibit the HIV-1 protease dimerization necessary to exhibit enzymatic activity, we synthesized and evaluated a new beta-sheet peptide (compound 1), containing 4-(2-aminoethyl)-6-dibenzofuranpropionic acid as a conformationally restricted linker and a non-peptidic beta-strand mimetic, 2-[3-([2-[(9-fluorenylmethoxy)carbonyl]hydrazino]carbonyl)-4-methoxyanilino]-2-oxoacetic acid (Fmoc-Hao-OH, compound 2). Kinetic analysis showed that compound 1 inhibited the dimerization of HIV-1 protease by a dissociative mechanism with a K(id) value of 5.4 microM at 37 degrees C (pH 5.0). However, compound 2 showed a small shift in the slope of the lines in the Zhang-Poorman plot (K(id)=9.1 microM), suggesting that compound 2 inhibits the dimerization of HIV-1 PR not only through a dissociative mechanism but also through an active-site directed mechanism partly. This is the first study of a non-peptidic inhibitor of HIV-1 protease dimerization.
Bioorg Med Chem Lett 2001 Sep 17
PMID:Design and synthesis of new inhibitors of HIV-1 protease dimerization with conformationally constrained templates. 1154 48

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.
Biochemistry 2001 Sep 18
PMID:Effectors of HIV-1 protease peptidolytic activity. 1155 Dec 11

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.
Anal Biochem 2001 Sep 15
PMID:Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? 1155 13

Ritonavir is an HIV-1 protease inhibitor that is often used to improve the systemic availability of concurrently administered protease inhibitors by impairing their metabolism through cytochrome P450 (CYP) 3A4. Pharmacodynamic relationships between plasma ritonavir concentrations and efficacy and toxicity have also been described. To date, published high-performance liquid chromatographic (HPLC) methods for the determination of ritonavir in human plasma are often complex, requiring the use of a buffered mobile phase that contains amine-modifiers (i.e. diethylamine, triethylamine). In the method herein, ritonavir was precipitated with acetonitrile plus barium hydroxide and zinc sulphate. Chromatographic separation was accomplished using a C-18 base-deactivated (250 x 4.6 mm I.D., 5 atm particle size) analytic column with a mobile phase composed of acetonitrile:water (52:48, v/v). Quantification was performed at 239 nm. Calibration curves were linear from 0.5-25 microg/ml (R2 > 0.999); percent errors, as a measure of accuracy, were < 12.7%. Intra- and inter-assay relative standard deviations (RSD) were below 12.8%. This method provides a rapid and simple means for the accurate and precise analysis of ritonavir in human plasma. Furthermore, the assay requires neither the use of a buffered mobile phase adjusted to a specific pH, nor the addition of amine modifiers. This method has been successfully used to determine plasma ritonavir concentrations in drug interaction studies.
Int J Clin Pharmacol Ther 2001 Sep
PMID:Rapid and sensitive high-performance liquid chromatographic method for the determination of ritonavir in human plasma. 1156 87

HIV-1-infected children are often treated with therapy regimens including protease inhibitors (PIs). We monitored the virologic response in a small group of pediatric patients undergoing therapy with regimens including the PI nelfinavir and determined whether new drug resistance mutations were present immediately after virologic failure. Seventeen reverse transcriptase inhibitor (RTI)-experienced children starting nelfinavir-containing therapy regimens were studied. After virologic failure, HIV-1 protease (PR) and RT sequences were examined for drug resistance mutations. Viral load levels decreased to <400 HIV RNA copies/ml in six patients and remained at <400 HIV RNA copies/ml in four patients. Three patients did not respond virologically; all three had mutations specific for one or more of their regimen drugs either before or soon after nelfinavir initiation. The virologic response was transient in eight patients whose viral loads did not decrease to <400 HIV RNA copies/ml. Genotypic data from seven of the eight patients revealed mutations specific for one or more of their regimen drugs after virologic rebound. PI resistance mutations occurred in eight patients: D30N in six, and L90M in three. In three patients, the only new mutation after failure was the RT mutation M184V. Despite virologic failure, sustained increases in CD4+ lymphocyte counts were noted in eight patients. We conclude that in this small group of pediatric patients, virologic failure occurred in all patients whose viral loads did not become undetectable after the switch to a nelfinavir-containing regimen. After failure, new drug resistance mutations were found in either PR or RT. Studies of larger cohorts are warranted to determine whether HIV-1 genotypic data can help in the formulation of effective salvage therapies in children.
AIDS Res Hum Retroviruses 2001 Sep 20
PMID:Emergence of drug resistance mutations in a group of HIV-infected children taking nelfinavir-containing regimens. 1160 42

Different proteins have been isolated from bovine milk including lactoferrin, lactoperoxidase, glycolactin, angiogenin-1, lactogenin, alpha-lactalbumin, lactoglobulin and casein. These proteins have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, protease and integrase, enzymes crucial to the HIV-1 life cycle. It was found that different milk proteins inhibited the three aforementioned HIV enzymes to different extents. Lactoferrin strongly inhibited HIV-1 reverse transcriptase but only slightly inhibited HIV-1 protease and integrase. On the other hand, alpha-lactalbumin, beta-lactoglobulin and casein inhibited HIV-1 protease and integrase to an appreciable extent but did not inhibit HIV-1 reverse transcriptase. Glycolactin and angiogenin-1 suppressed the activity of HIV-1 reverse transcriptase by a moderate extent but more powerfully inhibited HIV-1 protease and integrase. In comparison with the other milk proteins glycolactin was a strong inhibitor of HIV-1 protease and integrase and a moderate inhibitor of HIV-1 reverse transcriptase. Lactogenin was a strong inhibitor of HIV-1 integrase, a moderate inhibitor of HIV-1 reverse transcriptase and a weak inhibitor of HIV-1 protease.
Life Sci 2001 Sep 28
PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins. 1166 64

A new computational approach for the efficient docking of flexible ligands in a rigid protein is presented. It exploits the binding modes of functional groups determined by an exhaustive search with solvation. The search in ligand conformational space is performed by a genetic algorithm whose scoring function approximates steric effects and intermolecular hydrogen bonds. Ligand conformations generated by the genetic algorithm are docked in the protein binding site by optimizing the fit of their fragments to optimal positions of chemically related functional groups. We show that the use of optimal binding modes of molecular fragments allows to dock known inhibitors with about ten rotatable bonds in the active site of the uncomplexed and complexed conformations of thrombin and HIV-1 protease.
Biol Chem 2001 Sep
PMID:Fragment-Based flexible ligand docking by evolutionary optimization. 1168 19


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