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)

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).
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PMID:Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli. 207 Jul 93

The aspartyl protease of human immunodeficiency virus 1 (HIV-1) has been expressed in Escherichia coli at high levels, resulting in the formation of inclusion bodies which contain denatured insoluble aggregates of the protease. After solubilization of these inclusion bodies in guanidinium chloride, the protease was purified to apparent homogeneity by a single-step reverse-phase HPLC procedure. The purified, but inactive, protein was denatured in 8 M urea and refolded to produce the active protease. Enzyme activity was demonstrated against the substrate H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH, modeled after the cleavage region between residues 128 and 135 in the HIV gag polyprotein. With this substrate, a Vmax of 1.3 +/- 0.2 mumol/(min.mg) and KM of 2.0 +/- 0.3 mM were determined at pH 5.5. Pepstatin (Iva-Val-Val-Sta-Ala-Sta-OH) and substrate analogues with the Tyr-Pro residues substituted by Sta, by Phe psi [CH2N]Pro, and by Leu psi [CH(OH)CH2]Val inhibited the protease with KI values of 360 nM, 3690 nM, 3520 nM, and less than 10 nM, respectively. All were competitive inhibitors, and the tightest binding compound provided an active site titrant for the quantitative determination of enzymatically active HIV-1 protease.
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PMID:Substrate analogue inhibition and active site titration of purified recombinant HIV-1 protease. 218 16

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.
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PMID:A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease. 855 7

The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.
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PMID:Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors. 858 57

The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/L63I) (Mildner, A. M., Rothrock, D. J., Leone, J. W., Bannow, C. A., Lull, J. M., Reardon, I. M., Sarcich, J. L., Howe, W. J., Tomich, C.-S. C., Smith, C. W., Heinrikson, R. L., and Tomasselli, A. G. (1994) Biochemistry 33, 9405-9413), designed to better resist autolysis. Monitoring the intrinsic fluorescence of the two enzymes during urea-mediated denaturation has shown that at high NaCl concentration, both the conformational stability ( DeltaG0) and the transition midpoint (D1/2) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and far- and near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a two-state equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity.
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PMID:Conformational stability and catalytic activity of HIV-1 protease are both enhanced at high salt concentration. 862 2

A series of novel P1/P1'-substituted cyclic urea-based HIV-1 protease inhibitors was prepared. Three different synthetic schemes were used to assemble these compounds. The first approach uses amino acid-based starting materials and was originally used to prepare DMP 323. The other two approaches use L-tartaric acid or L-mannitol as the starting material. The required four contiguous R,S,S,R centers of the cyclic urea scaffold are introduced using substrate control methodology. Each approach has specific advantages based on the desired P1/P1' substituent. Designing analogs based on the enzyme's natural substrates provided compounds with reduced activity. Attempts at exploiting hydrogen bond sites in the S1/S1' pocket, suggested by molecular modeling studies, were not fruitful. Several analogs had better binding affinity compared to our initial leads. Modulating the compound's physical properties led to a 10-fold improvement in translation resulting in better overall antiviral activity.
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PMID:Preparation and structure-activity relationship of novel P1/P1'-substituted cyclic urea-based human immunodeficiency virus type-1 protease inhibitors. 866 59

High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.
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PMID:Cyclic HIV protease inhibitors: synthesis, conformational analysis, P2/P2' structure-activity relationship, and molecular recognition of cyclic ureas. 878 49

A tetrahedrally hydrogen-bonded structural water molecule, water 301, is seen in the crystal structure of nearly every HIV-1 protease/inhibitor complex. Although the urea oxygen of the designed inhibitor, DMP323, mimics and replaces water 301, other water molecules are seen in the protease/DMP323 crystal structure. As a first step toward understanding how water molecules may contribute to inhibitor potency and specificity, we have recorded water-NOESY and water-ROESY spectra of the protease/ DMP323 complex. Cross relaxation rates derived from these spectra, together with interproton distances calculated from the crystal structure of the complex, were used to classify the exchange cross peaks as follows: (A) a direct NOE with a water proton, (B) an indirect NOE with water through a labile protein proton, and (C) direct exchange of an amide proton with water. Type A and B cross peaks were analyzed using three models of water dynamics: (1) two-site exchange, with water molecules randomly hopping between bound and free states, (2) bound water with internal motion, and (3) free diffusion. Using the two-site exchange model to analyze the relaxation data of the type A cross peaks, it was found that the water molecules had short residence times, ca. 500 ps. in contrast with the > 9 ns residence time estimated for water 301 in the protease/P9941 complex [Grzesiek et al. (1994) J. Am. Chem. Soc. 116, 1581-1582]. The NMR data are consistent with the X-ray observation that two symmetry-related water molecules, waters 422 and 456, are bound at the DMP323 binding site. Hence, these water molecules may help to stabilize the structure of the complex. Finally, it was found that three buried and hydrogen-bonded Thr hydroxyl protons were in slow exchange with solvent. In contrast, it was found that the DMP323 H4/H5 hydroxyl protons and the Asp25/125 carboxyl protons, which form a buried hydrogen-bonded network at the catalytic site of the protease, are in rapid exchange with solvent, suggesting that solvent can penetrate into the buried protein/inhibitor interface on the millisecond to microsecond time scale.
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PMID:Mapping hydration water molecules in the HIV-1 protease/DMP323 complex in solution by NMR spectroscopy. 884 Nov 13

The maturation of the human immunodeficiency virus type 1 protease (PR) from the Gag-Pol polyprotein is dependent on the intrinsic proteolytic activity of the dimeric Gag-Pol. Herein, we report the kinetics and conformational stabilities of two unique fusion proteins of the protease. In one, X28-PR, a random sequences of 28 amino acids (X28) was linked to the N terminus of the mature protease. In the second construct, X28-delta TF*PR*delta Pol, X28 is fused to the protease which is flanked at both its termini by short sequences (delta) which correspond to the native sequences of the Gag-Pol precursor. Autoprocessing of the latter protein was prevented by inserting an Ala at the native protease cleavage sites. The measured kinetic parameters and the pH-rate profile of both enzymes are nearly identical to those of the mature protease. However, these fusion proteins are more sensitive to acid and urea denaturation than the mature protease. The decrease in the conformational stability of X28-PR and X28-delta TF*PR*delta Pol is reflected by increases in their apparent dissociation constants (Kd) from < 5 nM to approximately 180 and 25 nM, respectively. These results suggest that subunit interactions and hence the dimer stability of the protease domain in the Gag-Pol polyprotein differ from those of the mature protease. The high Kd of X28-PR further suggests that addition of non-native sequences to the N terminus of the protease destablizes the dimer.
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PMID:Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease. 884 Nov 42

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.
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PMID:Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy. 886 86


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