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)

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.
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PMID:Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins. 133 Oct 99

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.
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PMID:Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease. 221 28

Molecular dynamics simulations of human immunodeficiency virus (HIV)-1 protease with a model substrate were used to test if there is a stable energy minimum for a proton that is equidistant from the four delta oxygen atoms of the two catalytic aspartic acids. The crystal structure of HIV-1 protease with a peptidic inhibitor was modified to model the peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln for the starting geometry. A proton was positioned between the two closet oxygen atoms of the two catalytic aspartic acids, and close to the carbonyl oxygen of the scissile bond in the substrate. All crystallographic water molecules were included. Two molecular dynamics simulations were run: 30 ps with united-atom potentials and 40 ps using the more accurate all-atom potentials. The molecular dynamics used a new algorithm that increased the speed and allowed the elimination of a cut-off for non-bonded interactions and the inclusion of an 8 A shell of water molecules in the calculations. The overall structure of the protease dimer, including the catalytic aspartic acids, was stable during the course of the molecular dynamics simulations. The substrate and a water molecule, that is an important component of the binding site, were stable during the simulation using all-atom potentials, but more mobile when united-atom potentials were used. A Poincare map representation showed that the positions of the proton and its coordinating oxygen atoms were stable for 93% of both simulations, although many of the buried and poorly accessible water molecules exchanged with solvent. The proton has a stable minimum energy position and maintains coordination with all four delta oxygen atoms of the two catalytic aspartic acids and the carbonyl oxygen of the scissile bond of the substrate. Therefore, a loosely bound hydrogen ion at this position will not be rapidly exchanged with solvent, and will rebond to either a catalytic aspartic acid or possibly the substrate. The implications for the reaction mechanism are discussed.
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PMID:Molecular dynamics simulations of HIV-1 protease with peptide substrate. 770 Aug 67

Hydrogen bonding plays an important role in the stabilization of complexes between HIV-1 protease (HIV-1 PR) and its inhibitors. The adequate treatment of the protease active site protonation state is important for accurate molecular simulations of the protonation state is important for accurate molecular simulations of the protease-inhibitor complexes. We have applied the free energy simulation/thermodynamic cycle approach to evaluate the relative binding affinities of the S vs R isomers of the U85548E inhibitor of the protease. Several mono- and diprotonation states of the catalytic aspartic acid residues of the protease active site were considered in the course of molecular simulations. The calculated difference in binding free energy of the S vs R isomers strongly depended on the location of proton(s), but in all cases the binding free energy of the S inhibitor was higher. On the basis of our calculations, we propose that in the HIV-1 PR-inhibitor complex only one catalytic aspartic acid residue is protonated and that the binding free energy of the S isomer is ca. 2.8 kcal/mol higher than that of the R isomer. The accuracy of these predictions shall be evaluated when binding affinities of both isomers become available.
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PMID:Relative binding free energies of peptide inhibitors of HIV-1 protease: the influence of the active site protonation state. 783 38

Human immunodeficiency virus-1 (HIV-1) protease is catalytically active as a dimer of identical subunits that associate through noncovalent interactions. To investigate the forces stabilizing HIV-1 protease in its active form, we have studied the effects of pH and salts on structure and function of the enzyme. Enzymatic activity was measured by following the hydrolysis of a fluorogenic substrate. Dissociation of the dimer into its subunits was monitored by gel filtration, while conformational changes in the enzyme were probed by measurements of intrinsic tryptophan fluorescence. Mg2+ ions were capable of dissociating the dimeric enzyme with a concomitant red shift and increase in quantum yield of the tryptophan fluorescence, indicating increased accessibility of tryptophan to the aqueous environment. These structural changes also were associated with a loss of catalytic activity which was insensitive to substrate concentration, consistent with noncompetitive inhibition. Both structural and functional changes could be attributed to binding of Mg2+ ions to a site with an apparent dissociation constant of approximately 2 M. In contrast, increasing concentrations of Na ions up to 5 M were without effect. Increasing pH had similar effects on HIV-1 protease as increasing Mg2+ ions concentration, with concomitant dissociation into subunits, increase in quantum yield and red shift in tryptophan fluorescence, and loss in catalytic activity. The apparent pKa for these structural and functional transitions was 6.95 +/- 0.08. This value is consistent with that of an aspartic acid residue with an anomalously high pKa, which has been implicated in the catalytic activity of HIV-1 protease.
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PMID:Effect of pH and nonphysiological salt concentrations on human immunodeficiency virus-1 protease dimerization. 784 Sep 36

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.
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PMID:A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant. 788 51

Four kinds of retrovirus protease inhibitors (RPI-856 A, B, C and D) were isolated as white powder from the culture filtrate of a soil isolate, Streptomyces sp. AL-322 by column chromatography using Diaion HP-20, Sephadex LH-20, ODS reversed phase HPLC and SP-2SW ion exchange HPLC. The structures of these inhibitors were elucidated by physico-chemical properties, chemical reactions and spectral analyses, as valyl-ADPAA-leucyl-AOPBA-valyl-valyl-aspartic acid (RPI-856 A and B) and valyl-ADPAA-leucyl-AOPBA-valyl-valine (RPI-856 C and D) [ADPAA = 2-amino-2-(3,5-dihydroxyphenyl)acetic acid, AOPBA = 3-amino-2-oxo-4-phenylbutyric acid]. RPI-856 A and B, and RPI-856 C and D were both determined to be diasteromers each other on the asymmetric carbon in AOPBA. These four inhibitors strongly inhibited in vitro HIV-1 protease and HTLV-I protease both derived from recombinant Escherichia coli with IC50 of 10(-7) approximately 10(-8) M.
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PMID:Novel retrovirus protease inhibitors, RPI-856 A, B, C and D, produced by Streptomyces sp. AL-322. 804 53

The human immunodeficiency virus type 1 (HIV-1) protease is a potential target of acquired immune deficiency syndrome (AIDS) therapy. A highly potent, perfectly symmetrical phosphinate inhibitor of this enzyme, SB204144, has been synthesized. It is a competitive inhibitor of HIV-1 protease, with an apparent inhibition constant of 2.8 nM at pH 6.0. The three-dimensional structure of SB204144 bound to the enzyme has been determined at 2.3-A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel F(o) magnitude to - Fc parallel/sigma magnitude of F(o)), of 0.178. The inhibitor is held in the enzyme active site by a set of hydrophobic and hydrophilic interactions, including an interaction between Arg8 and the center of the terminal benzene rings of the inhibitor. The phosphinate establishes a novel interaction with the two catalytic aspartates; each oxygen of the central phosphinic acid moiety interacts with a single oxygen of one aspartic acid, establishing a very short (2.2-2.4 A) oxygen-oxygen contact. As with the structures of penicillopepsin bound to phosphinate and phosphonate inhibitors [Fraser, M. E., Strynadka, N. C., Bartlett, P. A., Hanson, J. E., & James, M. N. (1992) Biochemistry 31, 5201-14], we interpret this short distance and the stereochemical environment of each pair of oxygens in terms of a hydrogen bond that has a symmetric single-well potential energy curve with the proton located midway between the two atoms.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of human immunodeficiency virus-1 protease by a C2-symmetric phosphinate. Synthesis and crystallographic analysis. 834 1

The human immunodeficiency (HIV) codes for an aspartic protease known to be essential for retroviral maturation and replication. The HIV protease can recognize Phe-Pro and Tyr-Pro sequences as the virus-specific cleavage site. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treatment of acquired immunodeficiency syndrome (AIDS). HIV protease is formed from two identical 99 amino acid peptides. We replaced the two Cys residues by L-Ala to synthesize [Ala67,95]-HIV-1 protease by the solid phase method and then prepared [Tyr6,42, Nle36,46, (NHCH2COSCH2CO)51-52, Ala67,95] HIV-1 protease (NY-5 isolate) using the thioester chemical ligation method. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing an unnatural amino acid, (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) with a hydroxymethylcarbonyl (HMC) isostere. Among them, the conformationally constrained tripeptide kynostatin (KNI)-272 (iQoa-Mta-Apns-Thz-NHBut) was a highly selective and superpotent HIV protease inhibitor (Ki = 0.0055 nM). KNI-272 exhibited potent antiviral activities against both AZT-sensitive and -insensitive clinical HIV-1 isolates as well as HIV-2 with low cytotoxicity. After i.d. administration, bioavailability of KNI-272 was 42.3% in rats. Also, KNI-272 exhibited in vivo anti-HIV activities in human PBMC-SCID mice. The x-ray crystallography and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of HIV protease active site in the essentially same hydrogen-bonding mode as the transition state. This result implies that the HMC isostere is an ideal transition-state mimic and contributes to the high activity of KNI-272.
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PMID:Design and synthesis of substrate-based peptidomimetic human immunodeficiency virus protease inhibitors containing the hydroxymethylcarbonyl isostere. 878 65

A series of novel HIV-1 protease inhibitors was prepared in a short stereospecific manner. These compounds have only one P1 substituent interacting with the S1/S1' binding site of HIV-1 protease and only one hydroxyl group interacting with the catalytic aspartic acid domain, X-ray crystallography confirmed the desired R, R configuration of the final products.
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PMID:Synthesis of novel cyclic urea based HIV-1 protease inhibitors. 887 53


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