Molecular mechanics analysis of inhibitor binding to HIV-1 protease.

Crystallographic structures of HIV protease with three different peptide-mimetic inhibitors were subjected to energy minimization using molecular mechanics, the minimized structures analyzed and the inhibitor binding energies calculated. Partial charge assignment for the hydrogen bonded catalytic aspartic acids, Asp25 and -25', was in good agreement with charge calculations using semi-empirical molecular orbital methods. Root mean square deviations on minimization were small and similar for both subunits in the protease dimer. The surface loops, which had the largest B factors, changed most on minimization; the hydrophobic core and the inhibitor binding site showed little change. The distance-dependent dielectric of D(r) = 4r was found to be preferable to D(r) = r. Distance restraints were applied for the intermolecular hydrogen bonds to maintain the conformation of the inhibitor binding site. Using the dielectric of D(r) = 4r, the calculated interaction energy of the three inhibitors with the protease ranged from -53 to -56 kcal/mol. The psi groups of the inhibitors were changed to add or remove a 'transition state analogue' hydroxyl group, and the loss in energy on the removal of this group was calculated to be 0.9-1.7 kcal/mol. This would represent 19-36% of the total measured difference in binding energy between the inhibitors JG365 and MVT-101.     

Evaluating the potency of HIV-1 protease drugs to combat resistance

HIV-1 protease has been an important drug target for the antiretroviral treatment of HIV infection. The efficacy of protease drugs is impaired by the rapid emergence of resistant virus strains. Understanding the molecular basis and evaluating the potency of an inhibitor to combat resistance are no doubt important in AIDS therapy. In this study, we first identified residues that have significant contributions to binding with six substrates using molecular dynamics simulations and Molecular Mechanics Generalized Born Surface Area calculations. Among the critical residues, Asp25, Gly27, Ala28, Asp29, and Gly49 are well conserved, with which the potent drugs should form strong interactions. We then calculated the contribution of each residue to binding with eight FDA approved drugs. We analyzed the conservation of each protease residue and also compared the interaction between the HIV protease and individual residues of the drugs and substrates. Our analyses showed that resistant mutations usually occur at less conserved residues forming more favorable interactions with drugs than with substrates. To quantitatively integrate the binding free energy and conservation information, we defined an empirical parameter called free energy/variability (FV) value, which is the product of the contribution of a single residue to the binding free energy and the sequence variability at that position. As a validation, the FV value was shown to identify single resistant mutations with an accuracy of 88%. Finally, we evaluated the potency of a newly approved drug, darunavir, to combat resistance and predicted that darunavir is more potent than amprenavir but may be susceptible to mutations on Val32 and Ile84.     

A Potent Human Immunodeficiency Virus Type 1 Protease Inhibitor, UIC-94003 (TMC-126), and Selection of a Novel (A28S) Mutation in the Protease Active Site

We identified UIC-94003, a nonpeptidic human immunodeficiency virus (HIV) protease inhibitor (PI), containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranyl urethane (bis-THF) and a sulfonamide isostere, which is extremely potent against a wide spectrum of HIV (50% inhibitory concentration, 0.0003 to 0.0005 microM). UIC-94003 was also potent against multi-PI-resistant HIV-1 strains isolated from patients who had no response to any existing antiviral regimens after having received a variety of antiviral agents (50% inhibitory concentration, 0.0005 to 0.0055 microM). Upon selection of HIV-1 in the presence of UIC-94003, mutants carrying a novel active-site mutation, A28S, in the presence of L10F, M46I, I50V, A71V, and N88D appeared. Modeling analysis revealed that the close contact of UIC-94003 with the main chains of the protease active-site amino acids (Asp29 and Asp30) differed from that of other PIs and may be important for its potency and wide-spectrum activity against a variety of drug-resistant HIV-1 variants. Thus, introduction of inhibitor interactions with the main chains of key amino acids and seeking a unique inhibitor-enzyme contact profile should provide a framework for developing novel PIs for treating patients harboring multi-PI-resistant HIV-1.     

High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains

The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR(V82A) and PR(I84V) that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10-1.53A. The crystal structures of PR and PR(I84V) with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR(V82A) complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR(V82A) showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10A resolution structure of PR(V82A) with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.     

Mechanism of aspartyl proteinase action. VII. Noncovalent complexes of HIV-1 aspartyl proteinase with substrate and substrate-like inhibitors

A computer model of a noncovalent complex of HIV-1 aspartyl protease with substrate-like inhibitor JG-365 was a priori constructed by using the approaches of theoretical conformational analysis and molecular mechanics. The root mean square deviation of the calculated conformation of the inhibitor from the X-ray diffraction analysis data was 0.87 A. These results enabled the a priori calculation of the structure of noncovalent complex of HIV-1 protease with a hexapeptide fragment of its native specific substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val. The only possible orientation of the cleavable peptide bond in this and the nucleophilic water molecule relative to the catalytically active Asp residues of the enzyme (Asp25 and Asp125) was found that provides for the chemical transformation of the substrate to a tetrahedral intermediate. An action mechanism of enzymes of this class was proposed on the basis of the analysis of calculated distances. We showed that neither steric distortion of the cleavable bond nor the formation of unfavorable contacts in molecules of the enzymes and their substrates accompany the optimum orientation of substrate molecules at the active sites of HIV-1 aspartyl proteases and rhizopuspepsin.     

Unique thermodynamic response of tipranavir to human immunodeficiency virus type 1 protease drug resistance mutations

Drug resistance is a major problem affecting the clinical efficacy of antiretroviral agents, including protease inhibitors, in the treatment of infection with human immunodeficiency virus type 1 (HIV-1)/AIDS. Consequently, the elucidation of the mechanisms by which HIV-1 protease inhibitors maintain antiviral activity in the presence of mutations is critical to the development of superior inhibitors. Tipranavir, a nonpeptidic HIV-1 protease inhibitor, has been recently approved for the treatment of HIV infection. Tipranavir inhibits wild-type protease with high potency (K(i) = 19 pM) and demonstrates durable efficacy in the treatment of patients infected with HIV-1 strains containing multiple common mutations associated with resistance. The high potency of tipranavir results from a very large favorable entropy change (-TDeltaS = -14.6 kcal/mol) combined with a favorable, albeit small, enthalpy change (DeltaH = -0.7 kcal/mol, 25 degrees C). Characterization of tipranavir binding to wild-type protease, active site mutants I50V and V82F/I84V, the multidrug-resistant mutant L10I/L33I/M46I/I54V/L63I/V82A/I84V/L90M, and the tipranavir in vitro-selected mutant I13V/V32L/L33F/K45I/V82L/I84V was performed by isothermal titration calorimetry and crystallography. Thermodynamically, the good response of tipranavir arises from a unique behavior: it compensates for entropic losses by actual enthalpic gains or by sustaining minimal enthalpic losses when facing the mutants. The net result is a small loss in binding affinity. Structurally, tipranavir establishes a very strong hydrogen bond network with invariant regions of the protease, which is maintained with the mutants, including catalytic Asp25 and the backbone of Asp29, Asp30, Gly48 and Ile50. Moreover, tipranavir forms hydrogen bonds directly to Ile50, while all other inhibitors do so by being mediated by a water molecule.     

Application of the Free Energy Calculations to Study Drug-enzyme and Drug-dna Complexes

Applications of two free energy calculation approaches are presented to study drug-biomolecule complexes. The first method, the free energy perturbation (FEP) method and molecular dynamics simulations has been applied to study the JG-365 inhibitor bound to the HIV-aspartic protease. The FEP method has been applied to predict the consequence of replacing each of the seven peptide bonds in the JG-365 by trans-ethylene or fluoroethylene units. The necessary initial conformations of the inhibitor for "in water" perturbations have been found using neural network clustering approach applied to the long molecular dynamics trajectory of the inhibitor in water solution. The second method is applied to study binding free energies of some DNA-drug complexes and is based on analysis of long molecular dynamics trajectories by continuum solvent approach (MM/PBSA).     

Visualization of human immunodeficiency virus protease inhibition using a novel Förster resonance energy transfer molecular probe

The in vivo high‐throughput screening (HTS) of human immunodeficiency virus (HIV) protease inhibitors is a significant challenge because of the lack of reliable assays that allow the visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Förster resonance energy transfer (FRET) to visualize HIV‐1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV‐1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV‐1 protease leads to separation of AcGFP1 from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV‐1 protease inhibition can be determined by measuring the FRET signal's change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV‐1 protease inhibition. By cotransfecting HIV‐1 protease and the probe expression plasmids into 293T cells, we showed that the inhibition of HIV‐1 protease by inhibitors can be visualized or quantitatively determined within living cells through ratiometric FRET microscopy imaging measurement. It is expected that this new probe will allow high‐content screening (HCS) of new anti‐HIV drugs. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Crystallographic analysis of transition state mimics bound to penicillopepsin: difluorostatine- and difluorostatone-containing peptides.

Difluorostatine- and difluorostatone-containing peptides have been evaluated as potent inhibitors of penicillopepsin, a member of the aspartic proteinase family of enzymes. Isovaleryl-Val-Val-StaF2NHCH3 [StaF2 = (S)-4-amino-2,2-difluoro-(R)-3-hydroxy-6-methylheptanoic acid] and isovaleryl-Val-Val-StoF2NHCH3 [StoF2 = (S)-4-amino-2,2-difluoro-3-oxo-6-methylheptanoic acid] have measured Ki's of 10 x 10(-9) and 1 x 10(-9) M, respectively, with this fungal proteinase. The StoF2-containing peptide binds 32-fold more tightly to the enzyme than the analogous peptide containing the non-fluorinated statine ethyl ester. Each compound was cocrystallized with penicillopepsin, intensity data were collected to 1.8-A resolution, and the atomic coordinates were refined to an R factor [formula: see text] of 0.131 for both complexes. The inhibitors bind in the active site of penicillopepsin in much the same fashion as do other statine-containing inhibitors of penicillopepsin analyzed earlier [James, M. N. G., Sielecki, A. Salituro, F., Rich, D. H., & Hofmann, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6137-6141; James, M.N.G., Sielecki, A., & Hofmann, T. (1985) in Aspartic Proteinases and their Inhibitors (Kosta, V., Ed.) pp 163-177, Walter deGruyter, Berlin]. The (R)-3-hydroxyl group in StaF2 binds between the active site carboxyl groups of Asp33 and Asp213, making hydrogen-bonding contacts to each one. The ketone functional group of the StoF2 inhibitor is bound as a hydrated species, with the gem-diol situated between the two aspartic acid carboxyl groups in a manner similar to that predicted for the tetrahedral intermediate expected during the catalytic hydrolysis of a peptide bond [James, M. N. G., & Sielecki, A. (1985) Biochemistry 24, 3701-3713]. One hydrogen-bonding interaction from the "outer" hydroxyl group is made to O delta 1 of Asp33, and the "inner" hydroxyl group forms two hydrogen-bonding contacts, one to each of the carboxyl groups of Asp33 (O delta 2) and Asp213 (O delta 2). The only structural difference between the StaF2 and StoF2 inhibitors that accounts for the factor of 10 in their Ki's is the additional (R)-3-OH group on the tetrahedral sp3 carbon atom of the hydrated StoF2 inhibitor. The intermolecular interactions involving the fluorine atoms of each inhibitor are normal van der Waals contacts to one of the carboxyl oxygen atoms of Asp213 (F2-O delta 2 Asp213, 2.9 A). The observed stereochemistry of the bound StoF2 group in the active site of penicillopepsin has stimulated our reappraisal of the catalytic pathway for the aspartic proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I

Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).     

Active-site mobility in human immunodeficiency virus, type 1, protease as demonstrated by crystal structure of A28S mutant.

The mutation Ala28 to serine in human immunodeficiency virus, type 1, (HIV-1) protease introduces putative hydrogen bonds to each active-site carboxyl group. These hydrogen bonds are ubiquitous in pepsin-like eukaryotic aspartic proteases. In order to understand the significance of this difference between HIV-1 protease and homologous, eukaryotic aspartic proteases, we solved the three-dimensional structure of A28S mutant HIV-1 protease in complex with a peptidic inhibitor U-89360E. The structure has been determined to 2.0 A resolution with an R factor of 0.194. Comparison of the mutant enzyme structure with that of the wild-type HIV-1 protease bound to the same inhibitor (Hong L, Treharne A, Hartsuck JA, Foundling S, Tang J, 1996, Biochemistry 35:10627-10633) revealed double occupancy for the Ser28 hydroxyl group, which forms a hydrogen bond either to one of the oxygen atoms of the active-site carboxyl or to the carbonyl oxygen of Asp30. We also observed marked changes in orientation of the Asp25 catalytic carboxyl groups, presumably caused by the new hydrogen bonds. These observations suggest that catalytic aspartyl groups of HIV-1 protease have significant conformational flexibility unseen in eukaryotic aspartic proteases. This difference may provide an explanation for some unique catalytic properties of HIV-1 protease.     

Loss of Viral Fitness Associated with Multiple Gag and Gag-Pol Processing Defects in Human Immunodeficiency Virus Type 1 Variants Selected for Resistance to Protease Inhibitors In Vivo

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.     

The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease

Alanine mutations of the proposed catalytically essential residues in histoaspartic protease (HAP) (H34A, S37A and D214A) were generated to investigate whether: (a) HAP is a serine protease with a catalytic triad of His34, Ser37 and Asp214 [Andreeva N, Bogdanovich P, Kashparov I, Popov M & Stengach M (2004) Proteins55, 705-710]; or (b) HAP is a novel protease with Asp214 acting as both the acid and the base during substrate catalysis with His34 providing critical stabilization [Bjelic S & Aqvist J (2004) Biochemistry43, 14521-14528]. Our results indicated that recombinant wild-type HAP, S37A and H34A were capable of autoactivation, whereas D214A was not. The inability of D214A to autoactivate highlighted the importance of Asp214 for catalysis. H34A and S37A mutants hydrolyzed synthetic substrate indicating that neither His34 nor Ser37 was essential for substrate catalysis. Both mutants did, however, have reduced catalytic efficiency (P < or = 0.05) compared with wild-type HAP, which was attributed to the stabilizing role of His34 and Ser37 during catalysis. The mature forms of wild-type HAP, H34A and S37A all exhibited high activity over a broad pH range of 5.0-8.5 with maximum activity occurring between pH 7.5 and 8.0. Inhibition studies indicated that wild-type HAP, H34A and S37A were strongly inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride, but only weakly inhibited by pepstatin A. The data, in concert with molecular modeling, suggest a novel mode of catalysis with a single aspartic acid residue performing both the acid and base roles.     

Studies on flexibility and binding affinity of Asp25 of HIV-1 protease mutants

We have investigated and highlighted the behavior of binding residue, Asp25 by computational analysis, which play an important role in understanding docking process with drug molecule, Ritonavir (Norvir^®) and the flexibility nature of the Human Immunodeficiency Virus-1 (HIV-1) protease enzyme. It is well known that Ritonavir is a potent and a selective HIV-1 protease inhibitor. Molecular dockings were performed in order to gain insights regarding the binding mode of this inhibitor. In our analysis, we observed Ritonavir had different rank orders of scores against different mutant of this enzyme. Asp25 of the enzyme was found to be the active site for all the mutants. The results clearly suggest that Ritonavir is not able to appropriately bind at the active site of each HIV-1 protease mutant due to RMSD difference of the amino acid (Asp) at the position 25 of all mutants. These findings support the concept that 3D space of active site is a qualitative assessment for binding affinity of inhibitor with an enzyme. The investigation on the flexibility nature of Asp25 by normal mode analysis, show that binding residue posses less flexibility due to its solvation potential. The overall analysis of our study brings clarity to the binding behavior with respect to the different mutants with Ritonavir on the basis RMSD and also on the flexible nature of HIV-1 protease enzyme with respect to Asp25 position.     

Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25′) has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor–residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor–residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.     

Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.     

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.     

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.     

Analysis of two Aspergillus nidulans genes encoding extracellular proteases

Characterization of prtADelta mutants, generated by gene disruption, showed that the prtA gene is responsible for the majority of extracellular protease activity secreted by Aspergillus nidulans at both neutral and acid pH. The prtA delta mutation was used to map the prtA gene to chromosome V. Though aspartic protease activity has never been reported in A. nidulans and the prtADelta mutants appear to lack detectable acid protease activity, a gene (prtB) encoding a putative aspartic protease was isolated from this species. Comparison of the deduced amino acid sequence of PrtB to the sequence of other aspergillopepsins suggests that the putative prtB gene product contains an eight-amino-acid deletion prior to the second active site Asp residue of the protease. RT-PCR experiments showed that the prtB gene is expressed, albeit at a low level.