SEARCH · Search PubMed

Results for “PROTEASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources

PubMed2000

The cumulative occurrence of resistance mutations in the HIV-1 protease gene is associated with failure of salvage therapy with ritonavir and saquinavir in protease inhibitor-experienced patients.

Salvage therapy with ritonavir (RTV) and saquinavir (SQV) failed to achieve virological and immunological improvement in 24 HIV-infected patients who discontinued triple therapy with RTV or indinavir (IDV) because of failure or intolerance to treatment. Changes in the HIV-1 protease gene sequence were analyzed prospectively in 14 patients. No primary protease mutation was found prior to the use of protease inhibitors. After 7 months of treatment with IDV or RTV, primary resistance mutations at codons pol 46 and/or pol 82 were observed in 11 of 13 patients. After 16 weeks on RTV-SQV, novel primary mutations related to SQV emerged in 7 of 13 patients, together with an increase in the number of secondary resistance mutations. Our observations indicate that the cumulative occurrence of resistance mutations in the protease gene was associated with failure of antiretroviral therapy. The presence of mutations to a first protease inhibitor may represent a risk factor for the failure of a subsequent treatment with a second line protease inhibitor.

Adult↗

PubMed2000

Fusion of uninfected T-cells occurs with immature HIV-1 protease-mutant, but not morphologically similar protease inhibitor derived particles.

Protease inhibitors are widely used in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals and show a drastic effect on the reduction of virus load. We previously reported that doughnut-shaped, protease-defective gp120-containing HIV-1 particles from an L-2 cell clone, carrying a provirus with mutations at the pol (protease), env (gp41) and nef genes, rapidly and more effectively induces virus particle-mediated syncytia formation of uninfected T-cells, than a parental wild-type laboratory strain of HIV-1 (LAI). In this study, we examined the possibility of whether enhanced syncytia formation is mediated by morphologically similar doughnut-shaped particles obtained after treatment of LAI-infected cells with the protease inhibitors L-689, 502, DMP-323, RO-31-8959, and KNI-272. Utilizing such protease inhibitor-induced particles and a clone of MOLT-4 cells, we could not detect any enhancement of syncytia formation, over that seen with wild-type LAI particles. This result should alleviate concerns of patients on highly active antiretroviral therapy (HAART), that protease inhibitors might accelerate progression of the disease through enhanced production of defective, 'immature'-appearing particles.

Anti-HIV Agents↗

PubMed2003

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis.

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.

Animals↗

PubMed2000

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease.

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Anti-HIV Agents↗

PubMed2001

Genetic variability of HIV-1 protease from Nigeria and correlation with protease inhibitors drug resistance.

In Nigeria, the most populous country in Africa, the characterization of HIV-1 strains has been limited. In this study we evaluated the genetic diversity of the protease coding region, one of the anti-retroviral therapy target, and investigated the presence of mutations related to resistance to HIV protease inhibitors. We analyzed samples collected during 1996 and all patients were anti-retroviral drug naïves. Ten samples were evaluated by sequencing of the protease gene. The majority, 80%, were classified as subtype A and the two others were unclassified-divergent strains, something in between A and G subtypes. The gag region from these outliners were sequenced and the phylogenetic analysis classified them as subtype G. The protease amino acid consensus sequence of the Nigerian subtype A are in complete agreement with the consensus A differing from the USA subtype B consensus in 10 positions (L10V, I13V, K14R, I15V, K20I, M36I, R41K, P63L, H69K and L89M). The secondary substitutions associated with protease inhibitor resistance were observed in all Nigerian sequences at the positions L10V, M36I and L89M. The majority of sequence variation was concentrated in the interval between aminoacids 70-90 where the protease substrate binding region is located.

Amino Acid Sequence↗

PubMed1981

Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases.

Protease-nexin (PN), a component released by normal human fibroblasts into culture medium, forms covalent linkages with thrombin (Th) and the urinary plasminogen activator urokinase, apparently with their catalytic site serines. The present studies explored the function of PN by examining the interaction of protease-PN complexes with human fibroblasts and the consequences of this interaction. Th-PN and urokinase-PN complexes bind to cells via the PN portion of the complexes. The binding is selectively inhibited by heparin. Because PN has a heparin-binding site, this indicates that protease-PN complexes might bind to a cellular heparin-like site. After binding, the complexes are internalized. By inhibiting endocytosis with phenylarsine oxide, which does not affect cellular binding of Th-PN complexes, we showed that complexes must be internalized before they are degraded. Kinetic analysis of internalization and degradation of Th-PN showed that complexes are internalized more rapidly than they dissociate from the cell surface; by 120 min of incubation at 37 degrees C most cell-bound Th-PN complexes are degraded to amino acids. The results are summarized in a model showing how PN mediates the cellular binding, internalization, and degradation of serine proteases through formation of protease-PN complexes. This series of events may be involved in the regulation of serine protease activity at the cell surface and in the extracellular environment.

Amyloid beta-Protein Precursor↗

PubMed1993

Crystal structure of human immunodeficiency virus (HIV) type 2 protease in complex with a reduced amide inhibitor and comparison with HIV-1 protease structures.

The crystal structure of HIV-2 protease in complex with a reduced amide inhibitor [BI-LA-398; Phe-Val-Phe-psi (CH2NH)-Leu-Glu-Ile-amide] has been determined at 2.2-A resolution and refined to a crystallographic R factor of 17.6%. The rms deviation from ideality in bond lengths is 0.018 A and in bond angles is 2.8 degrees. The largest structural differences between HIV-1 and HIV-2 proteases are located at residues 15-20, 34-40, and 65-73, away from the flap region and the substrate binding sites. The rms distance between equivalent C alpha atoms of HIV-1 and HIV-2 protease structures excluding these residues is 0.5 A. The shapes of the S1 and S2 pockets in the presence of this inhibitor are essentially unperturbed by the amino acid differences between HIV-1 and HIV-2 proteases. The interaction of the inhibitor with HIV-2 protease is similar to that observed in HIV-1 protease structures. The unprotected N terminus of the inhibitor interacts with the side chains of Asp-29 and Asp-30. The glutamate side chain of the inhibitor forms hydrogen bonds with the main-chain amido groups of residues 129 and 130.

Amino Acid Sequence↗

PubMed1995

Three-dimensional structure of a mutant HIV-1 protease displaying cross-resistance to all protease inhibitors in clinical trials.

Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-A resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 A with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C. (1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The C alpha tracings of the two native structures are identical with a root-mean-square deviation of 0.5 A, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (approximately 2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 A) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding.

Acquired Immunodeficiency Syndrome↗

PubMed1998

HIV protease genotype and viral sensitivity to HIV protease inhibitors following saquinavir therapy.

OBJECTIVE: To examine the relationship between HIV protease genotype and altered protease inhibitor sensitivity of isolates from patients after therapy with saquinavir (SQV) in its hard gelatin formulation. DESIGN: Forty-one post-therapy isolates and corresponding baseline samples were obtained from 37 patients in four different clinical trials after therapy with SQV for 16-147 weeks. Post-therapy isolates were selected on the basis of preliminary sequence or drug sensitivity data. RESULTS: Fifteen out of 17 isolates without detectable Val-48 or Met-90 mutations retained sensitivity to SQV. (The remaining isolates showed only a marginal increase in median inhibitory concentration.) In addition, three out of 15 isolates with Met-90 retained sensitivity to all other protease inhibitors tested (indinavir, ritonavir, amprenavir, nelfinavir). Of the isolates showing reduced sensitivity to SQV, six out of 22 retained sensitivity to all other protease inhibitors, whereas only four out of 22 showed broad cross-resistance to all protease inhibitors tested. The reduction in sensitivity correlated closely with the presence of Val-48 or Met-90. Subsequent accessory substitutions were also linked to reduced sensitivity. However, significant linkage was observed only between mutations at residues 48 and 82 and between those at residues 82 and 74. CONCLUSIONS: Recruitment of Val-48/Met-90 mutations was not found to be synonymous with cross-resistance. Indeed, the majority of isolates with these mutations retained sensitivity to at least one protease inhibitor (Val-48, 86%; Met-90, 77%). The recruitment of accessory mutations may occur only after the selection of key resistance mutations. Furthermore, Met-90 was found to be a poor marker of cross-resistance in SQV-treated patients.

Amino Acid Substitution↗

PubMed2001

Variant human immunodeficiency virus type 1 proteases and response to combination therapy including a protease inhibitor.

The objective of this observational study was to assess the genetic variability in the human immunodeficiency virus (HIV) protease gene from HIV type 1 (HIV-1)-positive (clade B), protease inhibitor-naïve patients and to evaluate its association with the subsequent effectiveness of a protease inhibitor-containing triple-drug regimen. The protease gene was sequenced from plasma-derived virus from 116 protease inhibitor-naïve patients. The virological response to a triple-drug regimen containing indinavir, ritonavir, or saquinavir was evaluated every 3 months for as long as 2 years (n = 40). A total of 36 different amino acid substitutions compared to the reference sequence (HIV-1 HXB2) were detected. No substitutions at the active site similar to the primary resistance mutations were found. The most frequent substitutions (prevalence, >10%) at baseline were located at codons 15, 13, 12, 62, 36, 64, 41, 35, 3, 93, 77, 63, and 37 (in ascending order of frequency). The mean number of polymorphisms was 4.2. A relatively poorer response to therapy was associated with a high number of baseline polymorphisms and, to a lesser extent, with the presence of I93L at baseline in comparison with the wild-type virus. A71V/T was slightly associated with a poorer response to first-line ritonavir-based therapy. In summary, within clade B viruses, protease gene natural polymorphisms are common. There is evidence suggesting that treatment response is associated with this genetic background, but most of the specific contributors could not be firmly identified. I93L, occurring in about 30% of untreated patients, may play a role, as A71V/T possibly does in ritonavir-treated patients.

Adult↗

PubMed2006

Genotypic changes in human immunodeficiency virus type 1 protease associated with reduced susceptibility and virologic response to the protease inhibitor tipranavir.

Tipranavir is a novel, nonpeptidic protease inhibitor of human immunodeficiency virus type 1 (HIV-1) with activity against clinical HIV-1 isolates from treatment-experienced patients. HIV-1 genotypic and phenotypic data from phase II and III clinical trials of tipranavir with protease inhibitor-experienced patients were analyzed to determine the association of protease mutations with reduced susceptibility and virologic response to tipranavir. Specific protease mutations were identified based on stepwise multiple-regression analyses of phase II study data sets. Validation included analyses of phase III study data sets to determine if the same mutations would be selected and to assess how these mutations contribute to multiple-regression models of tipranavir-related phenotype and of virologic response. A tipranavir mutation score was developed from these analyses, which consisted of a unique string of 16 protease positions and 21 mutations (10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, and 84V). HIV-1 isolates displaying an increasing number of these tipranavir resistance-associated mutations had a reduced phenotypic susceptibility and virologic response to tipranavir. Regression models for predicting virologic response in phase III trials revealed that each point in the tipranavir score was associated with a 0.16-log10 copies/ml-lower virologic response to tipranavir at week 24 of treatment. A lower number of points in the tipranavir score and a greater number of active drugs in the background regimen were predictive of virologic success. These analyses demonstrate that the tipranavir mutation score is a potentially valuable tool for predicting the virologic response to tipranavir in protease inhibitor-experienced patients.

Drug Resistance, Viral↗

PubMed1983

[Intracellular proteases of Thermoactinomyces vulgaris. 2. Biochemical characterization of proteases in cell extracts].

The intracellular proteases (IP) of the cellular extract of Thermoactinomyces vulgaris are characterized as to the biochemical properties compared with the corresponding extracellular proteases (EP). According to that, the storage stability and the temperature and pH behaviour (optimum, stability) of both of the proteases are identical. Nevertheless, differences were detected between IP and EP after the action of several effectors and different substrates. As could be seen after a column chromatographic separation of the IP of the cellular extract, it is composed of at least 3 proteases, two of them are serine proteases which can be inhibited moreover unspecifically by p-chloromercuri benzoate. The purified proteases (IP) are very instable and therefore not yet characterized in detail.

Chromatography, High Pressure Liquid↗

PubMed1997

Protease-protease inhibitor interactions in Sertoli cell-germ cell crosstalk.

Peritubular cells, Sertoli cells, and germ cells of the seminiferous tubule synthesize and secrete several proteases and protease inhibitors. Experimental evidence suggests that the complex network of proteolytic enzyme activity and their regulation by protease inhibitors play an important role in male reproduction. Interaction between protease and protease inhibitors seems to play an important role in remodeling and restructuring of the seminiferous tubule during spermatogenesis. Controlled proteolytic activity is also involved in the migration of germ cells from the basal compartment to the lumen of the seminiferous epithelium, and in the release of spermatids during spermiation. The recently reported occurrence of Sertoli cell membrane-associated proteases indicate the possible involvement of regulatory peptide systems within the testis. This view is supported by the detection of all components of one of these paracrine systems, the kallikrein-kinin system, in cells of the seminiferous tubule.

Animals↗

PubMed1986

Degradation of myelin basic protein in myelin by protease in cerebrospinal fluid and effects of protease inhibitors.

Neutral protease is shown to be present in cell-free human cerebrospinal fluid. Incubation of heated human myelin with CSF at 25 degrees C resulted in a marked reduction of myelin basic protein (MBP) with time. Degradation products appeared at apparent mol wt 14 KDa and 12 KDa on polyacrylamide gel electrophoresis. Optimal pH of the protease was 7.0. This protease was activated by calcium ion. Degradation of MBP was inhibited by FOY305 (camostat mesilate), Trasylol, and Leupeptin, but not a specific calcium-activated neutral protease inhibitor, E-64-a. FOY305, which is a synthesized specific serine protease inhibitor, was the strongest inhibitor of all. The role of this protease in CSF has not been elucidated. In may be related to the physiological turnover of MBP, and may affect myelin maintenance in pathological conditions such as demyelination.

Humans↗

PubMed1998

Inhibition of tumour cell invasion by protease inhibitors: correlation with the protease profile.

The invasive potential of eight established human tumour cell lines of different origin has been studied in the Matrigel assay. Between 25% and 70% of the cells migrated through the Matrigel layer within 24 h, indicating that invasiveness varies with the cell type. Semiquantitative measurements of the proteases MMP-2 and MMP-9, and cathepsins B and L were performed in these cell lines and the cell culture media. High invasive potential was found in those cell lines expressing high levels of cathepsins B and L or matrix metal proteases (MMP), either alone or in combination. Overexpression of one of these enzymes is enough to explain a high invasive potential of a cell line. Selective protease inhibitors at 10 nM concentration in the culture medium were used to inhibit the migration of tumour cells in the Matrigel assay. The MMP inhibitor Batimastat reduced the invasive potential of all cell lines studied independently of the MMP expression. The effect of cysteine protease inhibitors was strongly correlated with the protease profile of the tumour cell line. Our findings support the hypothesis of a very complex activation cascade of matrix-degrading proteolytic enzymes and they underline the need to analyse the protease profile of any tumour before beginning an antiproteolytic tumour treatment.

Cathepsin B↗

PubMed1991

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography.

Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms. Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease. A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.

Amino Acid Sequence↗

PubMed2004

Expression of trypsin-like proteases and protease nexin-1 in mdx mouse muscles.

In order to examine the possible participation of trypsin-like proteases in the onset and progress of muscular dystrophy, we investigated the expression of the trypsin-like protease in muscular tissues in mdx mice. We found that the mRNAs of several trypsin-like proteases, including hepsin and t-PA, were expressed in the muscular tissues of mdx mice, but at levels not significantly different from normal mice. Since the enzymatic properties of dystrypsin, a muscle trypsin-like protease activated before onset of the disease, are similar to those of thrombin, we investigated the expression pattern of thrombin in mdx mouse muscles. The results showed that prothrombin mRNA is up-regulated in mdx mice at 20-30 days of age but not before the age of 15 days (preclinical). Since protease nexin-1 (PN-1) is known to be a physiological inhibitor of thrombin, we also examined the expression pattern of PN-1. We found that PN-1 transcription and translation is down-regulated in the muscular tissues of mdx mice, before the onset of clinical symptoms. These results suggest that thrombin may be involved in the progression of muscular dystrophy or the regeneration of muscle fibers after the onset of the disease and that the reduced level of PN-1 may enhance the activities stimulate the activities of muscle proteases, including dystrypsin, at a preclinical stage in mdx mice.

Aging↗

PubMed2005

Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide.

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.

Amino Acid Sequence↗