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Biomedical subjects

M A Levy

Publications and source records attributed to M A Levy.

At least 19 recordsLinked to original sources
PubMed2000

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

Cell Nucleus↗
PubMed2000

Evaluation of the use of accelerated infusions for the determination of pharmacokinetic linearity.

Accelerated infusions are potentially useful in the investigation of pharmacokinetic linearity. However, little information exists to validate this technique or to demonstrate its limitations. This investigation was performed to determine whether accelerated infusion regimens reliably estimate the range of pharmacokinetic linearity for molecules of varying pharmacokinetic properties, to evaluate the ability of accelerated infusions to identify pharmacokinetic nonlinearity, and to validate the accelerated infusion technique using compounds with known pharmacokinetic parameters. Simulations incorporating accelerated infusion as the input function resulted in the anticipated concentration-time profiles that contained an initial lag phase before reaching a linear slope. This lag phase increased with increasing distributional volume and in some instances was sufficiently great to obscure or prevent the linear portion of the profile. These simulations also revealed that clearance estimated from the apparently linear portion of the concentration-time profile can be erroneous under some conditions, as for large-volume compounds. Simulations of structured nonlinearity produced the predicted profiles for compounds with low to moderate volumes of distribution while demonstrating that modeling of data derived from compounds with large volumes of distribution may be inaccurate. Finally, experiments using accelerated infusions with various test compounds further demonstrated the usefulness of this technique while presenting limits imposed on the interpretation of the data. The results of this investigation indicate that the accelerated infusion may be used to determine pharmacokinetic linearity for compounds within certain pharmacokinetic boundaries, but that appropriate caution should be exercised in the extent of interpretation that should be extracted from such studies.

Algorithms↗
PubMed1999

Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases.

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.

Binding Sites↗
PubMed1999

A substrate combinatorial array for caspases.

An efficient strategy for the synthesis of a tetrapeptidyl substrate combinatorial array directed toward the caspases is described. Testing of this array with caspases 1 and 4 gave substrate hydrolytic profiles characteristic of each caspase, and permitted the identification of efficiently processed substrates. A comparison of this approach to that using a positional scanning library is presented.

Caspase 1↗
PubMed1999

Structure-based design of non-peptide, carbohydrazide-based cathepsin K inhibitors.

Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.

Cathepsin K↗
PubMed1999

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.

Cathepsin B↗
PubMed1999

Synergistic effects of concurrent challenge with bovine respiratory syncytial virus and 3-methylindole in calves.

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.

Animals↗
PubMed1998

Structure-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.

Benzoates↗
PubMed1998

Selective elevation of glutathione levels in target tissues with L-2-oxothiazolidine-4-carboxylate (OTC) protects against hyperoxia-induced lung damage in protein-energy malnourished rats: implications for a new treatment strategy.

It has become recognized that enhancing the antioxidant defense system during the early phase of rehabilitation is important to the survival of wasting protein-energy malnourished (PEM) patients. In this study, we compared the efficacy of dietary protein replenishment and supplementation with L-2-oxothiazolidine-4-carboxylate (OTC, 3.5 mg/d), a cysteine precursor, to protect against hyperoxia-induced lung damage in PEM rats. The PEM rats were produced by feeding weanling rats a protein-deficient diet (0.5% protein) for 14 d. PEM rats were then divided in three dietary treatment groups, 0.5% protein (-Pr), 0.5% protein plus the OTC supplement (+OTC), or 15% protein (+Pr) during 4 d of either hyperoxia (85% O2) or air exposure. Increased lung-to-body weight ratios, indicative of oxidative tissue damage, were observed following exposure to hyperoxia in -Pr and +Pr rats, but not in +OTC rats, even though the OTC supplement and the 15% protein diet contained a comparable amount of cysteine. Tissue reduced glutathione (GSH) status, GSH-dependent enzyme activity and antioxidant defense enzyme activities were monitored in the lung, liver and blood during 4 d of hyperoxia exposure. OTC supplementation enhanced GSH levels significantly in the lung of PEM rats, whereas protein repletion significantly elevated blood GSH concentrations. The protective effect of OTC was not a function of changes in activity of GSH-dependent enzymes or oxygen defense enzymes in the lung. These results indicate that a short-term strategy that selectively elevates GSH levels in the lung is more effective than protein repletion in protecting against hyperoxia-induced oxidative lung damage in PEM rats.

Animals↗
PubMed1997

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Binding Sites↗
PubMed1997

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Aldehydes↗
PubMed1996

Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification.

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F.H., Dodds, R., James I., Connor J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. (1996) J. Biol., Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not for by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (kobs/[I] = 273,000 m(-1).s(-1)). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Amino Acid Sequence↗
PubMed1996

3-Carboxy-20-keto steroids are dual uncompetitive inhibitors of human steroid 5 alpha-reductase types 1 and 2.

Steroidal 3-carboxy-20-ketones have been prepared within two structural series, the androsta-3,5-dienes and the estra-1,3,5-trienes, as potential inhibitors of types 1 and 2 steroid 5 alpha-reductase, the enzyme activity responsible for the final step in biosynthesis of dihydrotestosterone. These compounds are shown to be potent uncompetitive inhibitors of both human recombinant enzyme activities, defining a novel class of dual steroid 5 alpha-reductase inhibitors.

5-alpha Reductase Inhibitors↗
PubMed1995

Cloning, expression and functional characterization of type 1 and type 2 steroid 5 alpha-reductases from Cynomolgus monkey: comparisons with human and rat isoenzymes.

The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two known human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the suitability of this species at the level of the molecular targets, a Cynomolgus monkey prostate cDNA library was prepared and screened using human SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA sequences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with their human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corresponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Subsequently, both monkey cDNAs were individually expressed in a mammalian cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and rat enzymes, the monkey type 1 and type 2 SRs were most active at neutral and low pH, respectively. The results of inhibition studies with over 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocity and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest that the monkey SR isoenzymes are structurally and functionally comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of SR inhibitors.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗