Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 96
Filter
Add more filters










Publication year range
1.
Neuroscience ; 298: 293-301, 2015 Jul 09.
Article in English | MEDLINE | ID: mdl-25888933

ABSTRACT

We have previously demonstrated that multiple sclerosis (MS) patients have abnormal cerebrospinal fluid (CSF) levels of the key myelin-related molecules cobalamin (Cbl), epidermal growth factor (EGF), and normal cellular prions (PrP(C)s), thus confirming that some CSF abnormalities may be co-responsible for remyelination failure. We determined the levels of these three molecules in post-mortem spinal cord (SC) samples taken from MS patients and control patients. The control SC samples, almost all of which came from non-neurological patients, did not show any microscopic lesions of any type. All of the samples were supplied by the U.K. MS Tissue Bank. The Cbl, EGF, and PrP(C) levels were determined using enzyme-linked immunosorbent assays. The SC total homocysteine level was determined using a competitive immunoenzymatic assay. CSF samples, taken from a further group of MS patients, were used for the assay of holo-transcobalamin (holo-TC) levels. The Cbl, EGF, and PrP(C) levels were significantly decreased in MS SCs in comparison with controls and, paradoxically, the decreased Cbl levels were associated with decreased SC levels of homocysteine, a biochemical marker of Cbl deficiency. The trends of EGF and PrP(C) levels paralleled those previously found in CSF, whereas that of Cbl was the opposite. There was no significant difference in CSF holo-TC levels between the MS patients and the controls. Given that we have previously demonstrated that Cbl positively regulates central nervous system EGF levels, it is conceivable that the low EGF levels in the MS SC may be causally related to a local decrease in Cbl levels. Only PrP(C) levels were invariably decreased in both the SC and CSF regardless of the clinical course of the disease. These findings suggest that the simultaneous lack of Cbl, EGF, and PrP(C)s may greatly hamper the remyelination process in MS patients, because they are key molecules of the machinery for remyelination.


Subject(s)
Epidermal Growth Factor/metabolism , Multiple Sclerosis/pathology , Prions/metabolism , Spinal Cord/metabolism , Vitamin B 12/metabolism , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Multiple Sclerosis/cerebrospinal fluid , Severity of Illness Index , White Matter/pathology , Young Adult
2.
Bone ; 40(1): 122-31, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16962401

ABSTRACT

Cathepsin K is an osteoclast-derived cysteine protease that has been implicated as playing a major role in bone resorption. A substantial body of evidence indicates that cathepsin K is critical in osteoclast-mediated bone resorption and suggests that its pharmacological inhibition should result in inhibition of bone resorption in vivo. Here we report the pharmacological characterization of SB-462795 (relacatib) as a potent and orally bioavailable small molecule inhibitor of cathepsin K that inhibits bone resorption both in vitro in human tissue and in vivo in cynomolgus monkeys. SB-462795 is a potent inhibitor of human cathepsins K, L, and V (K(i, app)=41, 68, and 53 pM, respectively) that exhibits 39-300-fold selectivity over other cathepsins. SB-462795 inhibited endogenous cathepsin K in situ in human osteoclasts and human osteoclast-mediated bone resorption with IC50 values of approximately 45 nM and approximately 70 nM, respectively. The anti-resorptive potential of SB-462795 was evaluated in normal as well as medically ovariectomized (Ovx) female cynomolgus monkeys. Serum levels of the C- and N-terminal telopeptides of Type I collagen (CTx and NTx, respectively) and urinary levels of NTx were monitored as biomarkers of bone resorption. Administration of SB-462795 to medically ovariectomized or normal monkeys resulted in an acute reduction in both serum and urinary markers of bone resorption within 1.5 h after dosing, and this effect lasted up to 48 h depending on the dose administered. Our data indicate that SB-462795 potently inhibits human cathepsin K in osteoclasts, resulting in a rapid inhibition of bone resorption both in vitro and in vivo in the monkey. These studies also demonstrate the therapeutic potential of relacatib in the treatment of postmenopausal osteoporosis and serves to model the planned clinical trials in human subjects.


Subject(s)
Azepines/therapeutic use , Bone Resorption/drug therapy , Cathepsins/antagonists & inhibitors , Osteoclasts/drug effects , Sulfones/therapeutic use , Administration, Oral , Animals , Azepines/administration & dosage , Azepines/pharmacology , Biomarkers/blood , Biomarkers/urine , Cathepsin K , Cells, Cultured , Collagen Type I/blood , Collagen Type I/urine , Disease Models, Animal , Humans , Macaca fascicularis , Osteoclasts/enzymology , Peptides/blood , Peptides/urine , Sulfones/administration & dosage , Sulfones/pharmacology
3.
J Pept Res ; 63(3): 265-9, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15049838

ABSTRACT

The design, synthesis, enzymologic, and protein mass spectrometric characterization of benzodioxocin-3-one and N-acyl-3-amino-3-buten-2-one inhibitors of the cysteine protease cathepsin K are described. The benzodioxocin-3-one ring system is chemically unstable giving rise to a mixture of N-acyl-3-amino-3-buten-2-one and hemiketals. This mixture of N-acyl-3-amino-3-buten-2-one and hemiketals potently inhibits recombinant, human cathepsin K (IC50 = 36 nM) by a time-independent, irreversible mechanism. Formation of a covalent adduct between cathepsin K and inhibitor has been confirmed by mass spectrometry.


Subject(s)
Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Ketones/chemistry , Ketones/pharmacology , Cathepsin K , Cathepsins/chemistry , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/classification , Ketones/chemical synthesis , Molecular Structure
4.
J Neuroimmunol ; 127(1-2): 37-43, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12044973

ABSTRACT

We have previously demonstrated that the repeated intracerebroventricular (i.c.v.) microinjection of interleukin-6 (IL-6) prevented the myelinolytic lesions of cobalamin-deficient (Cbl-D) central neuropathy [or subacute combined degeneration (SCD)] in totally gastrectomized (TGX) rats. We therefore hypothesized that cobalamin (Cbl) may actually regulate IL-6 levels in rat cerebrospinal fluid (CSF). We measured IL-6 levels in the CSF of rats made Cbl-D by means of total gastrectomy (TG) or chronic feeding with a Cbl-D diet and killed at different times from the beginning of the experiment, and found that IL-6 levels significantly and progressively decreased over time. Chronic 2-month Cbl administration started 1 week after surgery prevented the decrease in IL-6 levels and, when it was started 2 months after surgery, it significantly increased IL-6 levels, but not to presurgical values. We also investigated whether IL-6 decrease might be ultimately due to the Cbl-deficiency-linked decrease in epidermal growth factor (EGF) synthesis. Repeated i.c.v. administrations of EGF to TGX rats did not modify CSF IL-6 levels. These results, together with those of a previous study showing the preventive effect of IL-6 treatment on SCD lesions, demonstrate that: (i) Cbl selectively regulates CSF IL-6 levels; and (ii) decreased IL-6 availability plays a role in the pathogenesis of the experimental SCD, in which no evidence of inflammatory and/or immunological reaction has been observed.


Subject(s)
Interleukin-6/cerebrospinal fluid , Vitamin B 12/pharmacology , Animals , Epidermal Growth Factor/cerebrospinal fluid , Epidermal Growth Factor/pharmacology , Gastrectomy , Injections, Intraventricular , Injections, Subcutaneous , Leptin/cerebrospinal fluid , Male , Rats , Rats, Sprague-Dawley , Vitamin B 12 Deficiency/cerebrospinal fluid , Vitamin B 12 Deficiency/immunology
5.
Bone ; 30(5): 746-53, 2002 May.
Article in English | MEDLINE | ID: mdl-11996914

ABSTRACT

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.


Subject(s)
Benzofurans/pharmacology , Bone Resorption/drug therapy , Bone Resorption/prevention & control , Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Osteoclasts/drug effects , Pyridines/pharmacology , Animals , Binding Sites/drug effects , Cathepsin K , Cathepsins/chemistry , Cathepsins/metabolism , Cysteine Proteinase Inhibitors/chemistry , Disease Models, Animal , Female , Humans , In Vitro Techniques , Male , Osteoclasts/cytology , Ovariectomy , Parathyroidectomy , Rats , Rats, Sprague-Dawley , Thyroidectomy
6.
J Bone Miner Res ; 16(10): 1739-46, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11585335

ABSTRACT

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.


Subject(s)
Bone Resorption , Cathepsins/antagonists & inhibitors , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Osteoclasts/drug effects , Animals , Biomarkers , Cathepsin K , Collagen , Collagen Type I , Female , Humans , Macaca fascicularis , Molecular Structure , Osteoclasts/physiology , Ovariectomy , Peptides , Primates , Rats
7.
Org Lett ; 3(17): 2725-8, 2001 Aug 23.
Article in English | MEDLINE | ID: mdl-11506619

ABSTRACT

[reaction: see text]. General stereocontrolled synthesis of all four (2,3)-stereoisomers of 2-substituted statines is described. The 2,3-syn and 2,3-anti isomers were synthesized via beta-ketoester reduction and aldol reactions, respectively. Peptides containing 2-substituted statines inhibit porcine pepsin with nanomolar IC50 values.


Subject(s)
Amino Acids/chemical synthesis , Aspartic Acid Endopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Amino Acids/chemistry , Animals , Protease Inhibitors/chemistry , Stereoisomerism , Swine
8.
J Med Chem ; 44(9): 1380-95, 2001 Apr 26.
Article in English | MEDLINE | ID: mdl-11311061

ABSTRACT

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.


Subject(s)
Azepines/chemical synthesis , Cathepsins/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Leucine/chemical synthesis , Administration, Oral , Animals , Azepines/chemistry , Azepines/pharmacokinetics , Azepines/pharmacology , Biological Availability , Cathepsin K , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Leucine/analogs & derivatives , Leucine/chemistry , Leucine/pharmacokinetics , Leucine/pharmacology , Mass Spectrometry , Models, Molecular , Molecular Structure , Osteoclasts/drug effects , Protein Binding , Rats , Stereoisomerism , Structure-Activity Relationship
9.
J Med Chem ; 44(5): 725-36, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11262083

ABSTRACT

Cathepsin K (EC 3.4.22.38), a cysteine protease of the papain superfamily, is predominantly expressed in osteoclasts and has been postulated as a target for the treatment of osteoporosis. Crystallographic and structure--activity studies on a series of acyclic ketone-based inhibitors of cathepsin K have led to the design and identification of two series of cyclic ketone inhibitors. The mode of binding for four of these cyclic and acyclic inhibitors to cathepsin K is discussed and compared. All of the structures are consistent with addition of the active site thiol to the ketone of the inhibitors with the formation of a hemithioketal. Cocrystallization of the C-3 diastereomeric 3-amidotetrahydrofuran-4-one analogue 16 with cathepsin K showed the inhibitor to occupy the unprimed side of the active site with the 3S diastereomer preferred. This C-3 stereochemical preference is in contrast to the X-ray cocrystal structures of the 3-amidopyrrolidin-4-one inhibitors 29 and 33 which show these inhibitors to prefer binding of the 3R diastereomer. The 3-amidopyrrolidin-4-one inhibitors were bound in the active site of the enzyme in two alternate directions. Epimerization issues associated with the labile alpha-amino ketone diastereomeric center contained within these inhibitor classes has proven to limit their utility despite promising pharmacokinetics displayed in both series of compounds.


Subject(s)
Cathepsins/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Ketones/chemical synthesis , Animals , Binding Sites , Cathepsin K , Chromatography, Liquid , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Furans/chemical synthesis , Furans/chemistry , Furans/pharmacokinetics , Humans , Ketones/chemistry , Ketones/pharmacokinetics , Mass Spectrometry , Models, Molecular , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Piperidines/pharmacokinetics , Pyrans/chemical synthesis , Pyrans/chemistry , Pyrans/pharmacokinetics , Pyrrolidinones/chemical synthesis , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacokinetics , Rats , Stereoisomerism , Structure-Activity Relationship
10.
J Bone Miner Res ; 16(3): 478-86, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11277265

ABSTRACT

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.


Subject(s)
Bone Resorption/metabolism , Cathepsins/metabolism , Osteoclasts/metabolism , Biochemistry/methods , Bone and Bones/embryology , Bone and Bones/enzymology , Cathepsin K , Cathepsins/analysis , Cathepsins/antagonists & inhibitors , Cell Adhesion , Cysteine Proteinase Inhibitors/pharmacology , Giant Cell Tumor of Bone/metabolism , Humans , Hydrogen-Ion Concentration , Kidney/embryology , Kidney/enzymology , Leucine/analogs & derivatives , Leucine/pharmacology , Linear Models , Oligopeptides/pharmacology , Pepstatins/pharmacology , Phenylmethylsulfonyl Fluoride/pharmacology , Protease Inhibitors/pharmacology , Protein Processing, Post-Translational , Substrate Specificity , Tumor Cells, Cultured
11.
J Biol Chem ; 276(15): 11507-11, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11148212

ABSTRACT

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K(i) = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situ cytochemical assay (IC(50) > 1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50) > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K(i) = 0.052 nm) and K (K(i) = 1.57 nm) was also active in both assays (IC(50) = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.


Subject(s)
Bone Resorption , Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Endopeptidases , Osteoclasts/drug effects , Cathepsin L , Cysteine Endopeptidases , Humans , Osteoclasts/cytology , Tumor Cells, Cultured
12.
Curr Opin Drug Discov Devel ; 3(4): 362-9, 2000 Jul.
Article in English | MEDLINE | ID: mdl-19649868

ABSTRACT

Cysteine proteases are attracting increased attention for therapeutic intervention by inhibitors because of an increased recognition of specific processing functions in both mammals and microbes. New advances in inhibitor design have been made for both reversible and irreversible classes. Reversible inhibitors may incorporate a covalent bond formation that can contribute an element of selectivity relative to serine protease inhibition. Ketones of low intrinsic reactivity are especially attractive agents, with potential for chronic therapeutic use. Effectively irreversible inhibition can be achieved with slow or single turnover substrates. This approach eliminates any reactivity toward non-enzyme nucleophiles. Only mechanistically related enzyme targets will be irreversibly blocked in an in vivo setting, thereby resulting in a safer type of inhibitor.

14.
Bioorg Med Chem ; 7(4): 581-8, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10353637

ABSTRACT

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.


Subject(s)
Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemical synthesis , Endopeptidases , Ketones/chemistry , Cathepsin B/antagonists & inhibitors , Cathepsin K , Cathepsin L , Cysteine Endopeptidases , Kinetics , Models, Chemical , Models, Molecular
15.
Bioorg Med Chem ; 7(4): 599-605, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10353639

ABSTRACT

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.


Subject(s)
Cathepsins/antagonists & inhibitors , Drug Design , Cathepsin K , Kinetics , Models, Molecular
16.
Biochemistry ; 38(48): 15893-902, 1999 Nov 30.
Article in English | MEDLINE | ID: mdl-10625455

ABSTRACT

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.


Subject(s)
Cathepsins/antagonists & inhibitors , Hydrazines/pharmacology , Protease Inhibitors/pharmacology , Binding Sites , Cathepsin K , Chromatography, High Pressure Liquid , Enzyme Reactivators , Hydrazines/chemistry , Kinetics , Leucine/analogs & derivatives , Leucine/chemistry , Leucine/pharmacology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Papain/antagonists & inhibitors , Spectrophotometry
17.
J Comb Chem ; 1(3): 207-15, 1999.
Article in English | MEDLINE | ID: mdl-10746010

ABSTRACT

To more rapidly prepare members of the 1,3-bis(acylamino)-2-butanone class of cysteine protease inhibitors, a solid-phase synthesis was developed. 1-Azido-3-amino-2,2-dimethoxybutane (4), which has the two amino groups differentiated and the ketone protected as a a ketal, served as a surrogate for the 1,3-diamino-2-butanone core. Amine (4) was coupled to the BAL-resin-linked carboxylic acids derived from alpha-amino acid esters. Evaluation of a small combinatorial array by measuring inhibition constants (Ki,appS) against cathepsins K, L, and B provided some structure-activity relationship trends with respect to selectivity and potency. Novel, potent inhibitors of cathepsins K and L were identified.


Subject(s)
Butanones/chemical synthesis , Cathepsins/antagonists & inhibitors , Combinatorial Chemistry Techniques/methods , Cysteine Proteinase Inhibitors/chemical synthesis , Endopeptidases , Butanones/chemistry , Butanones/pharmacology , Cathepsin K , Cathepsin L , Cysteine Endopeptidases , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , Kinetics , Models, Molecular , Structure-Activity Relationship
18.
J Med Chem ; 41(23): 4567-76, 1998 Nov 05.
Article in English | MEDLINE | ID: mdl-9804696

ABSTRACT

Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexes and the rational design of novel cathepsin K inhibitors. Unlike previously known crystal structures of papain-inhibitor complexes, our papain structures show ligand binding extending deep within the S'-subsites. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leucinal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, were refined to 2.2- and 2.5-A resolution with R-factors of 0.190 and 0. 217, respectively. The S'-subsite interactions with the inhibitors are dominated by an aromatic-aromatic stacking and an oxygen-aromatic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yielded a novel, symmetric inhibitor selective for cathepsin K. Simultaneous exploitation of both S- and S'-sites provides a general strategy for the design of cysteine protease inhibitors having high specificity to their target enzymes.


Subject(s)
Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemistry , Dipeptides/chemistry , Leupeptins/chemistry , Models, Molecular , Papain/chemistry , Binding Sites , Cathepsin K , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/metabolism , Dipeptides/metabolism , Drug Design , Leupeptins/metabolism , Papain/metabolism , Protein Structure, Tertiary
19.
J Med Chem ; 41(21): 3923-7, 1998 Oct 08.
Article in English | MEDLINE | ID: mdl-9767629

ABSTRACT

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.


Subject(s)
Benzoates/chemical synthesis , Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemical synthesis , Drug Design , Peptides/chemistry , Benzoates/chemistry , Benzoates/metabolism , Binding Sites , Cathepsin K , Cathepsins/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Models, Molecular , Molecular Mimicry , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...