Potent and selective inhibition of zinc aminopeptidase A (EC 3.4.11.7, APA) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the P1 position

Through the development of a new chemical strategy, aminophosphinic peptides containing a pseudoglutamyl residue (Glu Psi(PO2-CH2)Leu-Xaa) in the N-terminal position were synthesized and evaluated as inhibitors of aminopeptidase A (APA). The most potent inhibitor developed in this study, Glu Psi(PO2-CH2)Leu-Ala, displayed a Ki value of 0.8 nM for APA, but was much less effective in blocking aminopeptidase N (APN) (Ki = 31 microM). The critical role of the glutamyl residue in this phosphinic peptide, both in potency and selectivity, is exemplified by the P1 position analogue, Ala Psi(PO2-CH2)Leu-Ala, which exhibited a Ki value of 0.9 microM toward APA but behaved as a rather potent inhibitor of APN (Ki = 25 nM). Glu Psi(PO2-CH2)Leu-Xaa peptides are poor inhibitors of angiotensin converting enzyme (Ki values higher than 1 microM). Depending on the nature of the Xaa residue, the potency of these phosphinic peptides toward neutral endopeptidase 24-11 varied from 50 nM to 3 microM. In view of the in vivo role of APA in the formation of brain angiotensin III, one of the main effector peptides of the renin angiotensin system in the central nervous system, highly potent and selective inhibitors of APA may find important therapeutic applications soon.     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

Inhibition of rabbit lung angiotensin-converting enzyme by N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline

Two novel peptide analogs, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and the corresponding L-lysyl-L-proline derivative, have been demonstrated to be potent competitive inhibitors of purified rabbit lung angiotensin-converting enzyme: Ki = 2 and 1 X 10(-10) M, respectively, at pH 7.5, 25 degrees C, and 0.3 M chloride ion. Second-order rate constants for addition of these inhibitors to enzyme under the same conditions are in the range 1-2 X 10(6) M-1 s-1; first-order rate constants for dissociation of the EI complexes are in the range 1-4 X 10(-4) s-1. The association rate constants are similar to those measured for D-3-mercapto-2-methylpropanoyl-L-proline, captopril, but the dissociation rate constants are severalfold slower and account for the higher affinity of these inhibitors for the enzyme. The dissociation constant for the EI complex containing N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline is pH-dependent, and reaches a minimum at approximately pH 6: Ki = 4 +/- 1 X 10(-11) M. The pH dependence is consistent either with a model for which the protonation state of the secondary nitrogen atom in the inhibitor determines binding affinity, or one for which ionizations on the enzyme alone influence affinity for these inhibitors. The affinity of this inhibitor for the zinc-free apoenzyme is 2 X 10(4) times less than for the zinc-free apoenzyme is 2 X 10(4) times less than that for the holoenzyme. If considered as a "collected product" inhibitor, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline appears to derive an additional factor of 375 M in its affinity for the enzyme compared to that of the two products of its hypothetical hydrolysis, a consequence of favorable entropy effects.     

Investigating the stereochemistry of binding to HIV-1 protease with inhibitors containing isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid

A series of inhibitors containing all possible isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid was synthesized and tested for inhibition of HIV-1 protease. Incorporation of the (3S,4S) isomer of the t-butyloxycarbonyl protected amino acid into the sequence Glu-Phe resulted in a potent inhibitor of HIV-1 protease (Ki = 63 nM). This inhibitor is at least 47-times more potent than the inhibitors containing other isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid, indicating that the (3S,4S) isomer is the preferred isomer for binding to HIV-1 protease.     

Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.     

Inhibition kinetics of acetylcholinesterase with fluoromethyl ketones

A series of trifluoromethyl ketones that reversibly inhibit acetylcholinesterase and pseudocholinesterase were synthesized. By analogy to chymotrypsin and on the basis of data reported here, we propose that the active-site serine adds to the ketone to form an ionized hemiketal. The compound (5,5,5-trifluoro-4-oxopentyl)trimethylammonium bicarbonate (1) inhibits acetylcholinesterase with Ki = 0.06 X 10(-9)M and pseudocholinesterase with Ki = 70 X 10(-9)M. Replacement of the nitrogen of 1 by carbon (compound 2) increases Ki for 1 200-fold for acetylcholinesterase but does not significantly alter Ki for pseudocholinesterase. The Ki for the methyl ketone corresponding to 2 is 2 X 10(-4)M for both enzymes, as compared with 12 X 10(-9)M for the trifluoromethyl ketone (acetylcholinesterase). For both enzymes, a linear decrease in log Ki with decreasing pK of the inhibitor hydrate was observed with ketones containing from 0 to 3 fluorines. We attribute this effect to the stabilization of the hemiketal oxyanion. The reduction of the pK of the hemiketal by the trifluoromethyl group is an important contributing factor to the low Ki of trifluoromethyl ketones. The inhibition of acetylcholinesterase by tetramethylammonium chloride and trifluoroacetone was compared to the inhibition by 1, which is a composite of the two smaller inhibitors. The entropic advantage of combining the smaller inhibitors into one molecule is 1.1 X 10(3)M. Inhibitors with Ki less than or equal to 70 X 10(-9) M are slow binding (Morrison, 1982; Morrison & Walsh, 1988). The kinetic data do not require formation of a noncovalent complex prior to formation of the ketal, although such a complex(es) cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon

The kinetics and specificity of L-lactate transport into cardiac muscle were studied during a single transit through the isolated perfused rabbit heart using a rapid (15 s) paired-tracer dilution technique. Kinetic experiments revealed that lactate influx was highly stereospecific and saturable with an apparent Kt = 19 +/- 6 mM and a Vmax = 8.4 +/- 1.5 mumol/min per g (mean +/- S.E., n = 14 hearts). At high perfusate concentrations (10 mM), the inhibitors alpha-cyano-4-hydroxycinnamate (Ki = 7.3 mM), pyruvate (Ki = 6.5 mM), acetate (Ki = 19.4 mM) and chloroacetate (Ki = 28 mM) reduced L-lactate influx, and Ki values were estimated assuming a purely competitive interaction of the inhibitors with the monocarboxylate carrier. The monocarboxylic acids [14C]pyruvate and [3H]acetate were themselves transported, and sarcolemmal uptakes of respectively 38 +/- 1% and 70 +/- 8% were measured relative to D-mannitol. Perfusion of hearts for 10-30 min with 0.15 or 1.5 microM glucagon increased myocardial lactate production and simultaneously inhibited tracer uptake of lactate, pyruvate and acetate. It is concluded that a stereospecific lactate transporter exhibiting an affinity for other substituted monocarboxylic acids is operative in the sarcolemmal plasma membrane of the rabbit myocardium.     

Stereoselective inhibition of human placental aromatase

We have synthesized the (19R)- and (19S)-isomers (2 and 3 respectively) of 10 beta-oxiranylestr-4-ene-3,17-dione. The configurations and conformations of these compounds were established by X-ray crystallographic analysis. Each of these compounds is a powerful competitive inhibitor of human placental microsomal aromatase, and stereoselectivity of inhibition was observed (Ki values for 2 and 3 were 7 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of oxirane oxygen to the heme iron of the enzyme. The (19R)- and (19S)-10 beta-thiiranes (6 and 7) corresponding to 2 and 3 have been synthesized from the oxiranes by a stereospecific process. The thiiranes are very effective competitive inhibitors of placental aromatase, and show even greater stereoselectivity in binding than the oxiranes (Ki values for 6 and 7 were 1 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of thiirane sulfur to heme iron.     

Characterization of urinary proteinase inhibitors with segments of amino acids sequences identical to sequences of pancreatic secretory trypsin inhibitor

1. Slow migrating proteinase inhibitors were isolated from pathological human urine. 2. The N-terminal amino acid sequence including 23 amino acids was identical to the one in pancreatic secretory trypsin inhibitor. 3. The slow migrating proteinase inhibitors occurred in 3 forms with different electrophoretic mobility. 4. Time of flight mass spectrometry showed that the Mw of one of the forms was 6241 while the Mw of another form was 5923. 5. The Ki of complexes with trypsin was determined to be 1 x 10(-10) M, with chymotrypsin and plasmin Ki was 1 x 10(-7) M. Elastase, kallikrein and thrombin were not inhibited.     

Design of potent and specific inhibitors of carboxypeptidases A and B.

The combination in one molecule of functional groups that can interact specifically with different substrate binding areas at the active site of carboxypeptidases A and B has led to the development of potent and specific inhibitors of these enzymes. 2-Benzyl-3-mercaptopropanoic acid (SQ 14,603) has a Ki of 1.1 x 10(-8) M vs. carboxypeptidase A and a Ki of 1.6 x 10(-4) M vs. the B enzyme. 2-Mercaptomethyl-5-guanidinopentanoic acid (SQ 24,798) has a Ki of 4 x 10(-10) M vs. carboxypeptidase B and a Ki of 1.2 x 10(-5) M vs. carboxypeptidase A. It is proposed that the sulfhydryl groups of these inhibitors bind to the catalytically important zinc ions of these enzymes, and that, in conjunction with the benzyl and guanidinopropyl side chains, they are responsible for their specificity.     

Synthesis of 4,6-dideoxysucrose, and inhibition studies of Leuconostoc and Streptococcus D-glucansucrases with deoxy and chloro derivatives of sucrose modified at carbon atoms 3, 4, and 6

Starting from sucrose, 2,3,1',3',4',6'-hexa-O-benzoyl-6-deoxy-6-iodosucrose (1) was synthesized. Reaction of 1 with sulfuryl chloride in pyridine gave 2,3,1',3',4',6'-hexa-O-benzoyl-4-chloro-4,6-dideoxy-6-iodogalactosucr ose (2). Compound 2 was treated with tributyltin hydride in toluene in the presence of a radical initiator, alpha, alpha-azobis(isobutanonitrile) (AIBN), to remove iodine and chlorine groups and give hexa-O-benzoyl-4,6-dideoxysucrose. Benzoyl groups were removed by sodium methoxide in methanol to give 4,6-dideoxysucrose. Sucrose was modified at carbon atom 3, carbon atom 4, or carbon atoms 4 and 6, and these analogs were tested as inhibitors of the D-glucansucrases (D-glucosyltransferases) of Streptococcus mutans 6715 and Leuconostoc mesenteroides B-512F. Sucrose analogs used in this study are 4-deoxysucrose and 4-chloro-4-deoxygalactosucrose with S. mutans 6715 D-glucansucrases (GTF-S and GTF-I), and 3-deoxysucrose, 4-deoxysucrose, 4-chloro-4-deoxygalactosucrose, 6-deoxysucrose, and 4,6-dideoxysucrose with L. mesenteroides B-512F D-glucansucrase. The data indicate that 3-deoxysucrose, 4-deoxysucrose, and 4-chloro-4-deoxygalactosucrose are weak noncompetitive inhibitors for B-512F dextransucrase, with Ki values of 530, 201, and 202mM respectively. For the same enzyme, 6-deoxysucrose was a strong competitive inhibitor, with Ki of 1.60mM, and 4,6-dideoxysucrose was a good competitive inhibitor, with Ki of 20.3mM. 4-Deoxysucrose was a weak noncompetitive inhibitor for both GTF-I and GTF-S, with Ki values of 672 and 608mM, respectively. 4-Chloro-4-deoxygalactosucrose was also a weak noncompetitive inhibitor for GTF-I and GTF-S with Ki values of 391 and 308mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.     

Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition

The synthesis of 10 analogues of pepstatin modified so that statine is replaced by 4-amino-3-hydroxy-3,6-dimethylheptanoic acid (Me3Sta) or 4-amino-3-hydroxy-3-methyl-5-phenylpentanoic acid (Me3AHPPA) residues is reported. Both the 3S,4S and 3R,4S diastereomers of each analogue were tested as inhibitors of the aspartic proteases, porcine pepsin, cathepsin D, and penicillopepsin. In all cases the 3R,4S diastereomer (rather than the 3S,4S diastereomer) of the Me3Sta and Me3AHPPA derivatives was found to be the more potent inhibitor of the aspartic protease (Ki = 1.5-10 nM for the best inhibitors), in contrast to the results obtained with statine (Sta) or AHPPA derivatives, where the 3S,4S diastereomer is the more potent inhibitor for each diastereomeric pair of analogues. The Me3Sta- and Me3AHPPA-containing analogues are only about 10-fold less potent than the corresponding statine and AHPPA analogues and 100-1000-fold more potent than the corresponding inhibitors lacking the C-3 hydroxyl group. Difference NMR spectroscopy indicates that the (3R,4S)-Me3Sta derivative induces conformational changes in porcine pepsin comparable to those induced by the binding of pepstatin and that the (3S,4S)-Me3Sta derivatives do not induce the difference NMR spectrum. These results require that the C-3 methylated analogues of statine-containing peptides must inhibit enzymes by a different mechanism than the corresponding statine peptides. It is proposed that pepstatin and (3S)-statine-containing peptides inhibit aspartic proteases by a collected-substrate inhibition mechanism. The enzyme-inhibitor complex is stabilized, relative to pepstatin analogues lacking the C-3 hydroxyl groups, by the favorable entropy derived when enzyme-bound water is returned to bulk solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

Cathepsins have been found to have important physiological roles. The implication of cathepsin L in various types of cancers is well established. In a search for selective cathepsin L inhibitors as anticancer agents, a series of 2-cyanoprrolidine peptidomimetics, carrying a nitrile group as warhead, were designed. Two series of compounds, one with a benzyl moiety and a second with an isobutyl moiety at P(2) position of the enzyme were synthesized. The synthesized compounds were evaluated for inhibitory activity against human cathepsin L and cathepsin B. Although, none of the compounds showed promising inhibitory activity, (E)N-{(S)1-[(S)2-cyano-1-pyrrolidinecarbonyl]-3-methylbutyl}-2,3-diphenylacrylamide (24) with an isobutyl moiety at P(2) was found to show selectivity as a cathepsin L inhibitor (Ki 5.3 microM for cathepsin L and Ki > 100 microM for cathepsin B). This compound could act as a new lead for the further development of improved inhibitors within this inhibitor type.     

Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase

We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.     

Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

The interaction of angiotensin converting enzyme with six metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N alpha-(3-mercaptopropanoyl)-L-Arg, N alpha-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and three dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots were found to vary with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while five of seven tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp, and MK-422 were 8.3-, 5.5-, 4.7-, and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potent mechanism-based inhibitors for matrix metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.     

Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.     

Indomethacin inhibition of glutathione S-transferases

Indomethacin inhibited rat liver glutathione S-transferases (EC 2.5.1.18). Its inhibition was non-competitive with respect to 3,4-dichloronitrobenzene with an apparent Ki of 5.3 X 10(-5) M and uncompetitive with respect to glutathione with an apparent Ki of 4.0 X 10(-5) M. 4-Chlorobenzoic acid and 5-methoxy-2-methylindole-3-acetic acid, two metabolites of indomethacin, were weak inhibitors of the enzymes. On the other hand, meclofenamic acid was a competitive inhibitor of the enzymes with an apparent Ki of 3.0 X 10(-4) M. Possible significance of these findings in arachidonic acid metabolism is discussed.