Kinetic study of catalytic CO(2) hydration by water-soluble model compound of carbonic anhydrase and anion inhibition effect on CO(2) hydration

A kinetic study of CO(2) hydration was carried out using the water-soluble zinc model complex with water-soluble nitrilotris(2-benzimidazolylmethyl-6-sulfonate) L1S, [L1SZn(OH(2))](-), mimicking the active site of carbonic anhydrase, in the presence and absence of anion inhibitors NCS(-) and Cl(-). The obtained rate constants k(cat) for CO(2) hydration were 5.9x10(2), 1. 7x10(3), and 3.1x10(3) M(-1) s(-1) at 5, 10, and 15 degrees C, respectively: the k(cat)=ca. 10(4) M(-1) s(-1) extrapolated towards 25 degrees C has been the largest among the reported k(cat) using zinc model complexes for carbonic anhydrase. It was also revealed that NCS(-), Cl(-) and acetazolamide play a role of inhibitors by the decrease of k(cat): 7x10(2) and 2x10(3) M(-1) s(-1) for NCS(-) and Cl(-) at 15 degrees C, respectively. The sequence of their magnitudes in k(cat) is Cl(-) approximately acetazolamide>NCS(-), where the sequence Cl(-)>NCS(-) is confirmed for native carbonic anhydrase. The difference of k(cat) or k(obs) between NCS(-) and Cl(-) resulted from that between the stability constants K(st)=2x10(3) for [L1SZn(NCS)](2-) and 1x10(2) M(-1) for [L1SZnCl](2-) in D(2)O: for water-insoluble tris(2-benzimidazolylmethyl)amine L1, K(st)=1.8x10(4) for [L1Zn(NCS)](2-) and 1.5x10(3) M(-1) for [L1ZnCl](2-)in CD(3)CN/D(2)O (50% v/v). The crystal structure of anion-binding zinc model complexes [L1Zn(OH(2))](0.5)[L1ZnCl](0.5) (ClO(4))(1.5) 1(0.5)2(0.5)(ClO(4))(1.5) was revealed by X-ray crystallography. The geometry around Zn(2+) in 1 and 2 was tetrahedrally coordinated by three benzimidazolyl nitrogen atoms and one oxygen atom of H(2)O, or Cl(-).     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

β-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of Smilax Rhizoma (the dried rhizomes of Smilax china L.) showed potent inhibitory activity. The active compounds were identified as a trans/cis-resveratrol mixture, oxyresveratrol, veraphenol, and cis-scirpusin A. They were shown to non-competitively inhibit BACE1 with the Ki values of 5.4 x 10(-6), 5.4 x 10(-6), 3.4 x 10(-6), and 5.4 x 10(-6)M and IC(50) values of 1.5 x 10(-5), 7.6 x 10(-6), 4.2 x 10(-6), and 1.0 x 10(-5)M, respectively. The active compounds were less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.     

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.     

A facile synthesis of D-glucose-type gem-diamine 1-N-iminosugars: a new family of glucosidase inhibitors.

gem-Diamine 1-N iminosugars of D-glucose-type, a new type of glycosidase inhibitors, have been synthesized from siastatin B, isolated from Streptomyces culture. 2-Trifluoroacetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-trifluoroacetamido-5-hydroxymethylpiperidine -3,4-diol was proved to be a potent inhibitor for alpha-D- and beta-D-glucosidases (IC50 1.9x10(-7) and 4.2x10(-7) M, respectively). 2-Acetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-acetamido-5-hydroxymethylpiperidine-3,4-diol also affected these enzymes (IC50 2.9x10(-6) and 5.4x10(-6) M, respectively).     

Tonic regulation of vascular tone by nitric oxide and chloride ions in rat isolated small coronary arteries

We have investigated the involvement of Cl(-) in regulating vascular tone in rat isolated coronary arteries mounted on a small vessel myograph. Mechanical removal of the endothelium or inhibition of nitric oxide (NO) synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) led to contraction of rat coronary arteries, and these contractions were sensitive to nicardipine (10(-6) M). This suggests that release of NO tonically inhibits a contractile mechanism that involves voltage-dependent Ca(2+) channels. In arteries contracted with L-NAME, switching the bathing solution to physiological saline solution with a reduced Cl(-) concentration potentiated the contraction. DIDS (5 x 10(-6)-3 x 10(-4) M) caused relaxation of L-NAME-induced tension (IC(50) = 55 +/- 10 microM), providing evidence for a role of Cl(-). SITS (10(-5)-5 x 10(-4) M) did not affect L-NAME-induced tension, suggesting that DIDS is not acting by inhibition of anion exchange. Mechanical removal of the endothelium led to contraction of arteries, which was sensitive to DIDS (IC(50) = 50 +/- 8 microM) and was not affected by SITS. This study suggests that, in rat coronary arteries, NO tonically suppresses a contractile mechanism that involves a Cl(-) conductance.     

A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes

We describe a single-cell technique for measuring membrane potential, membrane resistance, and the efflux of rapidly penetrating solutes such as Cl and H2O. Erythrocytes from Amphiuma means were aspirated into a Sylgard (Dow Corning Corp.)-coated capillary. The aspirated cell separated a solution within the capillary from a solution in the bath. Each of these two solutions was contiguous with approximately 5% of the total membrane surface. Microelectrodes placed concentrically within the capillary permit the measurement of intracellular voltage, specific membrane resistance, and the electrical seal between the two solutions. The intracellular voltage averaged -17.7 mV (pH 7.6) and changed as either intra- or extracellular chloride was varied. The average specific membrane resistance measured by passing current across the exposed membrane surface was 110 ohm-cm2. 36Cl and tritiated H2O fluxes (0.84 +/- 0.05 x 10(-6) M . cm-2 . min-1 and 6.4 +/- 1.5 x 10(-3) M . cm-2 . min-1, respectively) were determined by noting the rate at which isotope leaves the cell and crosses the membrane exposed to the bath. Our measured values for the flux of 36Cl and tritiated H2O approximate reported values for free-floating cells. 36Cl efflux, in addition, is inhibited by 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid (SITS) and furosemide, known inhibitors of the anion exchange mechanism responsible for the rapid anion fluxes of red blood cells. One can also demonstrate directly that > 89% of 36Cl efflux is "electrically silent" by analyzing the flux in the presence of an imposed transcellular voltage.     

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.     

Manipulation of chloride flux affects histamine-induced contraction in rabbit basilar artery

Cl(-) efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl(-) flux in histamine-induced contraction in the rabbit basilar artery. Male New Zealand White rabbits (n = 16) weighing 1.8-2.5 kg were euthanized by an overdose of pentobarbital sodium. The basilar arteries were removed for isometric tension recording. Histamine produced a concentration-dependent contraction that was attenuated by the H(1) receptor antagonist chlorpheniramine (10(-8) M) but not by the H(2) receptor antagonist cimetidine (3 x 10(-6) M) in normal Cl(-) Krebs-Henseleit bicarbonate solution (123 mM Cl(-)). The histamine-induced contraction was reduced by the following manipulations: 1) inhibition of Na(+)-K(+)-2Cl(-) cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free HEPES solution to disable Cl(-)/HCO exchanger, and 3) blockade of Cl(-) channels with the use of niflumic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid, and indoleacetic acid 94 R-(+)-methylindazone. In addition, substitution of extracellular Cl(-) (10 mM) with methanesulfonate acid (113 mM) transiently enhanced histamine-induced contraction. Manipulation of Cl(-) flux affects histamine-induced contraction in the rabbit basilar artery.     

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.     

Influence of metal ions on structure and catalytic activity of papain

Papain is an endoprotease belonging to cysteine protease family. The catalytic activity of papain in presence of two different metal ions namely zinc and cadmium has been investigated. Both the metal ions are potent inhibitors of the enzyme activity in a concentration dependent manner. The enzyme loses 50% of its activity at 2 x 10(-4) M of CdCl2 and 4 x 10(-4) M of ZnCl2. It is completely inactivated above 1 x 10(-3) M concentration of either ZnCl2 or CdCl2. Of the two metal ions zinc with a ki value of 5 x 10(-5) M is a more potent inhibitor than cadmium which has a ki value of 8 x 10(-5) M. Both the metal ions have higher affinity for active site than the substrate. At concentrations above 1 x 10(-2) M of metal ions the inhibition is not reversible. Calorimetric studies showed decreased thermal stability of papain upon binding of these metal ions. Far UV circular dichroic spectral data showed only small changes in the beta-structure content upon binding of these metal ions. These data are also supported by decrease in the apparent thermal transition temperature of papain by 5 degrees C upon binding of metal ions indicating destabilization of the papain molecule. The mechanism of both partial and complete inactivation of papain in presence of these two metal ions both at lower and higher concentration has been explained.     

Purification,amino acid sequence,and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs

Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.     

Comparative properties of channel catfish (Ictalurus punctatus) and blue crab (Callinectes sapidus) acetylcholinesterases

1. Acetylcholinesterases (AChEs) from channel catfish and blue crabs were examined for substrate preference, KmS, effects of inhibitors, and pH and osmotic activity profiles. 2. Similarities were noted for substrate preference along with pH and osmotic optima. 3. Crab AChE had a lower Km (9 x 10(-5) vs 2 x 10(-4) M) and was more sensitive in terms of KI50S than fish AChE to eserine (2.6 x 10(-7) vs 3 x 10(-7) M), malathion (4.5 x 10(-5) vs 1.6 x 10(-4) M) and parathion (6.9 x 10(-5) vs 7 x 10(-4) M). 4. Fish AChE appeared easier to solubilize using Triton X-100.     

Bicarbonate secretion plays a role in chloride and water absorption of the European flounder intestine

Experiments performed on isolated intestinal segments from the marine teleost fish, the European flounder (Platichthys flesus), revealed that the intestinal epithelium is capable of secondary active HCO3(-) secretion in the order of 0.2-0.3 micromol x cm(-2) x h(-1) against apparent electrochemical gradient. The HCO3(-) secretion occurs via anion exchange, is dependent on mucosal Cl(-), results in very high mucosal HCO3(-) concentrations, and contributes significantly to Cl(-) and fluid absorption. This present study was conducted under in vivo-like conditions, with mucosal saline resembling intestinal fluids in vivo. These conditions result in a transepithelial potential of -16.2 mV (serosal side negative), which is very different from the -2.2 mV observed under symmetrical conditions. Under these conditions, we found a significant part of the HCO3(-) secretion is fueled by endogenous epithelial CO2 hydration mediated by carbonic anhydrase because acetazolamide (10(-4) M) was found to inhibit HCO3(-) secretion and removal of serosal CO(2) was found not to influence HCO3(-) secretion. Reversal of the epithelial electrochemical gradient for Cl(-) (removal of serosal Cl(-)) and elevation of serosal HCO3(-) resulted in enhanced HCO3(-) secretion and enhanced Cl(-) and fluid absorption. Cl(-) absorption via an anion exchange system appears to partly drive fluid absorption across the intestine in the absence of net Na(+) absorption.     

Features of thiamine diphosphate-dependent enzyme inhibition by oxodihydrothiochrome and its phosphorylated derivatives

Anticoenzyme action of new derivatives of thiamine: oxodihydrothiochrome and its mono- and diphosphoric esters has been studied in the experiments on mice. It is shown that the given compounds exert an inhibiting action on transketolase and pyruvate dehydrogenase and do not change activity of 2-oxoglutarate dehydrogenase in the animal organism. Antivitamin effect of the studied inhibitors is observed with the lower doses and in the earlier terms as compared with the other known inhibitors of thiamine-diphosphate-dependent enzymes. The preparations inhibit activity of the yeast pyruvate-decarboxylase by the mixed (with respect to thiamine-diphosphate) type (Ki for oxodihydrothiochrome and its mono- and diphosphoric esters: 2.3 x 10(-3), 7.2 x 10(-4), 5.6 x 10(-5) M, respectively). Possible mechanisms of the action of the mentioned compounds as thiamine antimetabolites are discussed.     

Effect of chlorpromazine, imipramine and lithium on MAO-A and MAO-B activity in rat brain mitochondria

Drugs with efficacy in psychiatric disorders affect the function of central neurotransmitter amines, which are inactivated primarily by monoamine oxidase (MAO). Effect of these drugs on the two types of MAO (MAO-A and MAO-B) has been studied in rat brain. The result showed that chlorpromazine (CPZ) and imipramine (IMI) at concentrations of 1x10(-2), 5x10(-3) and 2.5x10(-3) M inhibited rat brain mitochondrial MAO-A activity in vitro by 82, 50, 39 and 86, 74, 38 %, respectively. CPZ at concentrations of 5x10(-3), 2.5x10(-3), 1x10(-3) M inhibited rat brain mitochondrial MAO-B activity in vitro by 83, 55, 39 %, respectively, while IMI at concentrations of 5x10(-4), 2.5x10(-4), 1x10(-4) M inhibited the in vitro enzyme activity by 43, 35, 21 %, respectively. Lithium at concentration of 5x10(-3) M could not either inhibit MAO-A or MAO-B in the mitochondrial fraction of rat brain.     

Effect of mercuric chloride on electrical parameters and anion fluxes in the toad skin

The amphibian skin, widely used for studying the transepithelial passage of electrolytes, exhibits anion pathways relatively specific for Cl(-). We studied the effect of HgCl(2), 1.0 x 10(-4) M on its electrical parameters and unidirectional anion fluxes. In the presence of Cl(-), the transepithelial conductance (G) of the isolated skin of the Bufo arenarum toad increased considerably following exposure to HgCl(2), whereas short-circuit current (SCC)--reflecting transepithelial Na(+) transport-underwent only slight stimulation. Following the blockade of Na(+) intake by amiloride, 1.0 x 10(-4) M, the removal of Cl(-) from the solution bathing the epidermal border of the skin brought about a decrease in G, and gave rise to a gradient-induced SCC (SCCg) consistent with transepithelial passage of Cl(-) along its gradient. Addition of mercaptoethanol, 5.0 x 10(-3) M to the bath containing Hg(2+) fully reversed these effects. The increase in G was accompanied by an increase in the unidirectional (epidermal to dermal) fluxes of (36)Cl(-) and (131)I(-), and a decrease in the passage of (99m)TcO(4)(-). These results show the effects of HgCl(2) to be similar to those of theophylline, although exhibiting a different selectivity. Our data suggest that anion passage following exposure to HgCl(2) is, like that stimulated by theophylline, predominantly if not exclusively transcellular, and does not involve a significant opening of the tight junctions.     

Oxidized elafin and trappin poorly inhibit the elastolytic activity of neutrophil elastase and proteinase 3

Neutrophil proteinase-mediated lung tissue destruction is prevented by inhibitors, including elafin and its precursor, trappin. We wanted to establish whether neutrophil-derived oxidants might impair the inhibitory function of these molecules. Myeloperoxidase/H(2)O(2) and N-chlorosuccinimide oxidation of the inhibitors was checked by mass spectrometry and enzymatic methods. Oxidation significantly lowers the affinities of the two inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This decrease in affinity is essentially caused by an increase in the rate of inhibitory complex dissociation. Oxidized elafin and trappin have, however, reasonable affinities for NE (K(i) = 4.0-9.2 x 10(-9) M) and for Pr3 (K(i) = 2.5-5.0 x 10(-8) M). These affinities are theoretically sufficient to allow the oxidized inhibitors to form tight binding complexes with NE and Pr3 in lung secretions where their physiological concentrations are in the micromolar range. Yet, they are unable to efficiently inhibit the elastolytic activity of the two enzymes. At their physiological concentration, fully oxidized elafin and trappin do not inhibit more than 30% of an equimolar concentration of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin strongly impairs their activity. Inhibitor-based therapy of inflammatory lung diseases must be carried out using oxidation-resistant variants of these molecules.     

Furin inhibition by compounds of copper and zinc

Furin, a human subtilisin-related proprotein convertase (SPC), is emerging as an important pharmaceutical target because it processes vital proteins of many aggressive pathogens. Furin inhibitors reported as yet are peptide derivatives and proteins, with the exception of andrographolides, which are natural compounds. Here we report that the small and highly stable compounds M(chelate)Cl(2) (M is copper or zinc) inhibit furin and Kex2, with Cu(TTP)Cl(2) and Zn(TTP)Cl(2) as the most efficient inhibitors. (TTP is 4'-[p-tolyl]-2,2 ':6',2"-terpyridine.) Inhibition is irreversible, competitive with substrate, and affected by substituents on the chelate. The free chelates are not inhibitors. Solvated Zn(2+) is less potent than its complexes. This is true also for copper and Kex2. However, solvated Cu(2+) (k(on) of 25,000 +/- 2,500 s(-1)) is more potent than Cu(TTP)Cl(2) (k(on) = 140 +/- 13 s(-1) and allows recovery of furin activity prior to a second inhibition phase. A mechanism that involves coordination to the catalytic histidine is proposed for all inhibitors. Target specificity is indicated by the fact that these metal chelate inhibitors are much less potent toward Kex2, the yeast homologue of furin. For example, k(on) with Zn(TTP)Cl(2) is 120 +/- 20 s(-1) for furin, but only 1.2 +/- 0.1 s(-1) for Kex2.