alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-Phosphonopropionic acids inhibit carboxypeptidase A as multisubstrate analogues or transition-state analogues.

A series of phosphonic acid analogues of 2-benzylsuccinate were tested as inhibitors of carboxypeptidase A. The most potent of these, (2RS)-2-benzyl-3-phosphonopropionic acid, had a Ki of 0.22 +/- 0.05 microM, equipotent to (2RS)-2-benzylsuccinate and thus one of the most potent reversible inhibitors known for this enzyme. Lengthening by one methylene group to (2RS)-2-benzyl-4-phosphonobutyric acid increased the Ki to 370 +/- 60 microM. The monoethyl ester (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid was nearly as potent as (2RS)-2-benzyl-3-phosphonopropionic acid, with a Ki of 0.72 +/- 0.3 microM. The sulphur analogue of the monoethyl ester, 2-ambo-P-ambo-2-benzyl-3-(O-ethylthiophosphono)propionic acid, had a Ki of 2.1 +/- 0.6 microM, nearly as active as (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid. These phosphonic acids and esters could be considered to be multisubstrate inhibitors of carboxypeptidase A by virtue of their structural analogy with 2-benzylsuccinate. Alternatively, the tetrahedral hybridization at the phosphorus atom suggests that they could be mimicking a tetrahedral transition state for the enzyme-catalysed hydrolysis of substrate.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum

The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly-Pro-X-Gly-Pro-X-. Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by -Gly-Pro-X-. Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM). However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors. The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM. The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM). The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII). The alcohol analogue of XXV, 5-benzamido-4-hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM. Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII). Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

A metabolite of aspartame inhibits angiotensin converting enzyme

Aspartame (L-aspartyl-L-phenylalanine methyl ester, is a widely used artificIal sweetener. In humans and other animals aspartame is initially hydrolyzed to L-aspartyl-L-phenylalanine by intestinal esterases. L-Aspartyl-L-phenylalanine inhibits angiotensin converting enzyme purified from rabbit lungs with a Ki of 11 +/- 2 microM, equipotent to the IC50 of 12 microM for 2-D-methyl-succinyl-L-proline which has been reported to be an orally active antihypertensive agent in rats. Thus the possibility exists that L-aspartyl-L-phenylalanine inhibits angiotensin converting enzyme in humans consuming large quantities of aspartame. Both aspartame itself and the diketopiperazine formed from it, 3-carboxymethyl-6-benzyl-2,5-diketopiperazine, are weak inhibitors with Ki's greater than 1 mM.     

Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitors of angiotensin-converting enzyme containing a tetrahedral arsenic atom.

A series of tetrahedral oxo acids of Group VA and VIA elements and of silicon and boron were examined as inhibitors of angiotensin-converting enzyme. Arsenate is a competitive inhibitor with a Ki of 27 +/- 1 mM, at least 10-fold more potent than phosphate. Dimethylarsinate is a competitive inhibitor with a Ki of 70 +/- 9 mM, 2-fold more potent than dimethylphosphinate. Oxo acids of boron, silicon, antimony, sulphur and selenium are not inhibitors. On the basis of these results and the strong inhibition of this zinc metallopeptidase by substrate analogues containing a tetrahedral phosphorus atom, two substrate analogues containing a tetrahedral arsenic atom were prepared. 2-Arsonoacetyl-L-proline is a competitive inhibitor with a Ki of 18 +/- 7 mM, more than 2000-fold weaker than that of its phosphorus analogue 2-phosphonoacetyl-L-proline. 4-Arsono-2-benzylbutanoic acid is a mixed inhibitor with a Ki of 0.5 +/- 0.2 mM, indistinguishable in potency from its phosphorus analogue 2-benzyl-4-phosphonobutanoic acid.     

Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa.

We have previously used monoclonal antibodies to identify an epitope on the heavy chain of factor XIa that is a substrate-binding site for factor IX (Sinha, D., Seaman, F.S., and Walsh, P.N. (1987) Biochemistry 26, 3768-3775; Baglia, F.A., Sinha, D., and Walsh, P.N. (1989) Blood 74, 244-251). To define the factor XIa domain that binds factor IX, we have now screened a panel of factor XI heavy chain-derived synthetic peptides for their capacity to inhibit the formation of an activation peptide reflecting factor IX activation by factor XIa. Peptide Asn145-Ala176 (which is located in the second tandem repeat or A2 domain of the factor XI heavy chain) is a competitive inhibitor of factor IX activation by factor XIa with a Ki of 30 nM, whereas structurally similar peptides in the A1, A3, and A4 domains were required at 10-1000-fold higher concentrations for similar effects, and a synthetic peptide identical with a highly homologous region of the heavy chain A2 domain of prekallikrein (Tyr143-Ala176) had no effect on factor IX activation by factor XIa. Because detailed structural information is lacking, a potential three-dimensional structure for the factor XI A2 domain was calculated based on its sequence information in conjunction with previously determined structural constraints. The resulting structure depicted three juxtaposed beta-stranded stem-loops that, based on biological information, constitute a candidate surface for contact with factor IX. The A2 model was therefore used as a template in the rational design of three synthetic peptides (Ala134-Ile146 (peptide a), Leu148-Arg159 (peptide b), and Ile160-Leu172 (peptide c]. When peptides a and b or a and c were added together and the activation of factor IX by factor XIa was examined, a synergistic inhibitory effect was observed, compared with each peptide added individually, whereas peptides b and c showed additive effects. Our data suggest that the sequence of amino acids from Ala134 through Leu172 of the heavy chain of factor XI contains three antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of factor IX.     

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Choline transport in Fusarium graminearum A 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).     

4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

YC-1-like potentiation of NO-dependent activation of soluble guanylate cyclase by derivatives of protoporphyrin IX

The influence of protoporphyrin IX derivatives—2,4-di(1-methoxyethyl)-deuteroporphyrin IX disodium salt (dimegin) and hematoporphyrin IX (HP)—on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside was investigated. Dimegin and HP, like 1-benzyl-3-(hydroxymethyl-2-furyl)indazole (YC-1), produce synergistic effects on the activation of soluble guanylate cyclase by sodium nitroprusside. The synergistic activation of the enzyme by the combination of 10 μM sodium nitroprusside and 5 μM dimegin (or 5 μM HP) was 190 ± 19 and 134 ± 10%, respectively. The synergistic activation of guanylate cyclase by 3 μM YC-1 and 10 μM sodium nitroprusside was 255 ± 19%. Dimegin and HP had no effect on the activation of guanylate cyclase by YC-1; they did not change the synergistic effect of YC-1 (3 μM) and sodium nitroprusside (10 μM) on guanylate cyclase activity. The synergistic activation of NO-stimulated guanylate cyclase activity by dimegin and HP represents a new biochemical effect of these compounds that may have important pharmacotherapeutic and physiological significance. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 426–431.     

Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid.

Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.     

2-Acylimino-3H-thiazoline derivatives: a novel template for platelet GPIIb/IIIa receptor antagonists

In the course of our research for the low-molecular weight RGD peptide mimics, we have found that a rigid 2-acylimino-3H-thiazoline structure is suitable for the peptide backbone mimics. Introduction of amidinophenyl and beta-alanine moiety as arginine and aspartic acid side-chain surrogates to this backbone mimic resulted in a highly potent fibrinogen receptor antagonist 2-(4-amidinobenzoylimino)-3,4-dimethyl-N-(2-carboxyethyl)-3H-thiazoline-5-carboxamide (7c), namely PS-028 (Ki = 46.5 +/- 5.8 microM).     

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.     

Kinetics of D-glucose and 2-deoxy-D-glucose transport by Rhodotorula glutinis

The yeast Rhodotorula glutinis (Rhodosporidium toruloides) is capable of accumulative transport of a wide variety of monosaccharides. Initial velocity studies of the uptake of 2-deoxy-D-glucose were consistent with the presence of at least two carriers for this sugar in the Rhodotorula plasma membrane. Non-linear regression analysis of the data returned maximum velocities of 0.8 +/- 0.2 and 2.0 +/- 0.2 nmol/min per mg (wet weight) and Km values of 18 +/- 4 and 120 +/- 20 microM, respectively, for the two carriers. Kinetic studies of D-glucose transport also revealed two carriers with maximum velocities of 1.1 +/- 0.4 and 2.4 +/- 0.4 nmol/min per mg (wet weight) and Km values of 12 +/- 3 and 55 +/- 12 microM. As expected, 2-deoxy-D-glucose was a competitive inhibitor of D-glucose transport. Ki values for the inhibition were 16 +/- 8 and 110 +/- 40 microM. These Ki values were in good agreement with the Km values for 2-deoxy-D-glucose transport. D-Xylose, the 5-deoxymethyl analog of D-glucose, appears to utilize the D-glucose/2-deoxy-D-glucose carriers. This pentose was observed to be a competitive inhibitor of D-glucose (Ki values = 0.14 +/- 0.06 and 5.6 +/- 1.6 mM) and 2-deoxy-D-glucose (Ki values = 0.15 +/- 0.07 and 4.6 +/- 1.2 mM) transport.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.