Specificity of intestinal brush-border proline transport: cyanine dye studies

The ability of rabbit jejunal brush borders to transport inhibitors of the imino carrier was investigated in membrane vesicles by measuring their ability to depolarize the membrane potential. Membrane potentials were monitored using a voltage-sensitive cyanine dye. Piperidine and pyrrolidine carboxylic acids, which are potent inhibitors of Na+-dependent proline transport (Ki less than 0.5 mM) depolarize the potential in a Na+-dependent, saturable manner indicating transport. On the other hand, N-methylated amino acids, which are fair inhibitors (Ki 2-10 mM), do not depolarize the membrane to any significant extent, but they competitively inhibit the L-proline transport signal. This indicates that these analogs are nontransported inhibitors of the imino carrier. The poor inhibitors niacin and pipolinic acid (Ki greater than 60 mM) depolarize the membrane about twice as much as proline and with low Kf values. This suggests separate carriers for these substrates.     

A new class of powerful inhibitors of monamine oxidase A

It is well established that 1-methyl-4-phenylpyridinium (MPP), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and most of its analogs are good competitive inhibitors of monoamine oxidase A, with Ki values in the micromolar range, but they inhibit monoamine oxidase B only at much higher concentrations. We report here the finding that alkyl derivatives of MPP+ substituted at the 4' position of the aromatic ring are considerably more effective reversible inhibitors of the A type enzyme, with Ki values in the nanomolar range (0.075-1.6 microM). They inhibit the B type enzyme only at 2 to 3 orders of magnitude higher concentrations (32-374 microM).     

Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases

Aminopeptidase P (APP), dipeptidyl peptidase II (DP II), dipeptidyl peptidase IV (DP IV) and prolyl oligopeptidase (POP) are proline specific peptidases. Hence, they are able to cleave peptide bonds containing the imino acid proline. Amino acid pyrrolidides (Pyrr) and thiazolidides (Thia) are well-known product analogue inhibitors of DP IV and POP. For the first time we describe the influence of a thioxo amide bond, incorporated into these compounds, on the inhibition of the proline specific peptidases. Taking into account the substrate specificity of these peptidases, we have synthesized Xaa-psi[CS-N]-Pyrr and Xaa-psi[CS-N]-Thia of the amino acids Ala, Phe, Val and Ile. The inhibition constants were determined for the above mentioned proline specific peptidases isolated from different sources. As a result, the serine proteases DP II, DP IV and POP were inhibited competitively, whereas metal-dependent APP displayed a linear mixed-type inhibition with inhibition constants up to 10(-4) M. Thioxylation of Xaa-Pyrr and Xaa-Thia led to a slight decrease of inhibition of DP IV and POP compared to Xaa-Pyrr and Xaa-Thia, though the inhibition constants were still in the range up to 10(-7) M. As Xaa-Thia exist as two isomers, we investigated isomer specific inhibition with regard to DP IV. Thus, our studies have revealed that DP IV was only inhibited by the Z isomer of the Xaa-psi[CS-N]-Thia. For the first time, Xaa-Pyrr and Xaa-Thia were characterized as inhibitors of DP II with inhibition constants in the micromolar range. In contrast to DP IV inhibition, the Xaa-psi[CS-N]-Pyrr and Xaa-psi[CS-N]-Thia have proven to be more potent inhibitors of DP II than the corresponding Xaa-Pyrr and Xaa-Thia. Thus, these Xaa-psi[CS-N]-Thia are new potent inhibitors especially suitable for DP II with K(i) values ranging in the upper nanomolar concentration.     

Interaction between alpha-2 macroglobulin and trypsin with synthetic trypsin inhibitors]

The complex formed between trypsin (Tn) and alpha 2 Macroglobulin (alpha 2 M) retains the whole hydrolytic activity of the enzyme for synthetic substrates. Moreover synthetic inhibitors of low molecular weight stiel inhibit this activity. A comparative study of three inhibitors (Benzylamine, Butylamine, Benzamidine) has been carried out and shows that their behavior is similar. These inhibitors bind trypsin when it is bound to alpha 2 M and reciprocally alpha 2 M can bind Tn-inhibitor complex. Nevertheless the dissociation constant of the enzyme-inhibitor complex (Ki) is increased by alpha 2 M. In the case of Benzamidine the value of Ki is 2.22.10(-5) M for native enzyme and 13.4.10(-5) M for Tn-alpha 2 M and in the case of Butylamine this value increases from 0.5.10(-3) M to 2.95.10(-3) M. These variations of the Ki values are due to the modification of the accessibility of the inhibitor to the active site. Unpublished results show that the alpha 2 M molecule undergoes a deep structural modification in the course of the complex formation, which must lead to an increase of the value of Ki. This structural modification is probably irreversible so that the alpha 2 M complex has never been dissociated without altering the alpha 2 M molecule. The increase of the values of Ki cannot therefore result in an effective decrease of the association constant of the Tn-alpha 2 M complex.     

Inhibition of DNA polymerases by tripeptide derivative protease inhibitors.

Benzyloxycarbonyl(Z)-Leu-Leu-Leu-al and dansyl(Dns)-Leu-Leu-Leu-CH2Cl, well known as protease inhibitors, effectively inhibit the activities of DNA polymerases alpha, beta and gamma from rat liver and pol I from Escherichia coli, but the ability of these inhibitors to inhibit terminal deoxynucleotidyl transferase (TdT) is weak. The mode of inhibition by these tripeptide analogues is non-competitive with dNTP. The Ki values for Z-Leu-Leu-Leu-al and Dns-Leu-Leu-Leu-CH2Cl are 6.25 x 10(-5) M and 6.56 x 10(-5) M, respectively.     

Dipeptide-hydroxamates are good inhibitors of the angiotensin I-converting enzyme

The inhibition constants (Ki) and modes of inhibition have been determined for a series of dipeptide-hydroxamate compounds with bovine lung parenchyma angiotensin I-converting enzyme (peptidyldipeptide carboxy-hydrolase, E.C. 3.4. 15.1). The hydroxamido function was borne by aspartic, glutamic, or aminoadipic acid and extended by 2, 3 or 4 bond lengths from the proline amide bond. L-glu(NHOH)-L-pro (Ki = 3.4 microM) and D,L-aminoadipicyl (NHOH)-L-pro (Ki = 1.2 microM) were the best competitive inhibitors of the hydrolysis of benzoyl-gly-his-gly but were not effective as affinity ligands for purification of the enzyme.     

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.     

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.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Sulfamide derivatives as transition state analogue inhibitors for carboxypeptidase A

3-Phenyl-2-sulfamoyloxypropionic acid (2), 2-benzyl-3-sulfamoylpropionic acid (3), and N-(N-hydroxysulfamoyl)phenylalanine (5) have been synthesized and evaluated as inhibitors for carboxypeptidase A (CPA) to find that they inhibit the enzyme competitively with the Ki values in the microM range, suggesting that their binding modes to CPA are analogous to each other, and resemble the binding mode of N-sulfamoylphenylalanine (1) that has been established by the X-ray crystallographic method to form a complex with CPA in a manner reminiscent of the binding of a transition state in the catalytic pathway. It was concluded thus that they are a new type of transition state analogue inhibitors for CPA. (R)-N-Hydroxy-N-sulfamoyl-beta-phenylalanine (8) was shown to be also a potent CPA inhibitor (Ki = 39 microM), the high potency of which may be ascribed to the involvement of the hydroxyl in the binding of CPA, most likely forming bidentate coordinative bonds to the zinc ion in CPA together with the sulfamoyl oxygen atom.     

Prostaglandin 15-hydroxy dehydrogenase from human placenta.

The enzyme system prostaglandin 15-hydroxy dehydrogenase, which catalyzes the inactivation of all biologically active prostaglandins, has been purified 1270-fold from human placenta. Kinetic studies on the enzyme have provided information on a well-organized control mechanism to avoid prostaglandin accumulation and for a fast prostaglandin degradation. 15-Ketoprostaglandin E2 and 13,14-dihydro-15-ketoprostaglandin E2 inhibit prostaglandin 15-hydroxy dehydrogenase non-competitively with respect to prostaglandin E2. The rate equation of enzyme reaction for two substrates was used for determination of the equilibrium constant and Michaelis constants of the enzyme. The following kinetic constants for prostaglandin 15-hydroxy dehydrogenase have been found. The equilibrium constant with repect to prostaglandin E2 is 18 muM, the Michaelis constant Km for prostaglandin E2 is 1 muM for NAD+ 44muM. The inhibition constants for 15-ketoprostaglandin E2 ar Ki(slope) = 70 muM, Ki(intercept) = 150 muM, and for 13,14-dihydro-15-ketoprostaglandin E2 Ki(slope) = 80 muM, and Ki(intercept) = 150 muM. The maximal velocity for the forward reaction is V1 = 0.45 mumol/min. These kinetic data exclude a random or ping-pong mechanism, and also a Theorell-Chance type as suggested by Braithwaite and Jarabak. We propose, therefore, a sequential ordered mechanism. The isoelectric point for prostaglandin 15-hydroxy dehydrogenase is at pH 5.35, judged by isoelectric focusing.     

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.     

Peptide substrates and inhibitors of the HIV-1 protease

Oligopeptides containing the consensus retroviral protease cleavage sequence Ser/Thr-X-Y-Tyr/Phe-Pro are substrates for purified recombinant HIV-1 protease with Km's in the millimolar range. The minimum sequence containing the consensus pentapeptide which serves as a good substrate is a heptapeptide spanning the P4-P3' residues. Substitution of reduced Phe-Pro or Tyr-Pro dipeptide isosteres or the statine analog 3-hydroxy-4-amino-5-phenylpentanoic acid for the scissile dipeptide afforded inhibitors of HIV-1 protease with Ki values in the micromolar range, three orders of magnitude better in affinity than the corresponding substrates. Inhibitors of HIV-1 protease may provide a novel and potentially useful therapeutic approach to the treatment of acquired immune deficiency syndrome (AIDS).     

Phosphonamidate and phosphothioate dipeptides as potential inhibitors of VanX

In an effort to prepare novel inhibitors of VanX, N-[(1-aminoethyl)hydroxyphosphinyl]-D-alanine 1 and S-[(aminoethyl)hydroxyphosphinyl]-thiolacetic acid 2 were synthesized and evaluated as inhibitors of VanX. Phosphonamidate 1 was shown to be a partial competitive inhibitor of VanX with a Ki of 36+/-3 microM, and phosphothioate 2 was shown not to inhibit VanX.     

Synthesis and in vitro activities of new non-peptidic APA inhibitors.

Aminopeptidase A (APA) is involved in the maturation of angiotensin III, a peptide which seems to be implicated in blood pressure regulation at the brain level. Therefore APA inhibitors are potential new antihypertensive agents with possible novel applications. With the aim of enhancing the bioavailability and potency of EC 33, the APA inhibitor (Ki = 300 nM) initially used in the earlier studies, we have synthesized new non-peptidic inhibitors able to interact with the S1 and S'1 subsites of the targeted enzyme. Compound 10a, (3S,4S)-3-amino-4-mercapto-6-phenyl-hexane-1-sulfonic acid was obtained using an asymmetric synthesis. Inhibitor 10a exhibits a Ki value of 30 nm.     

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.     

A class of strong inhibitors of microsomal monooxygenases: the ellipticines.

Ellipticine (E) and its 9-hydroxy derivative inhibit strongly various liver monooxygenase activities mediated by microsomes from control and phenobarbital (PB), benzo[alpha]pyrene (BP) or Aroclor 1254 (Aroclor)-pretreated rats. The inhibition constants, Ki, are remarkably low, and often smaller than 1 micron, particularly in the case of microsomes containing cytochrome P-448. The inhibitory potency (I50) of 9-hydroxyellipticine (9-OHE) is larger (about ten-fold) than the one of classical inhibitors (metyrapone or 7,8-benzoflavone (7,8-BF)), whatever the activities studied and the induction of microsomes. Differences exist between the mechanisms of inhibition according to the form of cytochrome P-450 present in microsomes of differently pretreated rats; whichever the activities studied, one observes: (a) a competitive inhibition towards the activity of non-induced or PB-induced microsomes and (b) a non-competitive inhibition towards the activity of Aroclor or BP-induced microsomes, at variance with 7,8-BF. These results are in good agreement with the interaction properties of the ellipticines with microsomal cytochromes P-450.     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

Predictors of response to aromatase inhibitors

Aromatase inhibitors are now considered to be part of the endocrine treatment for most hormone receptor-positive breast cancer in post-menopausal women for both early and advanced disease. Despite the impressive efficacy of these agents, up to 50% of treated patients exhibit de novo or intrinsic resistance to aromatase inhibitors and hence identification of response predictors is essential to allow treatment to be directed towards responsive populations and for alternative or additional therapies to be offered to resistant patients. Emerging data seem to suggest a role for the conventional tumour markers of oestrogen receptor and progesterone receptor as possible predictors of response but, particularly in the adjuvant setting, the extent to which these are useful has not been fully elucidated. Data from both the neo-adjuvant and advanced disease settings suggest that response to aromatase inhibitors does not appear to be adversely affected by HER-2 overexpression. Within neo-adjuvant aromatase inhibitor studies, the proliferation marker Ki67 has shown a significant correlation with relapse-free survival, suggesting a role in prediction for measurement of Ki67 and other dynamic markers of response. Analysis of multiple gene expression changes over a short treatment period may also have potential clinical utility for prediction of response.     

The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase.

1. The oxygen kinetics of purified beef heart cytochrome c oxidase were investigated. 2. The effect of addition of various fixed concentrations of the inhibitors CO, HN3, HCOOH, HCN and H2S on the double reciprocal plot of respiration rate against oxygen concentration was studied. 3. CO is strictly competitive, azide and formate are uncompetitive, and cyanide and sulfide are non-competitive inhibitors towards oxygen. 4. Binding constants for the various inhibitors from secondary plots of the oxygen kinetics at pH 7.4 are: CO: Ki = 0.32 micronM, azide: Ki = 33 micronM; formate: Ki = 15 mM; cyanide: Ki = 0.2 micronM and sulfide: Ki = 0.2 micronM. 5. The possible significance of these results in the elucidation of the reaction mechanism is discussed.