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The oxidase form of xanthine dehydrogenase (XO; EC 1.1.3.22) has been purified approximately 200-fold from rat liver extracts using a three-step process of heat treatment, ammonium sulfate precipitation, and chromatography on benzamidine-Sepharose. The purified enzyme showed only minor contamination when analyzed by gel electrophoresis under either native or sodium dodecyl sulfate (SDS)-denatured conditions and appears to be intact based on its subunit size on SDS-polyacrylamide gel electrophoresis, its N-terminal amino acid sequence, and its ability to be converted to the NAD-dependent dehydrogenase form (XD; EC 1.1.1.204) by incubation with dithiothreitol. Isoelectric focusing analysis showed that the purified enzyme consists of two major, enzymatically active isoforms with average pI values of 6.13 and 6.23 and a minor enzymatically active isoform with an average pl value of 6.07. A similar purification of XD was achieved by preincubating the partially purified oxidase with dithiothreitol prior to affinity chromatography on benzamidine-Sepharose. The effects of benzamidine on the kinetic properties of purified rat XO were characterized at pH 8 and 9 and were compared to those of bovine milk XO. Benzamidine was found to be a weak competitive inhibitor of the purified rat enzyme with Ki values of 30 and 10 mM at pH 8 and 9, respectively. In contrast, the Ki values for benzamidine with bovine XO were more than 10-fold greater. The findings presented in this study show that benzamidine is a competitive inhibitor of XO and that affinity chromatography on benzamidine-Sepharose provides a simple, rapid, and effective means of purifying both the oxidase and dehydrogenase forms of rat XO.
We have synthesized N1-substituted benzamidines and poly-benzamidines with the aim to produce antitumor drugs retaining differential biological properties with respect to unsubstituted compounds. Antiproliferative activity on in vitro cultured human leukemic cells was exhibited by N1-substituted poly-benzamidines, while N1-substituted benzamidines were found to retain very low antitumor effects. Furthermore, our results suggest that N1-substituted benzamidines and some of poly-benzamidines exhibit low activity on trypsin and kallikrein. Taken together these data indicate that some N1-substituted poly-benzamidines could be of interest for experimental antitumor therapy, since are likely to retain low side effects due to alteration of proteinase activity.
Formation and dissociation of the benzamidine:beta-trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine:beta-trypsin complex minus the free proteinase) is 1.75 mM-1 cm-1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the beta-trypsin:BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 +/- 0.5 degree C. Under all the experimental conditions the beta-trypsin:BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine:beta-trypsin:BPTI adduct (7). The difference between the kinetic processes of beta-trypsin:BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S1 subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.
Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.
The inhibitory effect of mono-, bis-, tris- and tetra-benzamidine structures (benzamidine, DAPP, TAPB and TAPP, respectively) on the catalytic properties of human alpha-, beta- and gamma-thrombin (alpha-, beta- and gamma-thrombin, respectively) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C). The affinity of DAPP, TAPB and TAPP for alpha- and beta-thrombin is higher than that found for benzamidine association around neutrality, converging in the acidic pH limb; in contrast, benzamidine, DAPP, TAPB and TAPP show the same value of the association inhibition constant (Ki; M-1) for gamma-thrombin over the whole pH range explored. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to alpha-, beta- and gamma-thrombin, as well as for DAPP, TAPB and TAPP association to gamma-thrombin reflects the acidic-pK shift, upon inhibitor binding of a single ionizing group. On the other hand, values of Ki for DAPP, TAPB and TAPP binding to alpha- and beta-thrombin appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues over the same pH range. By considering molecular models of the serine proteinase:inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).
The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine beta-trypsin (beta-trypsin), human alpha-thrombin (alpha-thrombin) and porcine pancreatic beta-kallikrein-B (beta-kallikrein-B) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (Ki, M-1) for beta-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for alpha-thrombin and beta-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to beta-trypsin, alpha-thrombin and beta-kallikrein-B as well as for DAPP, TAPB and TAPP association to beta-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for DAPP, TAPB and TAPP binding to alpha-thrombin and beta-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to beta-trypsin, alpha-thrombin and beta-kallikrein-B has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).
We compared the effectiveness of two techniques involving the use of the enzyme trypsin to activate inactive renin in human plasma. Both these methods were developed to optimize activation with trypsin by preventing the possible destruction of activated renin by trypsin itself. In one method, an antitryptic agent such as benzamidine is added to plasma, concomitantly with trypsin (liquid phase). In the other a low concentration of Sepharose-bound (immobilized) trypsin is used. In six plasma samples we have found that trypsin (1.5 mg/ml) activation, with or without benzamidine (0.8 mg/ml), yielded similar values of activated renin (11.0 +/- 2.7 vs. 11.3 +/- 2.3 ng/ml/hr). However, the addition of immobilized trypsin to pool plasma pretreated with trypsin plus benzamidine caused a further increase in plasma renin activity (PRA); in contrast, the addition of trypsin and benzamidine to pool plasma pretreated with immobilized trypsin caused a decrease in PRA. In 17 plasma samples from patients with essential hypertension we found that the inactive renin values were always higher after treatment with immobilized trypsin than with trypsin plus benzamidine (9.0 +/- 0.7 vs. 6.1 +/- 0.5 ng/ml/hr, P less than 0.01); moreover, there was a positive correlation between the differences in the values of inactive renin measured with the two methods and the values obtained with immobilized trypsin (r = 0.64, P less than 0.01). Therefore, the activation with immobilized trypsin is more effective than that with liquid-phase trypsin, alone or in combination with benzamidine, in converting inactive renin in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
A series of substituted benzamidines has been examined for their inhibitory activity against the human serine proteases--trypsin, thrombin, plasmin, and C1s, a subunit of the first component of complement. The inhibition constants obtained for each enzyme were correlated with physical-chemical properties of the substituent group using the quantitative structure-activity relationship approach. This analysis indicated that plasmin and C1s are very similar in their interactions with substituted benzamidines. The binding of benzamidines in both enzymes was affected by electron donation from the substituent and its hydrophobicity. Thrombin-benzamidine interaction was affected only by the hydrophobicity of the substituent. Trypsin displayed a complex interaction with substituted benzamidines, and interaction was dependent on molar refractivity and molecular weight. Certain substituents deviated significantly from the interactions predicted by the analysis. These compounds, the (m- and p-amidinophenyl)pyruvic acids, when analyzed by computer modeling, suggested that direct interaction between the substituent and the enzyme surface is important in assessing the effect of substituent groups on inhibitory activity.
A series of N-[[(dialkylamino)alkoxyl]phenyl]benzamidines was synthesized and evaluated for hypoglycemic activity in the glucose-primed rat. Structure-activity relationship indicated that N'-phenyl-N-[4-[2(diisopropylamino)-ethoxy]phenyl]benzamidine dihydrobromide (7), N'-(4-chlorophenyl)-N-[4-[2-(diisopropylamino)ethoxy]phenyl]-benzamidine dihydrochloride (31), and N'-phenyl-N-[4-[(diisopropylamino)propoxy]phenyl]benzamidine dihydrobromide (11) are some of the more interesting compounds. A comparison of these hypoglycemic agents with classical standards (tolazamide, phenformin, and buformin) in several experimental models showed that the benzamidines seem to combine in one molecule some of the biological activities of the beta-cytotrophic sulfonylureas and some of the activities of the biguanides.
Considerable circumstantial evidence has been provided by in vitro studies that tryptase (EC 3.4.21.59), a neutral serine proteinase stored in large amounts in mast cell granules, may play an important pathogenetic role in mast cell-dependent diseases. However, a definitive role has not yet been ascribed to this trypsin-like enzyme with restricted substrate specificity as natural or synthetic inhibitors of tryptase applicable for in vivo studies are not available so far. Therefore, we have studied structure-activity relationships for inhibition of tryptase by benzamidine derivatives, compounds known to be potent inhibitors of various trypsin-like enzymes. Among the benzamidine derivatives 4-amidinophenylpyruvic acid exerts a striking inhibitory activity with a Ki of 0.71 mumol/l. Several additional inhibitors of tryptase with Ki values in the micromolar range were found among bis-benzamidines. Derivatives of N alpha-arylsulfonyl-omega-amidinophenyl-alpha-aminoalkylcarboxylic acids are only weak inhibitors of tryptase, although members of this group are potent and selective inhibitors of several other trypsin-like enzymes. Comparison of the inhibition of tryptase and trypsin revealed that the affinities of the benzamidine derivatives to both proteinases are closely correlated (correlation coefficient r = 0.702; n = 37; p < 0.001). These results demonstrate that 4-amidinophenylpyruvic acid may be useful as a pharmacologic tool for the investigation of the (patho)physiological role of tryptase. In addition, benzamidine derivatives may be applicable to probe the active site topography of tryptase isoenzymes.
After administration of benzamidine (1) or benzamidoxime (2), respectively, to rats and rabbits, plasma from rats and rabbits as well as urine from rats were examined for the presence of benzamidoxime (2) or benzamidine (1). Some of the samples were worked-up directly and the others after enzymatic pretreatment with beta-glucuronidase or arylsulfatase, respectively. HPLC analysis was employed for the detection of the metabolites. After administration of 1, an in vivo N-hydroxylation of an amidine to an amidoxime was demonstrated for the first time. The metabolite 2 could only be detected after enzymatic cleavage of the glucuronide or sulfate, respectively, and only in plasma at a low concentration. After administration of benzamidoxime (2), on the other hand, benzamidine (1) was detected in very high concentrations in all biological samples. Benzamidine was present in the free state but indications for glucuronidization and sulfatation were also detectable. These investigations suggest that the benzamidoxime (2) formed by an in vivo N-hydroxylation undergoes ready retro-reduction but that further transformations of the metabolite 2, such as conjugation to a glucuronide or a sulfate, respectively, prevent complete back reaction. Furthermore, benzamide (3) could be detected as a transformation product in urine after administration of either 1 or 2.
The genotoxic potentials of benzamidine and benzamidoxime were determined to study the toxicological relevance of the metabolic N-oxygenation (N-hydroxylation) of benzamidines to benzamidoximes. Benzamidoxime induced DNA single-strand breaks (in rat hepatocytes) and DNA amplification in SV40-transformed hamster cells. In the experiments performed, benzamidine itself was only marginally positive in the hepatocyte/DNA single-strand break assay. Since these cells possess an intact metabolization apparatus, the biological activities may be attributed to toxic and genotoxic metabolites formed by biotransformation. In the Salmonella typhimurium mutagenicity test (TA 98 and TA 100) benzamidoxime alone exhibited a low mutagenicity in the TA 98 strain in the presence of rabbit liver S-9 fractions. These results permit recognition of the metabolic N-hydroxylation of benzamidines to benzamidoximes as a process to toxication. Indirect evidence for the formation of a glucuronide of benzamidoxime has been obtained from in vitro experiments, but it could not be established that this process was a decisive factor in the genotoxicity of benzamidoxime.
The enzymic N-oxidation of a series of N-unsubstituted basic benzamidines (I) to a new type of metabolite, the amidoximes (II), is reported. Rabbit liver homogenates (9000 g supernatant) were used as enzyme source, and metabolites were identified by t.l.c. and mass spectral analysis using synthetic reference compounds. The microsomal NADPH- and oxygen-dependent hydroxylation of benzamidines was not detected after incubation of benzamidine in the presence of SKF 525-A, a known inhibitor of cytochrome P-450. Neither benzamidine or p-methoxybenzamidine is a good substrate for purified microsomal FAD-containing mono-oxygenase.
1. A simple and fast h.p.l.c. analysis of benzamidoxime formed by microsomal N-hydroxylation of benzamidine is presented which is well suited for the determination of the N-oxygenation activity of microsomal enzymes. 2. Optimal reaction conditions were determined. The apparent Km and Vmax values were, respectively, 1.61 mM and 0.38 nmol benzamidoxime/min per mg protein. 3. The effects of the inducers phenobarbital, 3-methylcholanthrene and benzamidine itself on hepatic benzamidine metabolizing activity in rabbits were determined. 4. Neither superoxide anion nor hydrogen peroxide is directly involved in the N-hydroxylation reaction. 5. The direct involvement of cytochrome P-450 in the N-hydroxylation of benzamidine is supported by the observation that inhibitors of cytochrome P-450, in particular carbon monoxide, markedly decreased the rate of N-oxygenation.
Findings on the pharmacology of derivatives of benzamidine characterized as potent competitive inhibitors of the clotting enzyme thrombin were presented including data on their toxicity. Bis-benzamidines and amidinophenylalanine amides were shown to exert pharmacodynamic effects in intact animals and isolated preparations (hypotension; influence on smooth muscle reactions to serotonin and histamine). Studies with 14C-4-amidinophenylpyruvic acid and 3H-N alpha-tosyl-(3-amidino)-phenylalanine piperidide on the pharmacokinetics in rabbits showed that benzamidine derivatives are suited, in principle, for use as anticoagulants in vivo. Pharmacokinetic properties of individual thrombin inhibitors have to be taken into account in order to reach and maintain adequate plasma levels. The antithrombotic effect of benzamidine-type thrombin inhibitors in animals experiments is directly related to their antithrombin activity.
Previous investigations have provided evidence for the participation of the cytochrome P-450 (P-450) enzyme system in the established N-hydroxylation of benzamidine to benzamidoxime by microsomal fractions from rabbit liver homogenates. In the present investigation, a representative mixture of P-450 isoenzymes was first isolated from the livers of untreated rabbits and then, together with purified NADPH-P-450 reductase, successfully used in a reconstituted enzyme system for the N-hydroxylation of benzamidine. In order to identify the participating isoenzyme, the P-450 mixture was separated by preparative high performance liquid chromatography on an anion exchange column. A P-450 fraction was obtained that was able to transform benzamidine with a specific activity > 3-fold higher than that of the P-450 mixture. The electrophoretic and spectral properties, as well as the inhibition by monoclonal antibodies against P-450 IIC3, show that the isolated P-450 fraction must consist of one or more variants of the isoenzyme P-450 IIC3. By means of reconstitution experiments with highly purified variants of P-450 IIC3 from rabbit liver and with purified variants of P-450 IIC expressed by recombinant Escherichia coli, the participation of the two variants P-450 IIC3 (6 beta H) and P-450 IIC3 (6 beta L) in the N-hydroxylation of benzamidine was unequivocally confirmed.
At pH 7.4 neither benzamidine (1) is ring-hydroxylated nor benzamidoxime (2) is N-hydroxylated, reduced or ring-hydroxylated by aerobic incubations with microsomal fractions (12000 g supernatant, microsomes) of rabbit liver homogenates and NADPH. Products of hydrolytic processes are also not detected. A very long incubation period and a pH 6.3 are necessary for the detection of a slight reduction of benzamidoxime (2) to benzamidine (1). Results are obtained by use of synthetic reference material and by newly developed HPLC methods. Thus, kinetic studies of the microsomal N-hydroxylation of benzamidine (1) to benzamidoxime (2) in the presence of N-methylbenzamidine (3) performed at pH 7.4 are not influenced by other transformations and provide evidence for the involvement of the same isoenzyme of cytochrome P-450 for both the N-hydroxylation of 1 and the N-dealkylation of 3.