Benzamidine Research Articles

Accelerated Screening of Protein-Ligand Interactions via Parallel T2-Weighted 19F-MRI.

In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a 19F-MRI coil. With this setup, we show that ligand binding can be detected by T2-weighted 19F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing 19F-T2-weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations-all recorded in parallel-the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein-ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques.

Benzamidine Conjugation Converts Expelled Potential Active Agents into Antifungals against Drug-Resistant Fungi.

Fungal infections present a growing global public health concern, necessitating the development of novel antifungal drugs. However, many potential antifungals, particularly the expelled potential active agents (EPAAs), are often underestimated owing to their limitations in cellular entry or expulsion by efflux pumps. Herein, we identified 68 EPAAs out of 2322 candidates with activity against a Candida albicans efflux pump-deficient strain and no inhibitory activity against the wild-type strain. Using a novel conjugation strategy involving benzamidine (BM) as a mitochondrion-targeting warhead, we successfully converted EPAAs into potent antifungals against various urgent-threat azole-resistantCandida strains. Among the obtained EPAA-BM conjugates, IS-2-BM (11) exhibited excellent antifungal activities and induced negligible drug resistance. Furthermore, IS-2-BM prevented biofilm formation, eradicated mature biofilms, and exhibited excellent therapeutic effects in a murine model of systemic candidiasis. These findings provide a promising strategy for increasing the possibilities of discovering more antifungals.

Deciphering the canonical blockade of activated Hageman factor (FXIIa) by benzamidine in the coagulation cascade: A thorough dynamical perspective.

The experimental inhibitory potency of benzamidine (BEN) paved way for further design and development of inhibitors that target β-FXIIa. Structural dynamics of the loops and catalytic residues that encompass the binding pocket of β-FXIIa and all serine proteases are crucial to their overall activity. Employing molecular dynamics and post-MD analysis, this study sorts to unravel the structural and molecular events that accompany the inhibitory activity of BEN on human β-FXIIa upon selective non-covalent binding. Analysis of conformational dynamics of crucial loops revealed prominent alterations of the original conformational posture of FXIIa, evidenced by increased flexibility, decreased compactness, and an increased exposure to solvent upon binding of BEN, which could have in turn interfered with the essential roles of these loops in enhancing their procoagulation interactions with biological substrates and cofactors, altogether resulting in the consequential inactivation of FXIIa. A sustained interaction of the catalytic triad residues and key residues of the autolysis loop impeded their roles in catalysis which equally enhanced the inhibitory potency of BEN toward β-FXIIa evidenced by a favorable binding. Findings provide essential structural and molecular insights that could facilitate the structure-based design of novel antithrombotic compounds with enhanced inhibitory activities and low therapeutic risk.

Investigation of the formation of drug-drug cocrystals and coamorphous systems of the antidiabetic drug gliclazide

The antidiabetic drug gliclazide (GLZ) has a slow absorption rate and a low bioavailability due to its poor solubility. GLZ is often prescribed along with an antihypertensive, as many diabetic patients have coexistent hypertension. Cocrystallization and coamorphization are attractive strategies to enhance dissolution rates and to reduce the number of medications a patient has to take. In this work the formation of cocrystals and coamorphous systems of GLZ with various antihypertensive drugs was studied, namely chlorothiazide (CTZ), hydrochlorothiazide (HTZ), indapamide (IND), triamterene (TRI) and nifedipine (NIF) as well as benzamidine (BZA) as a model for the amidine pharmacophore. TRI, IND and HTZ were found to form coamorphous systems with GLZ that are stable for at least six months at 22 ± 2 °C and 56% relative humidity. Coamorphous GLZ-TRI is also stable in dissolution medium. Coamorphization of GLZ-TRI with 15% sodium taurocholate gave a viable coamorphous formulation with an enhanced dissolution rate. Comilling of GLZ with BZA and cocrystallization from solution gave the amorphous and crystalline salt, respectively and the X-ray structure is reported. During attempts to obtain X-ray suitable cocrystals crystals of Na+GLZ− and IND 0.5H2O were obtained. Redetermination of the published structure of IND 0.5H2O revealed a unit cell with the length of the a axis doubled, a different space group and no disorder. Liquid-assisted grinding of a 1:1 mixture of GLZ and IND indicated the transformation of IND to a new solid-state form, while GLZ remained unaltered. Milling- and heating-induced solid-state transformations of IND are discussed.

Induction of proteases secreted by fusarium oxysporum F. SP. Dianthi in the presence of carnation root cell walls. Biochemical characterization of a serine protease

We determined that protease activity is secreted by Fusarium oxysporum f. sp. dianthi race 2 (Fod) in vitro. The cell wall extracted from resistant and susceptible cultivars of carnation (Dianthus caryophyllus L.) was used as an inducer. The activity increased in the presence of the two cultivars, and higher levels were detected with the susceptible cultivar. One of the three proteases secreted by Fod was purified using ion exchange (DEAE-cellulose), gel filtration (Sephadex G-75), and affinity chromatographies (Benzamidine Sepharose 6B). Using protease inhibitors, the purified enzyme was classified as a trypsin-like serine protease that was inhibited by phenylmethanesulfonyl fluoride, diisopropylfluorophosphate and the soybean trypsin inhibitor. In addition, the enzyme was biochemically characterized and determined to have an optimal pH of 8.0, an optimal temperature of 50°C, stability from pH 6 to 8, temperature stability between 40 to 60°C, a molecular weight of 54 kDa, and an isoelectric point of 4.4. The values of the Km and Vmax kinetic constants were found to be 0.31 mg/ml and 24.7 µmol/min, respectively.

Revealing the mechanism, pathways and kinetics of UV254nm/H2O2-based degradation of model active sunscreen ingredient PBSA

Transformation byproducts and pathways for the degradation of 2-phenylbenzimidazole-5-sulfonic acid (PBSA), a widely used organic active sunscreen agent, by UV254nm/H2O2 advanced oxidation process (AOP) were elucidated by mass spectrometry. Fourteen byproducts were identified, including environmentally hazardous benzamide (BD) and benzamidine (BZD). Beside of aromatic ring opening, desulfonation (−80Da) and hydroxylation (+16Da) were also two main reaction pathways. Degradation kinetics were compared with two PBSA structurally related compounds 2-phenylimidazole (2-PIZ) and benzimidazole (BZ) to estimate the application feasibility of the studied AOP. No apparent photolysis was noticed while the degradation of 1μM pollutants in the presence of 1mM H2O2 followed pseudo-first-order kinetics, with their observed rate constant (kobs) in the order of 2-PIZ>PBSA>BZ>BD>BZD, which is consistent with their respective second-order rate constant with hydroxyl radical. However, at high pollutant and oxidant concentrations, i.e., 50μM and 5mM, respectively, light attenuation contributed significantly to the change in kobs in the following manner, 2-PIZ∼BZ>PBSA∼BD>BZD. SO42− and NH4+ were the major inorganic mineral ions monitored in this study. Release of SO42− from PBSA degradation system reached 70% at 190min. At the same time interval, NH4+ were ⩽50% for all pollutants studied. Ultimate mineralization of PBSA, 2-PIZ, BZ, BD and BZD in terms of %TOC at 190min was found to be 23%, 38%, 45%, 57% and 62%, respectively. UV/H2O2 was shown to be efficient for the removal of organic sunscreen agents from water, however, special care toward the formation of environmentally hazardous byproducts should be considered.

Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues.

Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Glue n -BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Glue n -BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10-BA and Glue29-BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG-BA). Most importantly, Glue10-BA inhibited the protease activity of trypsin 13-fold more than TEG-BA. In sharp contrast, m Glue27-BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG-BA for trypsin, was inferior even to TEG-BA for trypsin inhibition.

Molecular mechanism analysis of Gloydius shedaoensis venom gloshedobin interaction with inhibitors by homology modeling

Snake venom thrombin-like enzymes (SVTLEs) are widely applied in the treatment of thrombotic diseases, however, the molecular mechanism of its inhibition by synthetic and natural proteinaceous inhibitors is not yet understood. Here we investigated effects of protease inhibitors including phenylmethylsulfonil fluoride (PMSF), benzamidine (BMD) and its derivates on the activity of recombinant gloshedobin, a SVTLE from the snake Gloydius shedaoensis. The molecular inhibition mechanism was postulated by separately docking inhibitors into three-dimensional model of gloshedobin using protein C activator from Agkistrodon contortrix contortrix venom (ACC-C, which bear 78% identity with gloshedobin) as template. The analysis indicated that the strongest inhibitor, PMSF, was via a covalent bond with the catalytic Ser195, while other inhibitors showing weaker inhibitory activity were via hydrogen bond with Ser195 or non-catalytic residues.

Kinase buffer with benzamidine, 1X

Kinase buffer with benzamidine, 1X

Specific conformational changes of plasminogen induced by chloride ions, 6-aminohexanoic acid and benzamidine, but not the overall openness of plasminogen regulate, production of biologically active angiostatins

The overall conformation of plasminogen depends upon the presence of anions and molecules such as AHA (6-aminohexanoic acid) and BZ (benzamidine). The purpose of the present study was to determine the effect of conformation on the initial and secondary cleavages of plasminogen to generate active angiostatins. Plasminogen was digested with the physiologically relevant neutrophil elastase in one of the four Tris/acetate buffers: buffer alone or buffer plus NaCl, AHA or BZ. The initial cleavage of Glu1-plasminogen was much slower in the tight NaCl-induced alpha-conformation, fastest in the intermediate BZ-induced beta-conformation and intermediate both in the control and in the AHA-induced open gamma-conformation. Although the buffer system determined the relative amounts of the initial cleavage products, the same four cleavage sites were utilized under all conditions. A fifth major initial cleavage within the protease domain was observed in the presence of BZ. N-terminal peptide cleavage required for angiostatin formation occurred as either the initial or the secondary cleavage. Angiostatins were generated fastest in the presence of BZ and slowest in the presence of NaCl. Both the initial and secondary cleavages were affected by the modifying agents, indicating that they influence the conformation of both Glu-plasminogen and the initial cleavage products. The angiostatins produced under the different conditions inhibited proliferation of human umbilical-vein endothelial cells. These results suggest that plasminogen conversion into active angiostatins is dependent more on the specific conformation changes induced by the various modifying reagents rather than on the overall openness of the molecule.

Corin, a Transmembrane Cardiac Serine Protease, as a Pro-Atrial Natriuretic Peptide Convertase

P92 Atrial natriuretic peptide (ANP) is a cardiac hormone that reduces high blood pressure by promoting salt excretion, decreasing blood volume and relaxing vessel tension. It is implicated in major cardiovascular diseases such as hypertension and congestive heart failure. In cardiomyocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated serine protease. Recently, we cloned a novel cardiac serine protease, corin, that has the predicted structure of a type II transmembrane protein. Northern and in situ hybridization detected corin mRNA in the heart where its expression was most abundant in cardiomyocytes of the atrium. Corin mRNA was also detected in developing kidney and bones. The overall expression pattern of corin mRNA was very similar to that of ANP, leading to our hypothesis that corin is the pro-ANP convertase. To test this hypothesis, we constructed expression vectors for both human corin and pro-ANP. Recombinant corin and pro-ANP were expressed in human 293 cells. Effects of corin on pro-ANP processing was examined by western analysis. The results showed that human corin, but not control proteases prothrombin and hepsin, converted pro-ANP to ANP in the cell-based experiments. To determine sequence specificity of corin-mediated cleavage in pro-ANP, we constructed a mutant pro-ANP R98G in which the predicted cleave site residue Arg98 was replaced with a Gly. In co-transfection experiments, corin processed wild-type pro-ANP but not mutant pro-ANP R98G, indicating that corin-mediated cleavage in pro-ANP is highly sequence specific. We also examined effects of protease inhibitors on processing of pro-ANP by corin. Our results showed that the activity of corin was inhibited by aprotinin, benzamidine and leupeptin but not soybean trypsin inhibitor. Similar effects of these proteases on pro-ANP processing were reported previously. Thus, corin matches all known characteristics of the long-sought pro-ANP convertase. Identification of corin as the pro-ANP convertase suggests a new regulatory mechanism for the ANP-mediated pathway that is important for controlling blood pressure.

Trypsinogen activation in rat pancreatic acinar cells hyperstimulated by caerulein

Inappropriate trypsinogen activation is discussed as an early intracellular event in the secretagogue-induced model of acute pancreatitis. However, the mechanisms by which trypsinogen is activated are not well characterized. In the present work, trypsinogen activation was studied in intact acinar cells using bis-(CBZ-arginyl)-Rhodamine 110 [(CBZ-Arg) 2-Rho 110] as a cell-permeant substrate for trypsin and also independently via the formation of trypsinogen activation peptide (TAP). Preincubation with 10 nM caerulein increased the Rho 110-substrate cleavage more than threefold. This proteolytic activity was fully sensitive to a benzamidine (BA)-type serine protease inhibitor. The appearance of enzymatic activity was paralleled by the formation of TAP. The lack of effect of the high-molecular soybean trypsin inhibitor indicates an intracellular substrate cleavage. The cathepsin B inhibitor CA-074 prevented neither the caerulein-induced formation of TAP nor the (CBZ-Arg) 2-Rho 110-cleaving activity. BA inhibited the Rho 110-substrate cleavage and significantly reduced the TAP formation. These results show that trypsinogen activation in caerulein-hyperstimulated acinar cells may occur independently of the activity of cathepsin B. On the contrary, the effect of BA suggests the role of a serine protease in trypsinogen activation.

Formation of pyrimidine‐5‐carboxaldehydes, 1‐aminoethylene‐2,2‐dicarboxaldehyde and 2‐amino‐4‐anilino‐1,3,5‐triazine from triformylmethane and amidines

AbstractTriformylmethane (1) reacted with benzamidine, guanidine and S‐methylisothiourea to give the corresponding 2‐substituted pyrimidines‐5‐carboxaldehydes. However, reactions of 1 with acetamidine (or for‐mamidine) and phenylbiguanide gave the unexpected 1‐aminoethylene‐2,2‐dicarboxaldehyde (5) and 2‐amino‐4‐anilino‐1,3,5‐triazine (9), respectively.

Trifluoromethylated Pyrimidines Starting from b-Trifluoroacetyl-lactams, -lactone and -cyclanone

A synthesis of trifluoromethylated pyrimidines from beta-trifluoroacetyl-lactams and -benzolactams is accomplished by reaction with benzamidine as bis-nucleophile. This condensation is also extended to cyclic trifluoromethylated 1,3-diketones and 3-aroyl-2-pyrrolidinones.

ChemInform Abstract: Heterocyclic Compounds from 2‐(Alkoxycarbonylcyanomethylene)‐1,3‐dioxolanes

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Lidocaine metabolite formation as a measure of liver function in hepatic transplantation

Transformation byproducts and pathways for the degradation of 2-phenylbenzimidazole-5-sulfonic acid (PBSA), a widely used organic active sunscreen agent, by UV254nm/H2O2 advanced oxidation process (AOP) were elucidated by mass spectrometry. Fourteen byproducts were identified, including environmentally hazardous benzamide (BD) and benzamidine (BZD). Beside of aromatic ring opening, desulfonation (−80 Da) and hydroxylation (+16 Da) were also two main reaction pathways. Degradation kinetics were compared with two PBSA structurally related compounds 2-phenylimidazole (2-PIZ) and benzimidazole (BZ) to estimate the application feasibility of the studied AOP. No apparent photolysis was noticed while the degradation of 1 μM pollutants in the presence of 1 mM H2O2 followed pseudo-first-order kinetics, with their observed rate constant (kobs) in the order of 2-PIZ > PBSA > BZ > BD > BZD, which is consistent with their respective second-order rate constant with hydroxyl radical. However, at high pollutant and oxidant concentrations, i.e., 50 μM and 5 mM, respectively, light attenuation contributed significantly to the change in kobs in the following manner, 2-PIZ ∼ BZ > PBSA ∼ BD > BZD. SO42− and NH4+ were the major inorganic mineral ions monitored in this study. Release of SO42− from PBSA degradation system reached 70% at 190 min. At the same time interval, NH4+ were ⩽50% for all pollutants studied. Ultimate mineralization of PBSA, 2-PIZ, BZ, BD and BZD in terms of %TOC at 190 min was found to be 23%, 38%, 45%, 57% and 62%, respectively. UV/H2O2 was shown to be efficient for the removal of organic sunscreen agents from water, however, special care toward the formation of environmentally hazardous byproducts should be considered.

Inhibition of in-vitro fertilization of mouse gametes by proteinase inhibitors.

Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD). 4'-nitrophenyl 4-guanidinobenzoate (NPGB), and 4'-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards alpha-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 microM for BD, 27 microM for ABD, 35 nM for MUGB, and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.

Antifertility activity of systemically administered proteinase (acrosin) inhibitors

Low molecular weight, synthetic proteinase inhibitors that inhibit spermassociated acrosin, were released systemically in female mice at a constant rate from minipumps. The release was timed so that, after mating, the minipumps were depleted of inhibitor before blastocyst implantation took place. Three of the inhibitors: 4-aminobenzamidine (ABD), 4-nitrophenyl-4-guanidinobenzoate (NPGB) and 4-methylumbelliferone-4-guanidinobenzoate (MUGB) caused a 50% decrease in fertility, the last two at very low concentrations. The fourth inhibitor, benzamidine (BD), which is also the weakest inhibitor of mouse acrosin and in vitro fertilization 1, had no effect. These results show that at least one of the processes leading to fertilization or early blastogenesis, is dependent on proteolytic activity and that the systemic application of proteinase inhibitors inhibits conception. MUGB possessed low toxicity and a high margin of safety, encouraging the development of phenol derivatives of guanidinobenzoic acid as contraceptive agents.

694 DIFFERENCES IN TRYPSIN SUBSTRATES AND INHIBITORS ON PMN CHEMOTAXIS AND PHAGOCYTOSIS

There are many apparent similarities between PMN chemotaxis and phagocytosis. Previous investigators have suggested that serine esterases are involved in the chemotactic response in so far as a variety of inhibitors and substrate analogues inhibit chemotaxis. We studied the effect of various trypsin inhibitors and a substrate on human PMN chemotaxis and phagocytosis. Reversible inhibition of chemotaxis was demonstrated with the low molecular weight trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE). The low molecular weight active site titrant, nitrophenyl-P-guanidino benzoate (NPGB) revealed irreversible inhibition of chemotaxis, whereas the low molecular weight inhibitor, benzamidine (BENZ) and the high molecular weight inhibitor, soybean trypsin inhibitor (STI), caused reversible inhibition. The various inhibitors and the substrate gave varying inhibition profiles. The following concentrations gave complete inhibition of chemotaxis: BAEE, 100mM; NPGB, 0.5mM; BENZ, 10mM; STI, 5mM. Neither the active site titrant, NPGB, nor the low molecular weight inhibitor, BENZ, affected phagocytosis as determined by the Maaloe technique at concentrations completely inhibiting chemotaxis. These results may suggest different underlying mechanisms initiating chemotaxis and phagocytosis.

Evidence against bifunctionality of benzamidine

Evidence is presented which is against the reported bifunctionality of benzamidine.