Pyridinium derivatives of histamine are potent activators of cytosolic carbonic anhydrase isoforms I, II and VII

A series of positively-charged derivatives has been prepared by reaction of histamine with substituted pyrylium salts. These pyridinium histamine derivatives were investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely the human isoforms hCA I, II and VII. Activities from the subnanomolar to the micromolar range were detected for these compounds as activators of the three isoforms, confirming the validity of current and previous designs. The substitution pattern at the pyridinium ring was the main factor influencing activity, the three isoforms showing different structural requirements for good activity, related with the number of pyridinium substituting groups and their nature, among various alkyl, phenyl and para-substituted styryl moieties. We were successful in identifying nanomolar potent and selective activators for each isozyme and also activators with a relatively good activity against all isozymes tested--valuable lead compounds for physiology and pathology studies involving these isozymes.     

Theoretical improvement of the specific inhibitor of human carbonic anhydrase VII

The selectivity of a known arylsulfonamides inhibitor for two isozymes II and VII of human carbonic anhydrases (hCAs) was studied by homology modeling, molecular docking and molecular dynamics methods. The results show that the selectivity of the inhibitor for two isozymes is due to the different side chain lengths between N67 of hCA II and Q64 of hCA VII. One more methene group in the side chain of Q64 of hCA VII makes it possible to form the hydrogen bond with the bromide atom of the known inhibitor. From the point of view, the modification to the known inhibitor was performed to obtain an inhibitor with higher selectivity. The complex conformations of the new designed inhibitor and two isozymes designate the formation of the hydrogen bond between the newly added group (hydroxypropyl group) and Q64 of hCA VII but N67 of hCA II. The results of the binding free energy from the MM/PBSA approach also prove the selectivity improvement of the new inhibitor in comparison with the known inhibitor. The work will help the design of the isozyme-specific inhibitors of hCA VII.     

Synthesis and some reactions of sulfides of the thiophene series

By the action of four equivalents of sodium in liquid ammonia on the bis(alkylmercapto)thiophenes I, II, and IX we have obtained the corresponding dimercaptothiophenes VII, IX, and VIII, which were characterizied in the form of their dibenzoyl derivatives XI–XIII. The dimercaptothiophenes VII and VIII and the o-alkylthiothiophenethiols III and IV form the internally complex compounds XIV–XVII with metal acetates.     

Kinetic isotope effects for concerted multiple proton transfer: a direct dynamics study of an active-site model of carbonic anhydrase II

The rate constant of the reaction catalyzed by the enzyme carbonic anhydrase II, which removes carbon dioxide from body fluids, is calculated for a model of the active site. The rate-determining step is proton transfer from a zinc-bound water molecule to a histidine residue via a bridge of two or more water molecules. The structure of the active site is known from X-ray studies except for the number and location of the water molecules. Model calculations are reported for a system of 58 atoms including a four-coordinated zinc ion connected to a methylimidazole molecule by a chain of two waters, constrained to reproduce the size of the active site. The structure and vibrational force field are calculated by an approximate density functional treatment of the proton-transfer step at the Self-Consistent-Charge Density Functional Tight Binding (SCC-DFTB) level. A single transition state is found indicating concerted triple proton transfer. Direct-dynamics calculations for proton and deuteron transfer and combinations thereof, based on the Approximate Instanton Method and on Variational Transition State Theory with Tunneling Corrections, are in fair agreement and yield rates that are considerably higher and kinetic isotope effects (KIEs) that are somewhat higher than experiment. Classical rate constants obtained from Transition State Theory are smaller than the quantum values but the corresponding KIEs are five times larger. For multiple proton transfer along water bridges classical KIEs are shown to be generally larger than quantum KIEs, which invalidates the standard method to distinguish tunneling and over-barrier transfer. In the present case, a three-way comparison of classical and quantum results with the observed data is necessary to conclude that proton transfer along the bridge proceeds by tunneling. The results suggest that the two-water bridge is present in low concentrations but makes a substantial contribution to proton transport because of its high efficiency. Bridging structures containing more water molecules may have lower energies but are expected to be less efficient. The observed exponential dependence of the KIEs on the deuterium concentration in H(2)O/D(2)O mixtures implies concerted transfer and thus rules out substantial contributions from structures that lead to stepwise transfer via solvated hydronium ions, which presumably dominate proton transfer in less efficient carbonic anhydrase isozymes.     

Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases

Multifunctional silica nanoparticles decorated with fluorescent and sulfonamide carbonic anhydrase (CA) inhibitors were prepared and investigated as multivalent enzyme inhibitors against the cytosolic isoforms hCA I and II and the transmembrane tumor‐associated ones hCA IX and XII. Excellent inhibitory effects were observed with these nanoparticles, with K_I values in the low nanomolar range (6.2–0.67 nM ) against all tested isozymes. A significant multivalency effect was seen for the inhibition of the monomeric enzymes hCA I and II compared to the dimeric hCA IX and hCA XII isoforms, where no multivalent effect was observed, suggesting that the multivalent binding is occurring through enzyme clustering.     

Carbonic anhydrase inhibitors: X-ray and molecular modeling study for the interaction of a fluorescent antitumor sulfonamide with isozyme II and IX

The X-ray crystal structure of the fluorescent antitumor sulfonamide carbonic anhydrase (CA, EC, 4.2.1.1) inhibitor (4-sulfamoylphenylethyl)thioureido fluorescein (1) in complex with the cytosolic isoform hCA II is reported, together with a modeling study of the adduct of 1 with the tumor-associated isoform hCA IX. Its binding to hCA II is similar to that of other benzesulfonamides, with the ionized sulfonamide coordinated to the Zn2+ ion within the enzyme active site, and also participating in a network of hydrogen bonds with residues Thr199 and Glu106. The scaffold of 1 did not establish polar interactions within the enzyme active site but made hydrophobic contacts (<4.5 A) with Gln92, Val121, Phe131, Val135, Leu198, Thr199, Thr200, and Pro202. The substituted 3-carboxy-amino-phenyl functionality was at van der Waals distance from Phe131, Gly132, and Val135. The bulky tricyclic fluorescein moiety was located at the rim of the active site, on the protein surface, and strongly interacted with the alpha-helix formed by residues Asp130-Val135. All these interactions were preserved in the hCA IX-1 adduct, but the carbonyl moiety of the fluorescein tail of 1 participates in a strong hydrogen bond with the guanidine moiety of Arg130, an amino acid characteristic of the hCA IX active site. This may account for the roughly 2 times higher affinity of 1 for hCA IX over hCA II and may explain why in vivo the compound specifically accumulates only in hypoxic tumors overexpressing CA IX and not in the normal tissues. The compound is in clinical studies as an imaging tool for acute hypoxic tumors.     

New Histamine-Related Five-Membered N-Heterocycle Derivatives as Carbonic Anhydrase I Activators

A series of histamine (HST)-related compounds were synthesized and tested for their activating properties on five physiologically relevant human Carbonic Anhydrase (hCA) isoforms (I, II, Va, VII and XIII). The imidazole ring of HST was replaced with different 5-membered heterocycles and the length of the aliphatic chain was varied. For the most interesting compounds some modifications on the terminal amino group were also performed. The most sensitive isoform to activation was hCA I (K_A values in the low micromolar range), but surprisingly none of the new compounds displayed activity on hCA II. Some derivatives (1, 3a and 22) displayed an interesting selectivity for activating hCA I over hCA II, Va, VII and XIII.     

The synthesis of naphthothiopyranopyranones and naphthothiopyranopyranthiones

The synthesis of fused tetracyclic naphthothiopyranopyranones from dihydronaphthothiopyranones I or II has been studied. Compounds I or II have been cyclised in good yield to the corresponding dioxaborin difluoride complexes III, IV, XIII and XIV by treatment with acetic or propionic anhydride and boron trifluoride etherate. These complexes and the Vilsmeier reagent reacted to produce fused tetracyclic 3-substituted naphthothiopyranopyranones V, VI, XV or XVI . The reaction of dioxaborin difluoride complexes III or IV with dimethylthioformamide (DMTF) afforded dimethylaminovinyldioxaborin difluoride complexes IX or X . Treatment of IX or X with hydrochloric acid solution gave naphthothiopyranopyranones VII or VIII . The reaction of VII, VIII, XV or XVI with DMTF/acetic anhydride yielded new products, which was identified as naphthothiopyranopyranthions XI, XII, XVII or XVIII .     

Molecular dynamics simulations of the mononuclear zinc-beta-lactamase from Bacillus cereus.

Herein, we report molecular dynamics simulations of the mononuclear form of the Bacillus cereuszinc-beta-lactamase. We studied two different configurations which differ in the presence of a zinc-bound hydroxide or a zinc-bound water and in the protonation state of the essential His210 residue. Contacts of the catalytically important residues (Asp90, His210, Cys168, etc.) with the zinc center are characterized by the MD analyses. The nature of the Zn-OH(2) --> His210 proton transfer pathway connecting the two configurations was studied by means of QM calculations on cluster models while the relative stability of the two configurations was estimated from QM/MM calculations in the enzyme. From these results, a theoretical model for the kinetically active form of the B. cereus metalloenzyme is proposed. Some mechanistic implications and the influence of mutating the Cys168 residue are also discussed.     

Synthesis of novel thiazoles bearing hydrazine,thiosemicarbazide, thiazole,and thiazolidinone moieties

Thiazolecarboxylate esters (I) and (II) react with hydrazine hydrate to give the acid hydrazides (III) and (IV), which then react with KSCN and PhNCS to give high yields of the thiosemicarbazides (V)-(VIII). Cyclocondensation of the thiosemicarbazide (V) with 3-phenyl-3-chloro-2-oxopropionic acid derivatives gives compounds with two thiazole moieties (IX)-(XIV). The reaction of the phenylthiosemicarbazides (VII) and (VIII) with chloroacetyl chloride and (or) chloroacetic acid affords the thiazolidinonethiazoles (XV) and (XVI).Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 12, pp. 2832–2836, December, 1991.     

Orientation and distance dependent chiral discrimination in the first step of the aminoacylation reaction: Integrated molecular orbital and semi-empirical method (ONIOM) based calculation

Aminoacylation is a vital step in natural biosynthesis process of peptide and is the key step in correlating the realm of protein with the RNA world. Incorrect aminoacylation might lead to misacylation of d-amino acid in the tRNA which might cause synthesis of a hetero-peptide rather than natural homopeptide leading to the altered functionality of the peptide. However, the accuracy of this process is remarkable and leads to the attachment of the correct enantiomer of the amino acid with their cognate tRNA. Thus, the chiral discrimination is stringent. In the present work, we presented a combined ONIOM (ab initio/semi-empirical) study of the chiral discrimination in the first step of aminoacylation reaction based on a model of crystal structure of the oligomeric complex of histidyl-tRNA synthetase (HisRS) from Escherichia coli complexed with ATP and histidinol and histidyl-adenylate. The study reveals that the molecular mechanism of the chiral discrimination involves the amino acid, ATP as well as surrounding residues of the synthetase. Several factors are noted to be responsible for discrimination and explain the high level of stereospecificity of the process. The chirality of the amino acid of the substrate and its (principally) electrostatic interaction with the ATP is important for discrimination. The distance and orientational changes involved in the approach of the d-His towards the ATP is energetically unfavorable. The charge distributions on the His and ATP are important for the discrimination. Removal of the charges in the model drastically reduces the discrimination. Restricted nature of the mutual orientation within the cavity of the active site where the His and ATP are located during the change in orientation for the approach to form the adenylate makes the resultant interaction profile as different for l-His and d-His also influences chiral discrimination. The analysis of the transition state structure revealed that alteration of the chirality of the His destabilize the transition state by removing the favorable electrostatic interaction between the Glu-83 and NH₃⁺ group of the His substrate. The proximity of the surrounding residues as present in the active site of the synthetase with the His and ATP (the separation is of nanometer range) has influence of discrimination. The study provides a molecular mechanism of the retention of biological homochirality.     

Quantifying the relative contribution of hydrogen bonding and hydrophobic environments, and coordinating groups, in the zinc(II)-water acidity by synthetic modelling chemistry

Ligands derived from the tripodal N4 ligand tris(pyridylmethyl)amine ((pyCH2)3N, tpa) of general formula (6-RNHpyCH2)nN(CH2py)(3-n)(R = H, n= 1-3 L(1-3); R = neopentyl, n= 1-3 L'(1-3)) were used to elucidate and quantify the magnitude of the effects exerted by hydrogen bonding and hydrophobic environments in the zinc-water acidity of their complexes. The pKa of the zinc-bound water molecule of [(L(1-3))Zn(OH2)]2+ and [(L'(1-3))Zn(OH2)]2+ 1'-3' was determined by potentiometric pH titrations in water (1-3) or water-ethanol (1:1) (1'-3'). The zinc(II) water acidity gradually increases as the number of -NH2 hydrogen bonding groups adjacent to the water molecule increases. Thus, the zinc-bound water of [(L3)Zn(OH2)]2+ and [(tpa)Zn(OH2)]2+ deprotonate with pKa values of 6.0 and 8.0, respectively. The pKa of the water molecule, however, is only raised from 8.0 in [(tpa)Zn(OH2)]2+ to 9.1 in [(bpg)Zn(OH2)]+ (bpa =(pyCH2)2N(CH2COO-)). Moreover, the acidity of the zinc-bound water of several of the five-coordinate zinc(II) complexes with the hydrogen bonding groups is greater than that of four-coordinate [((12)aneN3)Zn(OH2)]2+ (pKa = 7.0). This result shows that the magnitude of the effect exerted by the hydrogen bonding groups can be larger than that induced by changing one neutral by one anionic ligand, and/or even by changing the coordination number of the zinc(II) centre. The X-ray structure of [(L'2)Zn(OH)]ClO4 2' and [(L'3)Zn(OH)]ClO4.CH3CN 3'.CH3CN is reported, and show the neopentylamino groups forming N-H...O hydrogen bonds with the zinc-bound hydroxide. Although, which have hydrogen bonding and hydrophobic groups, have a zinc-bound water more acidic than [(tpa)Zn(OH2)]2+, their pKa is not always lower than that of 1-3. This result suggests that a hydrogen bonding microenvironment may be more effective than a hydrophobic one to increase the zinc-water acidity.     

Carbonic anhydrase inhibitors. Sulfonamide diuretics revisited--old leads for new applications?

Sulfonamide diuretics such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide were tested as inhibitors of the zinc enzyme carbonic anhydrases (CAs, EC 4.2.1.1). These drugs were discovered in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. We prove here that although acting as moderate to weak inhibitors of CA II, all these drugs considerably inhibit other isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar/subnanomolar inhibitors against such isoforms were detected, such as among others metolazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also resolved at high resolution, and the binding of this sulfonamide to the enzyme was compared to that of dichlorophenamide, sulpiride and a pyridinium containing sulfonamide. Indapamide binds to CA II in a manner not seen earlier for any other CA inhibitor, which might be important for the design of compounds with a different inhibition profile.     

Simultaneous determination and pharmacokinetic study of polyphyllin I,polyphyllin II,polyphyllin VI and polyphyllin VII in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC‐MS‐MS

For the first time, a rapid and specific LC‐MS‐MS method has been developed for the analysis of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma. The method was applied to study the pharmacokinetics of Rhizoma Paridis extracts containing polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII. The analysis was carried out on an Agilent Zorbax XDB‐C₁₈ reversed‐phase column (100 × 2.1 mm, 1.8 µm) by isocratic elution with acetonitrile and water (50:50, v/v). The flow rate was 0.25 mL/min. All analytes including internal standards were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were less than 6.66 and 9.15%. The extraction recovery ranged from 95.53 to 104.21% with RSD less than 8.69%. Stability studies showed that polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII were stable in preparation and analytical process. The validated method was successfully used to determine the concentration–time profiles of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII. Copyright © 2012 John Wiley & Sons, Ltd.     

Serendipitous fragment-based drug discovery: ketogenic diet metabolites and statins effectively inhibit several carbonic anhydrases

Acetoacetic acid and R-3-hydroxy-butyric acid (BHB) are "ketone bodies", metabolites produced during the ketogenic diet. We discovered that they inhibit in the submicromolar-micromolar range several carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in relevant physiologic processes such as lipogenesis and tumorigenesis. The BHB fragment is also present in the molecules of most statins, widely used drugs for inhibiting cholesterol biosynthesis through the 3-hydroxy-3-methyl-glutaryl-CoA reductase pathway. Three such statins, atorvastatin, fluvastatin and rosuvastatin, showed submicromolar-low nanomolar inhibition of the fifteen human isoforms hCA I-XIV. Our data point out that in addition to their cholesterol lowering properties, these drugs may exert a therapeutic effect by inhibiting lipogenesis through mitochondrial CA inhibition. The statins are also low nanomolar inhibitors of the tumor-associated isoforms CA IX and XII. Based on the BHB/statin scaffolds, antiepileptic, antiobesity and antitumor compounds with higher affinity for the various CA isoforms involved in epileptogenesis (CA VA, VB, VII), lipogenesis (CA III, CA VA, CA VB) and tumorigenesis (CA IX and CA XII) may be designed.     

Molecular dynamics study of crystalline water ices

The behavior of structures of H₂O crystalline ices Ih, Ic, XI, VII, VIII, VI is studied in molecular dynamics experiment using the potential offered by Poltev and Malenkov. The behavior of the system consisting of one of the two identical interpenetrating, but without any common hydrogen bonds, water frameworks comprising the ice VI structure is also simulated. As a result of simulations, the ice VII structure has collapsed, whereas other systems proved to be stable. The reasons of instability of the ice VII and previously studied ice IV structures in molecular dynamics experiments are discussed. Based on the simulation results of the above-mentioned ices and previous simulation of ices II, III, IX, IV, and XII, the general regularities of dynamic properties of water molecules in crystalline water ices are formulated. Unreliability of results obtained by molecular dynamics in the investigation of self-organizing processes in aqueous systems is shown.     

Evaluation of carbonic anhydrase and paraoxonase inhibition activities and molecular docking studies of highly water-soluble sulfonated phthalocyanines

The investigation of carbonic anhydrase and paraoxonase enzyme inhibition properties of water-soluble zinc and gallium phthalocyanine complexes ( 1 and 2 ) are reported for the first time. The binding of p-sulfonylphenoxy moieties to the phthalocyanine structure favors excellent solubilities in water, as well as providing an inhibition effect on carbonic anhydrase (CA) I and II isoenzymes and paraoxonase (PON1) enzyme. According to biological activity results, both complexes inhibited hCA I, hCA II, and PON1. Whereas 1 and 2 showed moderate hCA I and hCA II (off-target cytosolic isoforms) inhibitory activity (Ki values of 26.09 µM and 43.11 µM for hCA I and 30.95 µM and 33.19 µM for hCA II, respectively), they exhibited strong PON1 (associated with high-density lipoprotein [HDL]) inhibitory activity (Ki values of 0.37 µM and 0.27 µM, respectively). The inhibition kinetics were analyzed by Lineweaver–Burk double reciprocal plots. It revealed that 1 and 2 were noncompetitive inhibitors against PON1, hCA I, and hCA II. These complexes can be more advantageous than other synthetic CA and PON inhibitors due to their water solubility. Docking studies were carried out to examine the interactions between hCA I, hCA II, and PON1 inhibitors and metal complexes at a molecular level and to predict binding energies.     

The Thermal Stability of Ciprofloxacin Complexes with Magnesium(II), Zinc(II) and Cobalt(II)

The thermal behaviour of Mg(II), Zn(II) and Co(II) compounds of ciprofloxacin was studied by thermogravimetry (TG) and differential thermal analysis (DTA) in order to determine or to confirm some structural characteristics of substances. The complexes decompose in two steps: dehydration and pyrolytic decomposition of the anhydrous complexes to form metal oxide or metal fluoride. The dehydration process of one magnesium(II) compound takes place in two steps suggesting a marked difference in the bonding of water molecules. The different bonding mode of the ciprofloxacin molecules in both magnesium compounds leads to different residues of the thermal decompositions. This revised version was published online in August 2006 with corrections to the Cover Date.