Copolymers of glutamic acid and tyrosine are potent inhibitors of oocyte casein kinase II.

Polypeptides rich in glutamic acid are strong inhibitors purified from isolated nuclei of Xenopus laevis oocytes of casein kinase II. The presence of tyrosine in these peptides greatly enhances their inhibitory capacity. Using casein as a substrate, copolyglu:tyr (4:1) has an I50 value of 20 nM, 250 fold lower than that of polyglutamic acid which is 5 microM. A similar large difference is observed when a synthetic peptide is used as substrate. The inhibition of copolyglu:tyr is competitive with casein and can be completely reversed by high ionic strength. The relative inhibitory capacity of the polypeptides tested, in descending order, is copolyglu:tyr (4:1) greater than copolyglu:tyr (1:1) greater than polyglu greater than copolyglu:phe (4:1) greater than copolyglu:ala (6:4) greater than copolyglu:leu (4:1). The high affinity for tyrosine-containing acid peptides is shared by rat liver and yeast casein kinase II so that it seems to be a general property of these enzymes.     

New inhibitors of protein kinase CK2, analogues of benzimidazole and benzotriazole

Derivatives of 4,5,6,7-tetrabromobenzotriazole (TBBt) and 4,5,6,7-tetrabromobenzimidazole (TBBi) with IC(50) in the low micromolar range and with high selectivity belong to the most promising inhibitors of protein kinase CK2 (casein kinase 2). Treatment of various cell lines with TBBt, TBBi or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) affected cell viability with simultaneous induction of apoptosis. The inhibitory activity of newly synthesized hydroxyalkyl derivatives of TBBi and TBBt depends on the length of the alkyl chain. The hydroxypropyl substituted derivatives show higher or similar inhibitory activity than the parent compounds when tested with human protein kinase CK2. To test the distribution of this class of compounds in mammals, [(14)C] TBBi was synthesized.     

Lauryl gallate inhibits the activity of protein tyrosine kinase c-Src purified from human platelets

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu,Na,Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.     

Lauryl Gallate Inhibits the Activity of Protein Tyrosine Kinase c-Src Purified from Human Platelets

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu.Na, Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.     

Carbonic anhydrase inhibitors. Design of anticonvulsant sulfonamides incorporating indane moieties

A series of aromatic sulfonamides incorporating indane moieties were prepared starting from commercially available 1- and 2-indanamine, and their activity as inhibitors of two carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I and II was studied. The new sulfonamides incorporating acetamido, 4-chloro-benzoyl, valproyl, tetra-, and pentafluorobenzoyl moieties acted as very potent inhibitors of the slow red blood cell isozyme hCA I (K(i)s in the range of 1.6-8.5 nM), which usually has a lower affinity for such inhibitors, as compared to isozyme II. Some derivatives also showed excellent hCA II inhibitory properties (K(i)s in the range of 2.3-12 nM), but the anticonvulsant activity of these sulfonamides was rather low as compared to that of other sulfonamide/sulfamate CA inhibitors, such as methazolamide. Furthermore, the 2-amino/acetamido-indane-5-sulfonic acids prepared during this work also showed interesting CA inhibitory properties, with inhibition constants in the range of 43-89 nM against the two isozymes, being among the most potent sulfonic acid CA inhibitors reported so far.     

Carbonic anhydrase inhibitors: aromatic and heterocyclic sulfonamides incorporating adamantyl moieties with strong anticonvulsant activity

A series of aromatic/heterocyclic sulfonamides incorporating adamantyl moieties were prepared by reaction of aromatic/heterocyclic aminosulfonamides with the acyl chlorides derived from adamantyl-1-carboxylic acid and 1-adamantyl-acetic acid. Related derivatives were obtained from the above-mentioned aminosulfonamides with adamantyl isocyanate and adamantyl isothiocyanate, respectively. Some of these derivatives showed good inhibitory potency against two human CA isozymes involved in important physiological processes, CA I, and CA II, of the same order of magnitude as the clinically used drugs acetazolamide and methazolamide. The lipophilicity of the best CA inhibitors was determined and expressed as their experimental log k' IAM and theoretical ClogP value. Their lipophilicity was propitious with the crossing of the blood-brain barrier (log k' > IAM > 1.35). The anticonvulsant activity of some of the best CA inhibitors reported here has been evaluated in a MES test in mice. After intraperitoneal injection (30 mg kg(-1)), compounds A8 and A9 exhibited a high protection against electrically induced convulsions (> 90%). Their ED50 was 3.5 and 2.6 mg kg(-1), respectively.     

Superactivation of thermolysin by acylation with amino acid N-hydroxysuccinimide esters.

Synthesis of a series of active N-hydroxysuccinimide esters of aliphatic and aromatic amino acids has yielded a new class of reagents for the covalent modification of proteolytic enzymes such as thermolysin. The activities of aliphatic acyl amino acid thermolysins are from 1.7 to 3.6 times greater than that of the native enzyme when hydrolyzing durylacryloyl-Gly-Leu-NH2, the substrate employed most widely. By comparison, the aromatic acylamino acid derivatives are "superactive," their activities being as much as 70-fold greater. Apparently, the aromatic character of the amino acid introduced is a critical variable in the determination of the functional response. The increased activity is completely restored to that of the native enzyme by deacylation with nucleophiles, such as hydroxylamine, and the rate of restoration of native activity is a function of the particular acyl group incorporated. Preliminary evidence regarding the chemical properties of the modified enzyme suggests that tyrosine, rather than lysine, histidine, or arginine, may be the residue modified. The functional consequences of successive modification with different reagents, moreover, indicate that each of them reacts with the same protein residue. The competitive inhibitors beta-phenyl-propionyl-Phe and Zn-2+ do not prevent modification with these active esters. Hence, the site(s) of their inhibitory action differ(s) from that at which modification occurs. The structure of the substrate is also a significant variable which determines the rate at which each acyl amino acid thermolysin hydrolyzes peptides. Depending on the particular substrate, the activity of aromatic derivatives can be as much as 400-fold greater than that of the native enzyme, and the resultant activity patterns can be ordered in a series characteristic for each enzyme derivative.     

Polyhalogenobenzimidazoles: synthesis and their inhibitory activity against casein kinases

A series of novel polyhalogenated benzimidazoles have been prepared by exhaustive bromination of a variety of 2-substituted benzimidazoles. The efficacy of both new compounds and a number of their previously described cognates as inhibitors of casein kinases CK1, CK2 and G-CK was investigated. The type of N-1 alkyl substituent as well as introduction of a polyfluoroalkyl moiety at position 2 did not markedly influence the inhibitory efficacy toward CK2 of the respective 4,5,6,7-tetrabromobenzimidazole derivatives which conversely were almost ineffective toward CK1 and G-CK. However, 4,5,6,7-tetrabromobenzimidazoles substituted at position 2 with either chlorine, bromine or sulfur atom, while manifesting a still considerable inhibitory activity against CK2 (IC(50) in the 0.49-0.93 microM range) proved to be potentially powerful inhibitors also against CK1 (IC(50) in the 18.4-2.2 microM range).     

Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis).

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.     

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic anhydrase inhibitors. N-cyanomethylsulfonamides--a new zinc binding group in the design of inhibitors targeting cytosolic and membrane-anchored isoforms

A series of N-cyanomethyl aromatic sulfonamides and bis-sulfonamides was prepared by reaction of arylsulfonyl halides with aminoacetonitrile. The obtained derivatives incorporated various aryl moieties, such as 4-halogeno/alkyl/aryl/nitro-substituted-phenyl, pentafluorophenyl or 2-naphthyl. Moderate inhibitory activity was detected for some compounds against the cytosolic human isoform II of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA II, with inhibition constants of 90, 180 and 560n M for the 4-nitrophenyl-, 4-iodophenyl- and pentafluorophenyl-N-cyanomethylsulfonamides, respectively. Other derivatives acted as weak inhibitors of isoforms hCA I (KIs of 720 nM-45 microM), hCA II (KIs of 1000-9800 nM) and hCA IX (KIs of 900-10200 nM). Thus, the N-cyanomethylsulfonamide zinc binding group is less effective than the sulfonamide, sulfamate or sulfamide ones for the design of effective CA inhibitors.     

Carbonic anhydrase inhibitors: synthesis of sulfonamides incorporating dtpa tails and of their zinc complexes with powerful topical antiglaucoma properties

Reaction of diethylenetriamino pentaacetic acid (dtpa) dianhydride with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded bis-sulfonamides containing metal-complexing, polyamino-polycarboxylic acid moieties in their molecule. The corresponding mono-sulfonamide derivatives of dtpa were also obtained by an alternative method, from the free acid. Zn(II) complexes of these new sulfonamides were then prepared. Many of these derivatives showed nanomolar affinity towards isozymes I, II and IV of carbonic anhydrase (CA). Some of the best inhibitors were applied as 2% water solutions/suspensions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed.     

Protein phosphorylation in Rhodnius prolixus oocytes: identification of a type II casein kinase

A protein kinase activity in chorionated oocytes of Rhodnius prolixus phosphorylates in vitro vitellin (VT), the major yolk protein. Phosphatase inhibitors including NaF, sodium vanadate, beta-glycerophosphate and okadaic acid did not alter the protein phosphorylation profile to a visible extent. Among the exogenous protein substrates tested, casein was readily phosphorylated, but histones were not. Several different protein kinase activators, including cAMP, Ca2+ plus calmodulin, Ca2+ plus diolein and phosphatidylserine, were added to the reaction media but spermidine was the only effective one, inducing a 2-fold increase in the phosphorylation of VT. A strong inhibition was obtained with nanomolar levels of heparin. The enzyme could also accept GTP as the phosphate donor instead of ATP. These properties identify the major protein kinase activity as a type II casein kinase (CK II). The pH dependence and the effects of mono- and divalent cations on VT phosphorylation were also studied. Gel filtration revealed only one peak of protein kinase activity, with a molecular mass of 170 K, similar to values previously reported in the literature for CK IIs from other organisms.     

Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I,II, IX and XII

Imine derivatives were obtained by condensation of sulfanilamide with substituted aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride, leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (transmembrane, tumor-associated enzymes). We noted that the compounds incorporating secondary amine moieties showed a better inhibitory activity against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors were detected, whereas the activity against hCA I was less potent. The secondary amines incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the facile preparation and possibility to explore a vast chemical space.     

Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isoforms I and II and transmembrane,tumor-associated isoforms IX and XII with boronic acids

A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K_I of 3.7–4.5 μM, whereas the remaining derivatives showed inhibition constants in the range of 6.0–1560 μM for hCA I and of 6.0–1050 μM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K_Is of 7.6–12.3 μM) whereas the arylalkenyl and aryl–alkyl derivatives generally showed weaker inhibitory properties (K_Is of 34–531 μM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)₂ moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.     

Casein kinase activity in etiolated Cucumis sativus cotyledons

Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the α catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons.     

Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity.

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionate through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.     

Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II

A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.     

Carbonic anhydrase inhibitors: synthesis and inhibition against isozymes I, II and IV of topically acting antiglaucoma sulfonamides incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido moieties

Sulfonamides incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido moieties in their molecules were prepared by reaction of cis-5-norbornene-endo-2,3-dicarboxylic anhydride with aromatic/heterocyclic sulfonamides possessing free amino, hydrazino, or imino groups. Some of these compounds showed very good CA II and CA IV inhibitory properties, with affinities for the enzymes in the low nanomolar range. Some of the most active CA II inhibitors reported here have been formulated as aqueous solutions for topical administration as antiglaucoma agents in normotensive rabbits. Some of the derivatives incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido and aromatic sulfonamide moieties (as sodium salts) showed effective and longer lasting intraocular pressure (IOP) lowering as compared to dorzolamide, a widely used topical antiglaucoma drug. Compounds incorporating cis-5-norbornene-endo-2,3-carboximido moieties, although stronger in vitro CA inhibitors as compared to the corresponding cis-5-norbornene-endo-3-carboxy-2-carboxamido-;derivatives, showed no topical IOP lowering properties, probably due to their very poor water solubility.     

Carbonic anhydrase inhibitors. N-Cyanomethylsulfonamides—a new zinc binding group in the design of inhibitors targeting cytosolic and membrane-anchored isoforms

A series of N-cyanomethyl aromatic sulfonamides and bis-sulfonamides was prepared by reaction of arylsulfonyl halides with aminoacetonitrile. The obtained derivatives incorporated various aryl moieties, such as 4-halogeno/alkyl/aryl/nitro-substituted-phenyl, pentafluorophenyl or 2-naphthyl. Moderate inhibitory activity was detected for some compounds against the cytosolic human isoform II of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA II, with inhibition constants of 90, 180 and 560 nM for the 4-nitrophenyl-, 4-iodophenyl- and pentafluorophenyl-N-cyanomethylsulfonamides, respectively. Other derivatives acted as weak inhibitors of isoforms hCA I (K_Is of 720 nM–45 μM), hCA II (K_Is of 1000–9800 nM) and hCA IX (K_Is of 900–10200 nM). Thus, the N-cyanomethylsulfonamide zinc binding group is less effective than the sulfonamide, sulfamate or sulfamide ones for the design of effective CA inhibitors.