Effects of L-1-methyl-histidine and the muscle dipeptides carnosine and anserine on the activities of muscle calpains

1. Carnosine, anserine and L-1-methyl-histidine activated muscle calpain II assayed at 2.5 mM Ca2+. 2. At 5 microM Ca2+, none of these compounds activated calpain II sufficiently to bring its activity up to the level measured at 2.5 mM Ca2+. 3. Carnosine increased, whereas both anserine and L-1-methyl-histidine decreased the inhibitory effect of calpastatin on calpain II. 4. These results suggest that although the compounds are not potent activators of calpain II, the ratio of the dipeptides in muscle may have an effect on calpain II-calpastatin interaction.     

The biological activity of the histidine-containing diketopiperazines cyclo(His-Ala) and cyclo(His-Gly)

Two cyclic dipeptides, cyclo(His-Ala) and cyclo(His-Gly,) were synthesized from their linear counterparts and their structures elucidated using standard elucidation techniques. Molecular modeling and predictive NMR results indicated that the majority of energetically favourable conformers adopted a boat conformation with respect to the diketopiperazine ring. Cyclo(His-Ala), at concentrations of 100 microM inhibited the growth, in vitro, of various cancer cell lines, including HT-29, MCF-7 and HeLa carcinoma cells while cyclo(His-Gly) inhibited the growth of MCF-7 cells. While the antibacterial potential of these two compounds was limited, both cyclic dipeptides significantly inhibited the growth of C. albicans. Both compounds at a concentration of 100 microM resulted in a decrease in heart rate, coronary flow rate and left ventricular systolic pressure in the isolated rat heart. Inhibition of thrombin, amounting to a 63.3% and 36.7% reduction in the rate of fibrin formation, was noted for cyclo(His-Ala) and cyclo(His-Gly), respectively. While cyclo(His-Ala) showed no notable effects on platelet aggregation, cyclo(His-Gly) significantly inhibited both pathways tested with greatest effects on thrombin-induced platelet aggregation, yielding an IC(50) of 0.0662 mM (R(2)=0.989). The results of the anticancer and hematological studies indicate that histidine-containing diketopiperazines have potential as a novel group of cytotoxic agents with antithrombotic effects.     

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.     

Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against oral pathogens

Microorganisms embedded in a biofilm are significantly more resistant to antimicrobial agents and the defences of the human immune system, than their planktonic counterpart. Consequently, compounds that can inhibit biofilm formation are of great interest for novel therapeutics. In this study, a screening approach was used to identify novel cyclic dipeptides that have anti-biofilm activity against oral pathogens. Five new active compounds were identified that prevent biofilm formation by the cariogenic bacterium Streptococcus mutans and the pathogenic fungus Candida albicans. These compounds also inhibit the adherence of microorganisms to a hydroxylapatite surface. Further investigations were conducted on these compounds to establish the structure–activity relationship, and it was deduced that the common cleft pattern is required for these molecules to act effectively against biofilms.     

Increasing structure diversity of prenylated diketopiperazine derivatives by using a 4-dimethylallyltryptophan synthase

To create structural diversity of prenylated diketopiperazine derivatives, acceptance of cyclic dipeptides was tested using FgaPT2, a prenyltransferase from Aspergillus fumigatus, which catalyses the conversion of l-tryptophan to 4-dimethylallyl-l-tryptophan. It could be shown that seven tryptophan-containing cyclic dipeptides were accepted by FgaPT2 at high protein concentrations and regiospecifically converted to their C4 prenylated derivatives. The structures of the enzymatic products were elucidated by NMR and LC-MS analyses. This substrate promiscuity of a dimethylallyltryptophan synthase towards cyclic dipeptides increases the potential of the fungal indole prenyltransferases as tools for the production of biologically active compounds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides

Fourteen tryptophan-containing cyclic dipeptides 1a-14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b-14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses. The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.     

Structure and catalytic mechanism of a cyclic dipeptide prenyltransferase with broad substrate promiscuity

Fungal indole prenyltransferases (PTs) typically act on specific substrates, and they are able to prenylate their target compounds with remarkably high regio- and stereoselectivity. Similar to several indole PTs characterized to date, the cyclic dipeptide N-prenyltransferase (CdpNPT) is able to prenylate a range of diverse substrates, thus exhibiting an unusually broad substrate promiscuity. To define the structural basis for this promiscuity, we have determined crystal structures of unliganded CdpNPT and of a ternary complex of CdpNPT bound to (S)-benzodiazepinedione and thiolodiphosphate. Analysis of the structures reveals a limited number of specific interactions with (S)-benzodiazepinedione, which projects into a largely hydrophobic surface. This surface can also accommodate other substrates, explaining the ability of the enzyme to prenylate a range of compounds. The location of the bound substrates suggests a likely reaction mechanism for the conversion of (S)-benzodiazepinedione. Structure-guided mutagenesis experiments confirm that, in addition to (S)-benzodiazepinedione, CdpNPT can also act on (R)-benzodiazepinedione and several cyclic dipeptides, albeit with relaxed specificity. Finally, nuclear magnetic resonance spectroscopy demonstrates that CdpNPT is a C-3 reverse PT that catalyzes the formation of C-3β prenylated indolines from diketopiperazines of tryptophan-containing cyclic dipeptides.     

Enzymatic conversion-based method for screening cyclic dipeptide-producing microbes

We developed a method for screening cyclic dipeptide-producing microbes by enzymatic conversion. In this method, cyclic dipeptides are detected by the combination of: (i) conversion of cyclic dipeptides to dehydro cyclic dipeptides by cyclo(Leu-Phe) oxidase and (ii) detection of the dehydro derivative by UV spectrophotometry using TLC or HPLC analysis based on the absorbance change caused by the conversion. Using this method, the actinomycete strain A8 was isolated as a cyclic dipeptide-producing strain. The cyclic dipeptides were purified from the microbial extract by enzymatic detection-guided fractionation, and their structures were determined to be cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) by spectroscopic methods.     

大黄鱼共生菌Bacillus coagulans LL1103的代谢产物研究

[目的]研究大黄鱼共生菌Bacillus coagulans LL1103的次生代谢产物及其抑菌活性。[方法]利用色谱层析技术对B.coagulans LL1103发酵液乙酸乙酯和正丁醇提取物进行分离纯化;运用波谱技术鉴定化合物的结构;采用微量肉汤稀释法对化合物进行抗菌活性测定。[结果]从大黄鱼内生菌B.coagulans LL1103发酵液中分离并鉴定9个环二肽类化合物,分别为:(1)环(4-羟基-脯氨酸-亮氨酸)、(2)环(异亮氨酸-丙氨酸)、(3)环(脯氨酸-缬氨酸)、(4)环(脯氨酸-苯丙氨酸)、(5)环(脯氨酸-亮氨酸)、(6)环(甘氨酸-丙氨酸)、(7)环(脯氨酸-甘氨酸)、(8)环(脯氨酸-丙氨酸)和(9)环(络氨酸-甘氨酸)。活性评价显示化合物3、7和9对大肠埃希菌(Escherichia coli)具有较强抑制作用,最低抑菌浓度(minimal inhibitory concentration,MIC)分别为8.0、4.0和16.0μg/mL。[结论]从大黄鱼共生菌B.coagulans LL1103的发酵液中分离得到3个对大肠埃希菌(E.coli)具有较好抑制活性的化合物。     

Synthesis of [beta-(4-pyridyl-1-oxide)-L-alanine4]-angiotensin I as a potential suicide substrate for protein-tyrosine kinases

An asymmetric synthesis of [beta-(4-pyridyl-1-oxide)-L-alanine4]-angiotensin I (1a), which is a potential suicide substrate (mechanism-based inhibitor) for protein-tyrosine kinases, has been performed. Deprotonation of 6 with n-butyllithium in THF gave the anion 7, which was alkylated with 4-(chloromethyl)pyridine-1-oxide to afford intermediate 9 as a crystalline solid. Hydrolysis of 9 afforded a mixture of 11 and 12 in a ratio of 96:4 as estimated by conversion to the diastereomeric dipeptides 13 and 14 followed by HPLC analysis. The 96:4 mixture of 11 and 12 was used in the solid phase synthesis of the target angiotensin analog 1a and its diastereomer 1b, which were separated and tested for inhibitory activity against two thymocyte protein-tyrosine kinases: p40 and p56lck. Neither peptide displayed significant inhibitory activity toward p40 and both served as weak competitive inhibitors of p56lck.     

Exploration of cornea permeable calpain inhibitors as anticataract agents

To explore cornea permeable calpain inhibitors, four compounds displaying different characteristics were designed and synthesized based on the known potent calpain inhibitor, peptidyl aldehyde SJA6017. Two approaches were adopted; an improvement in the physicochemical properties, and conversion of the active aldehyde. The water-soluble peptidyl aldehyde 1 containing a pyridine ring at the P3 site showed a modest inhibition against calpains and an improvement of corneal permeability in comparison with SJA6017. Replacement of the aldehyde of SJA6017 by an alpha-ketoamide provided compound 2 that was approximately equipotent with SJA6017, but it was extremely water-insoluble. However, compound 3, in which the aldehyde was converted into a cyclic hemiacetal, proved to be a less potent calpain inhibitor than SJA6017, but demonstrated excellent transcorneal permeability. Further modification generating the cyclic hemiacetal 4 containing a thiourea linker between the P3 and P2 sites exhibited potent inhibitory activities, high cornea permeability and excellent efficacy in the rat lens culture cataract model.     

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.     

On the mechanisms of oligopeptide reactions in solution and clay dispersion.

Mechanisms of the reactions of representative dipeptides (Gly2, Gly-Ala), oligopeptides (Gly3, Gly4) and the polypeptide (poly-Gly)n) in solution and clay suspensions at 85 degrees C were investigated. The reaction products and their yields were analysed and determined by means of HPLC. Interestingly, hydrolysis, where water molecules act as the reactant, was not the main reaction, even for oligopeptides. Formation of cyclic dipeptides prevailed in the reactions of dimers as well as oligopeptides. The breakdown of oligopeptide molecules proceeded via an intramolecular cyclization reaction. For example, the reaction of Gly3 led to the formation of equal amounts of cyclic dipeptide, c(Gly)2 and Gly. The presence of clay (montmorillonite) significantly increased yields in the reactions of dipeptides but it did not have much effect on the reactions of oligopeptides. However, an opposite effect of clay, protection of poly(Gly)n against decomposition, was proven.     

Identification of small molecule inhibitors of beta-amyloid cytotoxicity through a cell-based high-throughput screening platform

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of beta-amyloid (Abeta) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Abeta. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Abeta with IC(50) values of <10 microM and showed little or no toxicity at concentrations up to 30 microM, none inhibit the calpain enzyme directly.     

海洋真菌与细菌发酵物中的环二肽

从红树林真菌1356攻株发酵液中分离到4个环二肽,它们是环Pro-Tyr,环Pro-Ile,环Gly-Phe和环Ala-Phe。另从海洋细菌110菌株发酵液中分离到5个环二肽,它们是环Leu-Ile,环Val-Leu,环Ala-Val,环Ala-Ile和环Ala-Leu。这些环二肽多数是新发现的微生物产物。本比较了分离的环二肽与已经报道的环二肽在来源、结构、功能等方面的区别。     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.     

Thermally induced cyclization of L‐isoleucyl‐L‐alanine in solid state: Effect of dipeptide structure on reaction temperature and self‐assembly

Thermal treatment of short‐chain oligopeptides is able to initiate the process of their self‐assembly with the formation of organic nanostructures with unique properties. On the other hand, heating can lead to a chemical reaction with the formation of new substances with specific properties and ability to form structures with different morphology. Therefore, in order to have a desired process, researcher needs to find its temperature range. In the present work, cyclization of _L‐isoleucyl‐_L‐alanine dipeptide in the solid state upon heating was studied. Kinetic parameters of this reaction were estimated within the approaches of the nonisothermal kinetics. The correlation between side chain structure of dipeptides and temperature of their cyclization in the solid state was found for the first time. This correlation may be used to predict the temperature, at which dipeptide self‐assembly changes to chemical reaction. The differences in self‐assembly of linear and cyclic dipeptides were demonstrated using atomic force microscopy. The effect of dipeptide concentration in a source solution and an organic solvent used on self‐assembly of dipeptides was shown. The new information obtained on the thermal properties and self‐assembly of linear and cyclic forms of _L‐isoleucyl‐_L‐alanine may be useful for the design of new nanomaterials based on oligopeptides, as well as for the synthesis of cyclic oligopeptides.     

Identification of novel neuroprotective agents using pharmacophore modeling

In addition to its endocrine function, for which it was named, thyrotropin-releasing hormone (TRH) has substantial neuroprotective actions as well as other physiological effects. We have developed a number of modified TRH analogues as well as cyclic dipeptides structurally related to a major metabolic product of TRH, which have enhanced neuroprotective activity but none of the other major physiological effects of TRH. The extensive structure-activity data developed with these compounds were used to develop a pharmacophore model. Subsequently, a web-based pharmacophore searching program was used to query several large three-dimensional databases. Of the 219 compounds identified whose structures met the pharmacophore model, 15 were chosen for study in a classical model of neuronal cell death in vitro; five of these, 2-6, showed neuroprotective activity. Thus, pharmacophore modeling developed from neuroprotective small peptides can be used to identify novel lead compounds as neuroprotective agents.