Potent trypsin-resistant hGH-RH analogues.

A series of analogues of hGH-RH-(1-29)-NH2 designed to have metabolic stability has been synthesized. Standard Boc-SPPS was employed, modified to permit the guanidinylation of amino side-chains after chain assembly but before release from the resin. [Dat1, Har(11, 12, 20, 21, 29), Ala15, Nle27, Asp28]-, [Dat1, Har(11, 20, 29), Orn12, Ala15, Nle27, Asp28]-, and [Dat1, Gap(11,12, 21, 29), Ala15, Har20, Nle27, Asp28]-hGH-RH-(1-29)-NH2 were completely resistant to trypsin and about 50 times as potent as hGH-RH-(1-29)-NH2 itself when injected subcutaneously in rats. These peptides are candidates for clinical application in the therapy of GH deficiency.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria.

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

A compendium of cyclic sugar amino acids and their carbocyclic and heterocyclic nitrogen analogues

This compendium focuses on functionalised sugar amino acids (SAAs) and their 3- to 6-membered nitrogen heterocyclic and carbocyclic analogues. The main benefit of using SAAs and their related nitrogen and carbon congeners in the production of peptidomimetics and glycomimetics is that their properties can be readily altered via modification of their ring size, chemical manipulation of their numerous functional groups and fine-tuning of the stereochemical arrangement of their ring substituents. These building blocks provide access to hydrophilic and hydrophobic peptide isosteres whose physical properties allow entry to a region of chemotherapeutic space which is still under-explored by medicinal chemists. These building blocks are also important in providing amino acids whose inherent conformational bias leads to predisposition to secondary structure upon oligomerisation in relatively short sequences. These foldamers, particularly those containing ω-amino acids, provide an additional opportunity to expand access to the control of structures by artificial peptides. The synthesis and biological evaluation of these building blocks in glycomimetics and peptidomimetics systems keep expanding the reach of the glycosciences to the medical sciences, provide a greater outlook onto the wide range of cellular functions of saccharides and their derivatives involved and greater insight into the nature of oligosaccharide and protein folding.     

Anticonvulsant activity of topiramate and phenytoin in a rat model of ischemia-induced epilepsy

Topiramate, a structurally novel anticonvulsant, and phenytoin were evaluated in a rat model of ischemia-induced epilepsy. In this model a transient global cerebral ischemia is induced by cardiac compression. By precisely controlling the experimental conditions the procedure causes reproducible neurological deficits that include audiogenic epileptic seizures. The seizures can be broadly separated into three types reflecting the degree of severity: wild running, clonic seizures, and tonic extension seizures of the forelimbs and hindlimbs. Topiramate and phenytoin blocked all three types of seizures. Calculated ED₅₀ values for topiramate 1 hr after oral administration were 8.2, 13.0 and 36.1 mg/kg for blockade of tonic extension seizures, clonic seizures and wild running, respectively. Corresponding ED₅₀ values for phenytoin were 5.0, 10.8 and 20.7 mg/kg. These results support the concept that the anticonvulsant activity of these drugs is due primarily to an ability to block the spread of seizures.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Anticonvulsant effects of magnesium sulfate in hippocampal-kindled rats

The objective of the present study was to determine whether magnesium sulfate has anticonvulsant actions in the hippocampal-kindled rat model of epilepsy. Fully kindled rats received acute intraperitoneal injections of magnesium sulfate (270 mg/kg), phenytoin (20 mg/kg) or saline in random order. Electrical seizure duration, behavioral seizure stage and duration of postictal EEG depression were examined 15, 30 and 60 min after injection. In an additional group of rats, kindled seizures were measured before and after chronic (2 h) intraperitoneal injections of magnesium sulfate versus saline. There was a significant decrease in electrical seizure duration (p<0.01) and behavioral seizure stage (p<0.01) with acute magnesium sulfate injections compared to saline injections. Phenytoin had no statistically significant effects on hippocampal-kindled seizures. Chronic magnesium sulfate treatment significantly reduced behavioral seizure stage at 2, 24, and 48 h postinjection (p<0.05), but did not affect seizure duration. There was a significant time by treatment effect for magnesium sulfate on postictal EEG depression (p<0.01). We conclude that in this model of hippocampal epilepsy-induced (kindled) rats, magnesium sulfate has significant anticonvulsant effects.     

Plant sulfur metabolism--the reduction of sulfate to sulfite

Until recently the pathway by which plants reduce activated sulfate to sulfite was unresolved. Recent findings on two enzymes termed 5'-adenylylsulfate (APS) sulfotransferase and APS reductase have provided new information on this topic. On the basis of their similarities it is now proposed that these proteins are the same enzyme. These discoveries confirm that the sulfate assimilation pathway in plants differs from that in other sulfate assimilating organisms.     

Design and synthesis of cyclic sulfonamides and sulfamates as new calcium sensing receptor agonists

The design, synthesis and calcimimetic properties of various cyclic sulfonamides and sulfamates are described. The latter were prepared from the corresponding o-alkenylarenesulfonamides via copper- or rhodium-catalyzed intramolecular aziridination. The size of the cyclic sulfonamide rings as well as the position of the crucial (R)-naphthylethylamine substituent significantly affected calcimimetic activity. The most active compounds were the six- and seven-membered sulfonamides 30a and 31a and sulfamate 34a.     

Circular-dichroic spectra of vasopressin analogues and their cyclic fragments.

The circular dichroic spectra of [Arg8]vasopressin, [Mpr1, Arg8]vasopressin, [Mpr1, D-Arg8]-vasopressin, pressinamide, deaminopressinamide, tocinamide, deaminotocinamide, [Leu4, D-Arg8]-vasotocin, [Mpr1, Leu4, D-Arg8]vasotocin and [Phe2, Lys8]vasopressin have been studied. All these substances showed a characteristic positive dichroic band at about 225 nm due to the presence of tyrosine in sequence position 2. The intensity of this band was affected by interactions between the tyrosine side-chain and other structural elements in the molecule, such as the Na-amino group, the side-chain of phenylalanine in position 3 and the linear C-terminal peptide. Analysis of the response of this band to structural modifications of the molecule and change in the solvent (particularly comparing neutral aqueous solutions with hexafluoroacetone solutions) allowed some conformational conclusions. The linear C-terminal tripeptide is probably situated over the cyclic portion of the molecule both in vasopressin and oxytocin substances. Its steric interaction with the tyrosine side-chain seems to be particularly efficient in molecules containing D-arginine in position 8. In the vasopressin series the stacking interaction of neighbouring aromatic amino acid residues furthermore limits the conformational freedom of the tyrosine side-chain and also probably distorts the dihedral angles of residues 1-3 in comparison with oxytocin. The interactions of phenylalanine and arginine with tyrosine relatively decrease the conformational effects of the primary amino group. Consequently the local conformation of vasopressin in the region of the tyrosine residue is more rigid and less sensitive to changes in medium than that of oxytocin. The circular dichroic spectra did not show any basic conformational differences in the backbone peptide chain of oxytocin and vasopressin substances. A weak negative disulphide band at about 290 nm could be observed in the spectra of both series of substances.     

A sulfate,sulfite and thiosulfate incorporating system inCandida utilis

Sulfate, sulfite and thiosulfate incorporation in the yeastCandida utilis is inhibited by extracellular sulfate, sulfite and thiosulfate and by sulfate analogues selenate, chromate and molybdate. The three processes are blocked if sulfate, sulfite, thiosulfate, cysteine and homocysteine are allowed to accumulate endogenously. Incorporation of the three inorganic sulfur oxy anions is inactivated by heat at the same rate. Mutants previously shown to be defective in sulfate incorporation are also affected in sulfite and thiosulfate uptake. Revertants of these mutants selected by plating in ethionine-supplemented minimal medium recovered the capacity to incorporate sulfate, sulfite and thiosulfate. These results taken together with previous evidence demonstrate the existence of a common sulfate, sulfite and thiosulfate incorporating system in this yeast.     

Linear and cyclic beta-casomorphin analogues with high analgesic activity.

We investigated the antinociceptive efficacy of casomorphin (CM) derivatives using the vocalization test. Male Wistar rats received chronic microcannulae into the right lateral ventricle. One week later we examined the analgesic effect of CM derivatives 10, 30, 60, and 90 min after intraventricular injection (5 microliters). The analgesic effect was calculated as the individual percent increase in the pain threshold and was compared to controls (saline treatment). The substitution of D-lysine and D-ornithine in position 2 in connection with a cyclization through ring closure of the 2 position side chain amino group to the C-terminal glycine-COOH group resulted in high analgesic potency. The substitution of D-Pro4 was without any effect in the ineffective linear derivatives and decreased the effectiveness in the highly effective cyclic derivatives. The cyclic [D-Orn2]CM-5 and the cyclic [D-Lys2]CM-5 are the CM derivatives with the highest antinociceptive activity. The cyclic [D-Orn2]CM-5 is greater than 1000 times more effective than morphine. We conclude, on the basis of studies of receptor binding and in vitro investigations, that mu receptor activity alone is not responsible for the analgesic activity. The delta receptor and possibly also the kappa receptor could modulate the nociceptive effectiveness.     

Design,Synthesis and Anticonvulsant Activity of the Potent Adenosine Kinase Inhibitor GP3269

Abstract

The pyrrolopyrimidine nucleoside GP3269 (12) was shown to be a potent and selective inhibitor of human adenosine kinase (IC₅₀ = 11 nM) and to exhibit anticonvulsant activity in rats after oral administration. Synthesis of GP3269 was accomplished in 4 steps from 4-chloro-5-iodopyrrolopyrimidine (9) and the protected 5-deoxy-1-α-chlororibose (8) using a base-catalyzed nucleoside coupling reaction and the Suzuki reaction to replace the 5-iodo substituent with phenyl.     

Potent side-chain to side-chain cyclized dermorphin analogues containing a carbonyl bridge.

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method and ring formation was achieved via a ureido group incorporating the side chain amino functions of D-Aaa2 (D-Lys, D-Orn) and Aaa4 (Lys, Orn, Dab, Dap). The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Most showed very high agonist potency in the GPI assay. The peptide containing D-Lys in position 2 and Dab in position 4 was 210 times more active than enkephalin, and that containing Orn and Dab, respectively, was 150 times more active than enkephalin. The latter peptide was also very active in the MVD assay, and showed an IC50 MVD/GPI ratio of 0.816. NMR spectra of selected peptides were recorded, and structural parameters were determined. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. With the help of the NMR spectra each peptide was described as an ensemble of conformations. The conformations have been interpreted with regard to the opioid activities, and comparisons have been made with a model proposed earlier for enkephalin analogues.     

Synthesis of some fully 3' and 4'-C-branched-chain sugar nucleoside analogues.

The synthesis of some new 3'-azido-3'-C-substituted pyrimidine nucleoside analogues is described. The key step is the geminal disubstitution of an appropriated ketone carbohydrate, via the regioselective ring opening of the corresponding tosyl-epoxide derivative.     

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10.

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10 were synthesised and tested in androgen-sterilised constant-oestrus rats. The agonist, [D-Ser(Bu^t)⁶, Azgly¹⁰]-luliberin, induced ovulation at a dose of 6ng/rat i.v., 10μg/rat p.o. and was at least five times as potent as [D-Ser(Bu^t)⁶, des-Gly-NH₂¹⁰, Pro-ethylamide⁹]-luliberin. [D-Ser(Bu^t)⁶, Azgly¹⁰]-luliberin (1μg/rat) also prevented HCG-induced increases in ovarian and uterine weight in immature rats and was a highly potent antitumour agent when given to rats bearing DMBA-induced mammary tumours. The antagonist, [D-Phe², D-Phe⁶, Azgly¹⁰]-luliberin at a dose of 15μg/rat completely inhibited ovulation induced by luliberin (0.5μg/rat), whereas [D-Phe², D-Phe⁶]-luliberin lost activity below 125μg/rat.     

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti‐Leishmania activities. In the present study, the total syntheses of backbone‐cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)^2,15]‐Gm (one disulfide bond) and [Thr^2,6,11,15,d ‐Pro⁹]‐Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu¹ and Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60 °C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N‐terminal amino acid pGlu¹ and the amidated C‐terminal tripeptide Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr^2,6,11,15,d ‐Pro⁹]‐Gm analogue remains the best active Gm‐derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Antiviral activity of some natural and synthetic sugar analogues

A number of natural and synthetic sugar analogues have been tested for their antiviral activity, using an influenza virus strain as a model. Hemagglutinating titres (HA) and cytopathic effect (CPE) were surveyed to estimate the virus production. It was found that introduction of the benzyl group into these sugars generally causes them to become antivirally active. Substitution with methyl, acetyl, uridyl and thiocyanyl groups or derivatization with azido, isopropylidene and benzylidene groups were without effect. All sugars containing the 2-deoxy-2-acetamido group were inactive.     

Antitumoral cyclic peptide analogues of chlamydocin

A series of cyclic tetrapeptides bearing the bioactive alkylating group on an ε-amino-lysyl function have been examined for their antitumoral activity on L1210 and P388 murine leukemia cell lines. One analogue belonging to the chlamydocin family and bearing a β-chloroethylnitrosourea group was found to be potent at inhibiting L1210 cell proliferation and had a higher therapeutic index than the reference compound bis-β-chloroethylnitrosourea (BCNU) on the in vivo P388-induced leukemia model.     

Computer simulations of cyclic enkephalin analogues

Molecular dynamics simulations and energy minimization studies of cyclic enkephalin analogues incorporating retro-inverso modifications have been carried out. The dynamic trajectories are analyzed in terms of the relative mobility of the 14-membered rings, conformational transitions among equilibrium states, and hydrogen-bonding patterns. The cyclization of the molecules reduces the motion of the ring structures substantially. Time-correlated conformational transitions resulting in the reorientation of peptide units are observed. Hydrogen bonds form principally C7 structures. Because of the incorporation of retro-inverso residues, C6 and C8 structures are also formed. Starting conformations for energy minimizations were obtained from the molecular dynamics simulations and from a systematic search of the conformational space available to the molecules. Several minimum energy backbone and side-chain conformations were found for each analogue. The effect of retro-inverso residues on hydrogen-bonding patterns and backbone conformations is discussed.