Synthesis and biological evaluation of norcantharidin analogues: towards PP1 selectivity

Simple modifications to the anhydride moiety of norcantharidin have lead to the development of a series of analogues displaying modest PP1 inhibition (low muM IC(50)s) comparable to that of norcantharidin (PP1 IC(50)=10.3+/-1.37 microM). However, unlike norcantharidin, which is a potent inhibitor of PP2A (IC(50)=2.69+/-1.37 microM), these analogues show reduced PP2A inhibitory action resulting in the development of selective PP1 inhibitory compounds. Data indicates that the introduction of two ortho-disposed substituents on an aromatic ring, or para-substituent favours PP1 inhibition over PP2A inhibition. Introduction of a p-morphilinoaniline substituent, 35, affords an inhibitor displaying PP1 IC(50)=6.5+/-2.3 microM; and PP2A IC(50)=7.9+/-0.82 microM (PP1/PP2A=0.82); and a 2,4,6-trimethylaniline, 23, displaying PP1 IC(50)=48+/-9; and PP2A IC(5) 85+/-3 microM (PP1/PP2A=0.56). The latter shows a 7-fold improvement in PP1 versus PP2A selectivity when compared with norcantharidin. Subsequent analysis of 23 and 35 as potential PP2B inhibitors revealed modest inhibition with IC(50)s of 89+/-6 and 42+/-3 microM, respectively, and returned with PP1/PP2B selectivities of 0.54 and 0.15. Thus, these analogues are the simplest and most selective PP1 inhibitors retaining potency reported to date.     

The first two cantharidin analogues displaying PP1 selectivity

High pressure Diels-Alder reactions of furan and dimethylmaleate, and thiophene and maleimide resulted in two cantharidin analogues, 3 and 6 possessing PP1 selectivity (>40- and >30-fold selectivity) over PP2A. Both compounds exhibited moderate PP1 activity, 3 IC(50) 50 microM and 6 IC(50) 12.5 microM. Interestingly, the corresponding mono-ester derivatives of 3 showed no such selectivity.     

Synthesis of lipid A monosaccharide analogues containing acidic amino acid: exploring the structural basis for the endotoxic and antagonistic activities

For elucidation of the structural and conformational requirements on the endotoxic and antagonistic activity of lipid A derivatives, we designed and synthesized lipid A analogues containing acidic amino acid residues in place of the non-reducing end phosphorylated glucosamine. Definite switching of the endotoxic or antagonistic activity was observed depending on the difference of the acidic groups (phosphoric acid or carboxylic acid) in the lipid A analogues.     

Ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit—Potential anti-metastatic drugs

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.     

The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: rational modifications imparting PP1 selectivity

Based on the results from previously reported molecular modeling analyses of the interactions between the inhibitor microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural modifications intended to impart PP1 selectivity. The synthesis of several first generation analogues followed by inhibition assays revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues is more selective for PP1 than any known small molecule inhibitor.     

Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation

A wide range of piperine analogues has been synthesised in order to undertake a structure-activity study of their ability to stimulate melanocyte proliferation. Results demonstrate that an aromatic ring containing at least one ether function and a carbonyl group containing side chain is essential for this activity. A number of highly active piperine analogues have been identified, for instance 1-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienoic acid methyl ester (5a), 1-E,E-piperinoyl-isobutylamine (4f) and 1-(3,4-methylenedioxyphenyl)-pentanoic acid cyclohexyl amide (20). A selection of analogues has also been evaluated for their effect on melanocyte morphology and melanogenesis. The piperine analogues altered cell morphology by increasing dendrite formation leading to bi-, tri- and quadripolar cells. These same analogues were found to increase total melanin in cell cultures, although melanin content per cell was not significantly altered from control in the presence of these compounds.     

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.     

Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A-subunit of PP2A.

Proteins of approximately 35, 55 and 65kDa were purified from cauliflower extracts by microcystin-Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A-subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5,the casein phosphatase activity of plant PP5 was activated >10-fold by arachidonic acid, with half-maximal stimulation occurring at approximately 100 microM lipid.     

Alanine-scanning mutagenesis of HM-1 killer toxin and the essential residues for killing activity

Each of the aromatic, acidic and basic amino acid residues in HM-1 were separately substituted with alanine by site-directed mutagenesis. The mutant genes were successfully expressed in HM-1 resistant Saccharomyces cerevisiae. HM-1 gene analogues corresponding to the aromatic substitutions resulted in lower production of HM-1 analogues. In the case of the acidic amino acid residue and basic amino acid residue substitutions, some analogues were produced in the same amount as and exhibited similar killing activity to that of the wild type HM-1. But the H35A HM-1 analogue had completely lost the killing activity, and D44A, K21A, K46A, R82A, R85A and R86A HM-1 showed highly decreased killing activities. These results strongly indicate the importance of histidine-35, aspartic acid-44, lysine-21, lysine-46, and C-terminal arginine residues in HM-1 for the killing activity.     

Design and synthesis of carbocyclic versions of furanoid nucleoside phosphonic Acid analogues as potential anti-hiv agents

Novel 5'-norcarbocyclic adenine and guanine phosphonic acid analogues with 6',6'-difluorine moiety were designed and synthesized from commercially available epichlorohydrin 5. A regioselective Mitsunobu reaction successfully proceeded from an allylic functional group 16b at low reaction temperature in polar cosolvent to give purine phosphonate analogues 17 and 24, respectively. The purine nucleoside phosphonate and phosphonic acid analogues were subjected to antiviral screening against HIV-1. Adenine analogue 21 and its SATE prodrug 29 show significant anti-HIV activity in MT-4 cell lines.     

Side-chain homologation of nodulisporic acid: synthesis of potent new dienyl derivatives

A series of new, diene-modified nodulisporic acid analogues (2) bearing diverse functionality at the 3"- and 4"-sites was efficiently prepared from the 3"-aldehyde 3. Biological evaluation of these synthetic nodulisporic acid analogues for systemic flea efficacy identified potent compounds and further clarified the structural requirements for ectoparasite activity.     

Luliberin analogues exhibiting a cytotoxic effect on tumor cells in vitro

Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized.     

Analogues of thiolactomycin as potential anti-malarial and anti-trypanosomal agents

A series of analogues of the naturally occurring antibiotic thiolactomycin (TLM) have been synthesised and evaluated for their ability to inhibit the growth of the malaria parasite, Plasmodium falciparum. Thiolactomycin is an inhibitor of Type II fatty acid synthase which is found in plants and most prokaryotes, but not an inhibitor of Type I fatty acid synthase in mammals. A number of the analogues showed inhibition equal to or greater than TLM. The introduction of hydrophobic alkyl groups at the C3 and C5 positions of the thiolactone ring lead to increased inhibition, the best showing a fourteenfold increase in activity over TLM. In addition, some of the analogues showed activity when assayed against the parasitic protozoa, Trypanosoma cruzi and Trypanosoma brucei.     

Metabolism of DDT analogues by a Pseudomonas sp.

A Pseudomonas sp. rapidly metabolized several nonchlorinated analogues of DDT, with the exception of 2,2-diphenylethanol, as the sole carbon source. Several of the mono-p-chloro-substituted diphenyl analogues were also metabolized as the sole carbon source by the bacterium. The resulting chlorinated aromatic acid metabolites were not further metabolized. The isolate was unable to metabolize p,p'-dichlorodiphenyl analogues as the sole carbon source.     

Synthesis of new bile acid analogues and their metabolism in the hamster: 3 alpha, 6 alpha-dihydroxy-6 beta-methyl-5 beta-cholanoic acid and 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid

This paper reports the chemical synthesis of two new bile acid analogues, namely, 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid from 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acid and describes their metabolism in the hamster. A Grignard reaction of the oxo acid with methyl magnesium iodide in tetrahydrofuran gave two epimeric dihydroxy-6-methyl-cholanoic acids which were separated as the methyl esters by silica gel column chromatography. The configuration of the 6-methyl groups was assigned by proton nuclear magnetic resonance spectroscopy and was supported by the chromatographic properties of the new compounds. The metabolism of the two new bile acid analogues was studied in the hamster. After intraduodenal administration of the 14C-labeled analogues into bile fistula hamsters, both compounds were absorbed rapidly from the intestine and secreted into bile. Intravenous infusion studies revealed that these compounds were efficiently extracted by the liver; the administered analogues became major biliary bile acids, present as either the glycine or taurine conjugates. These compounds are useful to study the effect of methyl-substituted bile acids on cholesterol and bile acid metabolism and may possibly possess cholelitholytic properties.     

Regulation of GSK3 isoforms by phosphatases PP1 and PP2A

Dephosphorylation of phospho GSK3 isoforms, from COS-7 cells, was determined in vitro and in cultured cells in the absence or the presence of okadaic acid and lithium. Our results indicate a preferential dephosphorylation of phospho GSK3α by PP2A phosphatase, whereas dephosphorylation of phospho GSK3β mainly takes place by PP1 phosphatase.     

One-pot chemoenzymatic preparation of coenzyme A analogues

A rapid and stoichiometric method for the synthesis of analogues of coenzyme A is described. The method links the enzymes pantothenate kinase, phosphopantotheine adenylyltransferase, and dephosphocoenzyme A kinase in vitro to generate a variety of CoA analogues from chemically synthesized pantothenic acid derivatives. The Escherichia coli CoA biosynthetic enzymes were overexpressed as hexa-histidine-tagged proteins, providing an abundant source of pure active catalysts for the reaction. The synthesis of five novel CoA derivatives is reported and the method is shown to be robust and tolerant of a number of different pantothenic acid structures, which indicates that the procedure should be widely applicable.     

Synthesis of two biologically active fluorescent probes of thymopentin

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.     

Development of highly potent and selective dynorphin A analogues as new medicines.

Dynorphin A (Dyn A), a 17 amino acid peptide H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH, is a potent opioid peptide which interacts preferentially with kappa-opioid receptors. Research in the development of selective and potent opioid peptide ligands for the kappa-receptor is important in mediating analgesia. Several cyclic disulphide bridge-containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A(1-11)-NH2 cyclic analogues incorporating the sulphydryl-containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the mu- and delta-receptors) as the parent Dyn A(1-11)-NH2 peptide in the guinea-pig brain (GPB), but exhibited a much lower activity in the guinea-pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure-activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at kappa-opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand-based drug design.     

Effect of Glutamate Analogues on Brain Tumor Cell Lines

Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.