Diamino-C,N-diarylpyridine positional isomers as inhibitors of lysophosphatidic acid acyltransferase-beta

2,6-Diamino-4,N-diarylpyridines were identified as potent, isoform selective inhibitors of the enzymatic activity of lysophosphatidic acid acyltransferase-beta (LPAAT-beta).     

The structural requirements of histone deacetylase inhibitors: SAHA analogs modified at the C5 position display dual HDAC6/8 selectivity

Histone deacetylase (HDAC) proteins have emerged as important targets for anti-cancer drugs, with four small molecules approved for use in the clinic. Suberoylanilide hydroxamic acid (Vorinostat, SAHA) was the first FDA-approved HDAC inhibitor for cancer treatment. However, SAHA inhibits most of the eleven HDAC isoforms. To understand the structural requirements of HDAC inhibitor selectivity and develop isoform selective HDAC inhibitors, SAHA analogs modified in the linker at the C5 position were synthesized and tested for potency and selectivity. C5-modified SAHA analogs displayed dual selectivity to HDAC6 and HDAC8 over HDAC 1, 2, and 3, with only a modest reduction in potency. These findings are consistent with prior work showing that modification of the linker region of SAHA can alter isoform selectivity. The observed HDAC6/8 selectivity of C5-modified SAHA analogs provide guidance toward development of isoform selective HDAC inhibitors and more effective anti-cancer drugs.     

Expression of antimicrobial peptide genes encoding Enbocin and Gloverin isoforms in the silkworm, Bombyx mori

Antimicrobial peptides, Enbocin and Gloverin isoforms from the silkworm Bombyx mori, were analyzed for expression of these peptide genes. Tissue-specific expression of Enbocin and Bmgloverin isoform genes was observed mainly in the fat body upon injection of Escherichia coli. Peptidoglycan and lipopolysaccharide triggered expression of these genes in vivo. On the other hand, lipid A activated Bmgloverin isoform genes but not Enbocin isoform genes. These results illustrate the fact that expression of Enbocin and Bmgloverin isoform genes is inducible by bacteria and that the effects of bacterial cell wall components on the activation of these peptide genes are not necessarily the same. In addition, selective activation of the Enbocin2, Bmgloverin2, and Bmgloverin4 genes by BmRelB rather than BmRelA was observed, providing additional evidence for the occurrence of selective activation of antimicrobial peptide genes by a Rel protein. These results suggest complex regulatory mechanisms in insect antimicrobial peptide genes by bacterial cell wall components.     

Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide as potent and isoform selective ROCK2 inhibitors

ROCK1 and ROCK2 are highly homologous isoforms. Accumulated studies indicate that they have distinct different functions, and the development of isoform selective ROCK inhibitors will pave new roads for the treatment of various diseases. In this work, a series of amide-chroman derivatives were synthesized and biologically evaluated in order to develop potent and isoform selective ROCK2 inhibitors. Remarkably, (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide ((S)-7c) possessed ROCK2 inhibitory activity with an IC₅₀ value of 3?nM and 22.7-fold isoform selectivity (vs. ROCK1). Molecular docking indicated that hydrophobic interactions were the key element for the high potency and isoform selectivity of (S)-7c. The binding free energies predicted by MM/GBSA were in good agreement with the experimental bioactivities, and the analysis of individual energy terms suggested that residue Lys105 in ROCK1 or Lys121 in ROCK2 was the key residue for the isoform selectivity of (S)-7c.     

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.     

Physicochemical properties of malate dehydrogenase from the bacterium Rhodopseudomonas palustris strain f8pt

Electrophoretically homogenous isoforms of malate dehydrogenase with different quaternary structure were prepared from Rhodopseudomonas palustris strain f8pt cultured photolithoheterotrophically on malate and acetate. By selective inhibition of the tricarboxylic acid cycle or glyoxylate cycle, it was shown that the dimeric isoform of the enzyme is responsible for Krebs cycle functioning and the tetrameric isoform is involved in functioning of the glyoxylate cycle.     

An integrase of endogenous retrovirus is involved in maternal mitochondrial DNA inheritance of the mouse

The mechanism of maternal mitochondrial DNA (mtDNA) inheritance in animals can be said to be the selective elimination of sperm mtDNA via the elimination factor of the egg and a sperm mitochondria-specific factor. In 2005, we clarified that t-tpis (Spag1 isoform 1) is a mitochondria-specific translocator and the sperm factor, and furthermore estimated that the elimination factors of the egg are the divalent cation-dependent endonuclease and s-tpis (Spag1 isoform 2 and isoform 3) as the elimination system-specific chaperone [K. Hayashida, K. Omagari, J. Masuda, H. Hazama, Y. Kadokawa, K. Ohba, S. Kohno, The sperm mitochondria-specific translocator has a key role in maternal mitochondrial inheritance, Cell Biol. Int. 29 (2005) 472-481]. This time, using a recombinant Spag1 isoform 1 protein, a pull-down assay of ovary cytosol was performed and the elimination factors searched for. Surprisingly, an endogenous retroviral integrase fragment (Eri15) was identified using mass spectrometry of the electrophoresis band of the pull-down protein. Eri15 was detected as a complex of ∼500 kDa with Spag1 isoform 2 or isoform 3 in native PAGE of the ovary cytosol. This strongly suggested that Eri15 is selectively transported into the sperm mitochondria matrix by Spag1 isoform 2 and 3 via Spag1 isoform 1 and that sperm mtDNA is destroyed, thus causing the establishment of maternal mtDNA inheritance.     

Biological functions of cyclooxygenase 2: control of its expression and activity

Cyclooxygenase (COX) plays a pivotal role in the biosynthesis of prostanoids. The identification of an inducible isoform, COX-2, besides the constitutive one, COX-1, suggested that the latter was dedicated to cytoprotection, whereas the former was responsible for inflammation and neoplasia. This oversimplification was amended due to the synthesis of new selective inhibitors of COX-2 and to the deletion of the COX-2 gene, which demonstrated the involvement of the inducible isoform in physiological as well as in pathological functions. Moreover, several reports describe effects of nonsteroidal anti-inflammatory drugs which do not result from the inhibition of COX activity.     

Novel HDAC6 isoform selective chiral small molecule histone deacetylase inhibitors

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC₅₀ values 10–200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.     

A novel 18 beta-glycyrrhetinic acid analogue as a potent and selective inhibitor of 11 beta-hydroxysteroid dehydrogenase 2

Using 18beta-glycyrrhetinic acid as a template, the synthesis of a series of secondary amides at the 30-position is described and the effects of these modifications on the SAR of the 11beta-hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. An isoform selective inhibitor has been discovered and compound 5, N-(2-hydroxyethyl)-3beta-hydroxy-11-oxo-18beta-olean-12-en-30-oic acid amide, is highlighted as a very potent and selective inhibitor of 11beta-hydroxysteroid dehydrogenase 2 with an IC(50)=4 pM.     

A new class of selective and potent inhibitors of neuronal nitric oxide synthase.

The synthesis and SAR of a series of 6-(4-(substituted)phenyl)-2-aminopyridines as inhibitors of nitric oxide synthase are described. Compound 3a from this series shows potent and selective inhibition of the human nNOS isoform, with pharmacokinetics sufficient to provide in vivo inhibition of nNOS activity.     

Evaluation of class I HDAC isoform selectivity of largazole analogues

Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole–thiazoline and the warhead moiety. In order to elucidate class I-specific structure–activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.     

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.     

Structural basis for the specificity of the nitric-oxide synthase inhibitors W1400 and Nomega-propyl-L-Arg for the inducible and neuronal isoforms

The high level of amino acid conservation and structural similarity in the immediate vicinity of the substrate binding sites of the oxygenase domains of the nitric-oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, and nNOSoxy) make the interpretation of the structural basis of inhibitor isoform specificity a challenge and provide few clues for the design of new selective compounds. Crystal structures of iNOSoxy and nNOSoxy complexed with the inhibitors W1400 and Nomega-propyl-l-arginine provide a rationale for their isoform specificity. It involves differences outside the immediate active site as well as a conformational flexibility in the active site that allows the adoption of distinct conformations in response to interactions with the inhibitors. This flexibility is determined by isoform-specific residues outside the active site.     

Synthesis and SAR of succinamide peptidomimetic inhibitors of cathepsin S

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.     

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F(2t)-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites--produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,--was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F(2t)-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.     

Synthesis, molecular modeling studies, and selective inhibitory activity against monoamine oxidase of N,N'-bis[2-oxo-2H-benzopyran]-3-carboxamides

A novel series of N,N'-bis[2-oxo-2H-1-benzopyran]-3-carboxamide derivatives have been synthesized and investigated for the ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). Some of the synthesized compounds show good selective inhibitory activity against the MAO-A isoform. Both the MAO-A and -B isoforms, deposited in the Protein Data Bank as the 2BXR and 1GOS models, respectively, were considered in a computational study performed with docking techniques on the most active and selective inhibitors.     

Virtual screening-driven identification of human carbonic anhydrase inhibitors incorporating an original, new pharmacophore

Combinated ligand- and pharmacophore-based virtual screening approaches were used to discover novel potential pharmacophores acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). A free database of commercially available compounds was screened through drug-like filters using a four-point pharmacophore, and followed by docking calculation within the active site of an X-ray structure of isoform CA II. One compound, bearing a trifluoro-dihydroxy-propanone moiety, showed an interesting, selective inhibitory activity in low micromolar range against this isoform versus CA I. The chemical originality of this new pharmacophore can represent an important bioisosteric alternative to the sulfonamido-based functionalities, thus leading to the development of a new class of CAIs.     

The identification of novel p38α isoform selective kinase inhibitors having an unprecedented p38α binding mode

A novel series of p38 MAP kinase inhibitors with high selectivity for the p38α isoform over the other family members including the highly homologous p38β isoform has been identified. X-ray co-crystallographic studies have revealed an unprecedented kinase binding mode in p38α for representative analogs, 5c and 9d, in which a Leu108/Met109 peptide flip occurs within the p38α hinge region. Based on these findings, a general strategy for the rational design of additional promising p38α isoform selective inhibitors by targeting this novel binding mode is proposed.