URB754 has no effect on the hydrolysis or signaling capacity of 2-AG in the rat brain

Previous studies indicate that in brain tissue the endocannabinoid 2-AG is inactivated by monoglyceride lipase (MGL)-catalyzed hydrolysis, and a recent report has indicated that MGL activity could be specifically inhibited by URB754 . In the present study, URB754 failed to inhibit 2-AG hydrolysis in rat brain preparations. In addition, brain cryosections were employed to assess whether URB754 could facilitate the detection of 2-AG-stimulated G protein activity. Nevertheless, whereas pretreatment with PMSF readily allowed detection of 2-AG-stimulated G protein activity, URB754 was ineffective. In contrast to previous claims, brain FAAH activity was also resistant to URB754. Thus, in our hands URB754 was not able to block the endocannabinoid-hydrolyzing enzymes and cannot serve as a lead structure for future development of MGL-specific inhibitors.     

正碳离子样的过渡态类似物研究进展

综述了应用酶催化过渡态理论设计的几类正碳离子样的过渡态类似物作为酶的抑制剂的研究进展。其中包括类异戊二烯的正离子过渡态类似物,唾液酸转移酶的过渡态类似物,3-脱氧-D-甘露糖-2-辛酮糖酸酯-8-磷酸酯(KDO8P) 合成酶的过渡态类似物,尿苷二磷酸酯葡糖醛酸基转移酶(UGT) 的过渡态类似物。     

Refined homology model of monoacylglycerol lipase: toward a selective inhibitor

Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue 2-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.     

Preparation and biological activity of 2-[4-(thiazol-2-yl)phenyl]propionic acid derivatives inhibiting cyclooxygenase.

A series of 2-[4-(thiazol-2-yl)phenyl]propionic acids substituted at various positions were prepared by the reaction of diethyl 2-methyl-2-(4-thiocarbamoylphenyl)malonates with alpha-bromoaldehyde diethyl acetals or alpha-haloketones followed by hydrolysis of esters. The inhibition of prostaglandin H synthetase (cyclooxygenase) was assayed by use of an enzyme preparation from guinea pig polymorphonuclear leukocytes. Examination of the structure-activity relationship of these compounds indicated that the substitution pattern with halogens at position 3 (R1) of the benzene ring and a methyl group in position 4 (R2) and/or 5 (R3) of the thiazole ring were favorable for inhibitory activity. The compounds bearing bulky alkyl or polar functional groups at the R2 position were weak inhibitors. The potent inhibitors of cyclooxygenase were tested for their ability to reduce carrageenin-induced inflammation of rat paws. These derivatives had strong anti-inflammatory activity based on their strong inhibition of cyclooxygenase, with some exceptions, including those with a thiomethyl group at R1.     

Structure-activity relationships of alpha-human atrial natriuretic peptide (alpha-hANP) analogs: role of charged groups in alpha-hANP.

To determine whether the addition of a methylene unit in the side chain of the Asp or Arg residue in alpha-human atrial natriuretic peptide (alpha-hANP) influences its biological activity, analogs of alpha-hANP, [Glu13]-alpha-hANP (7-28) (1), [Aad13]-alpha-hANP (7-28) (2), and [Harn]-alpha-hANP(7-28) (where n is any possible combination of 11, 14 and 27) (3-9), where the original Asp or Arg residue was replaced by a homo-amino acid, were synthesized by the solid-phase synthesis method. All the analogs were evaluated for their receptor binding, cyclic guanosine monophosphate (cGMP) accumulation activity in rat vascular smooth muscle cells (VSMC), and for vasorelaxant activity employing rat aorta. 1 and 2 were 0.9 and 0.03 times as potent as alpha-hANP (7-28), respectively, in binding. Har-containing analogs (3-9) were as potent as alpha-hANP (7-28) in binding. Among the Har-containing analogs, [Har11,14]-alpha-hANP (7-28) (6) and [Har11,27]-alpha-hANP (7-28) (7) were remarkably vasorelaxant active, being 4.2 and 5.3 times potent than alpha-hANP (7-28), respectively, in spite of relatively lower cGMP accumulation activity in the case of 7. The roles of the chargeable amino acid residues in biological activity are discussed.     

Identification of a bioactive impurity in a commercial sample of 6-methyl-2-p-tolylaminobenzo[d][1,3]oxazin-4-one (URB754)

The compound URB754 was recently identified as a potent inhibitor of the endocannabinoid-deactivating enzyme monoacylglycerol lipase (MGL) by screening of a commercial chemical library. Based on HPLC/MS, NMR and EI/MS analyses, the present paper shows that the MGL-inhibitory activity attributed to URB754 is in fact due to a chemical impurity present in the commercial sample, identified as bis(methylthio)mercurane. Although this organomercurial compound is highly potent at inhibiting MGL (IC50 = 11.9 +/- 1.1 nM), its biological use is prohibited by its toxicity and target promiscuity.     

Molecular Docking and Design of Novel Heterodimers of Donepezil and Huperzine Fragments as Acetylcholinesterase Inhibitors

To provide hints for the design of new acetylcholinesterase(ACh E) inhibitors with higher potency and specificity, the binding modes of novel heterodimers comprised of donepezil and huperzine A fragments with ACh E were explored by employing the docking simulations. The results show that the binding mode of S-17b(the most potent inhibitor in Ref. 2, i.e., Bioorg. Med. Chem. 2013, 21, 676-683) is clearly different from that of donepezil, while the binding modes of other heterodimers in Ref. 2 are the same as that of donepezil. In addition, based on the binding mode and structure modification of S-17 b, two novel inhibitors(S-17b1 and S-17bb1) with much higher inhibitory potency than S-17 b were obtained. Our design strategy was to replace the hupyridone moiety of S-17 b with the bulky group, and to replace the dimethoxyindanone moiety of S-17 b with more hydrophobic and bulky group with a highly positive charge, which would result in generating potent and selective AChE inhibitors.     

Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented.     

Synthesis of some pseudo-peptide analogs of thiol proteinase inhibitors

Some pseudo-peptide analogs of thiol proteinase inhibitors were synthesized by a conventional solution method. Among them, Suc-Ala-Val-Val-Ala-psi-(CH2-NH)-Ala-pNA (peptide 1) and Suc-Ala-Val-Val-psi-(CH2-NH)-Ala-Ala-pNA (peptide 2) showed a stronger inhibitory activity compared with parent peptide such as Suc-Ala-Val-Val-Ala-Ala-pNA. In particular, peptide 2 was about 10-fold as active as the parent peptide (IC50 = 8 microM). Inserting psi-(CH2-NH) possibly makes the inhibitor less susceptible to papain and, as a result, produces more potent inhibition.     

Mechanistic studies on the alkyltransferase activity of serotonin N-acetyltransferase

BACKGROUND: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) catalyzes the first, rate-limiting step in the biosynthesis of the circadian hormone melatonin (5-methoxy-N-acetyltryptamine) from serotonin. Our recent discovery that, in addition to catalyzing the acetyl transfer from acetyl-coenzyme A (acetyl-CoASH) to serotonin, AANAT is also a robust catalyst for the alkyl transfer reaction between CoASH and N-bromoacetyltryptamine has not only opened up a new way to develop cell-permeable AANAT acetyltransferase inhibitors that are valuable in vivo tools in helping elucidate melatonin's (patho)physiological roles, but has also raised a question - how does AANAT accelerate the alkyl transfer reaction? In this study, mechanistic aspects of the AANAT-catalyzed alkyl transfer reaction were explored by employing CoASH and a series of N-haloacetyltryptamines that were also evaluated for their AANAT acetyltransferase inhibitory activities. RESULTS: Investigation of various N-haloacetyltryptamine analogs showed a similar leaving group effect on the enzymatic and non-enzymatic reaction rates. Steady-state kinetic analyses demonstrated that AANAT alkyltransferase obeys a sequential, ternary complex mechanism, with random substrate binding. Rate versus pH profiles revealed the catalytic importance of an ionizable group with pK(a) of approximately 7. All those N-haloacetyltryptamines that serve as substrates of AANAT alkyltransferase are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. In particular, N-chloroacetyltryptamine was also shown to be a potent inhibitor of intracellular melatonin production in a pineal cell culture assay. CONCLUSIONS: This is the first detailed investigation of the alkyltransferase activity associated with an acetyltransferase. Our results indicate that AANAT does not accelerate the alkyl transfer reaction by simple approximation effect as previously proposed for the similar alkyl transfer reaction catalyzed by other acyltransferases. This study has general implications for developing novel inhibitors by taking advantage of the promiscuous alkyltransferase activity associated with several acyltransferases.     

Synthesis and structure-activity relationship of N-arylrolipram derivatives as inhibitors of PDE4 isozymes

Structure activity studies of N-phenylrolipram derivatives have led to the identification of highly potent PDE4 inhibitors. The potential of these inhibitors for cellular activity was routinely assessed in an assay of fMLP induced oxidative burst in human eosinophils. Since first generation PDE4 inhibitors have been plagued with a number of unwanted side effects, parallel structure activity studies for competition with the [3H]-rolipram binding site in rat brain were performed. In this fashion 5-[4-(3-cyclopentyloxy-4-methoxyphenyl)-2-oxo-pyrrolidin-1-yl]-3-(3-methoxybenzyloxy)benzoic acid N',N'-dimethylhydrazide (22) was identified as a potent inhibitor of PDE4 which exhibits >1000 fold selectivity versus PDE3, and is a nanomolar inhibitor in all the cellular assays tested. Studies on the stereoselectivity of PDE4 inhibition of this class of rolipram based compounds revealed, that for example (S)-11 is a more potent inhibitor than (R)-11. This effect can also be observed in primary human cells where the (S)-enantiomer is about 10 fold more potent than the corresponding (R)-enantiomer.     

Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.     

Quantitative structure-activity relationship study of N-(3-oxo-3,4-dihydro-2H-benzo[1,4]thiazine-6-carbonyl)guanidines as potent Na/H exchange inhibitors

We have previously reported that N-(4-isopropyl-2,2-dimethyl-3-oxo-3,4-dihydro-2H-benzo[1,4]oxazine-6-car bonyl)guanidine (4b) methanesulfonate salt (KB-R9032) is a potent and highly water-soluble Na/H exchange inhibitor. In a series of studies on Na/H exchange inhibitors, we designed and synthesized N-(3-oxo-3,4-dihydro-2H-benzo[1,4]thiazine-6-carbonyl)guanidines (5) as more potent inhibitors with high water-solubility. The design strategy for 5 was based on a quantitative structure-activity relationship (QSAR) study, involving the proportional relationship between the biological activity and hydrophobicity of the ring structure of compounds 4. As expected, compounds 5 showed more potent activity than 4. It was found by using the QSAR analysis that 5 were about five-fold more potent than 4. The increase in potency of compounds 5 well agreed with our previous QSAR analysis result. The most potent derivative was the methanesulfonate salt 5d of the 4-isopropyl derivative (IC50=0.0091 microM). And in addition to the in vitro study, 5d showed significant protective activity against a rat acute myocardial infraction model.     

Synthesis and matrix metalloproteinase-12 inhibitory activity of ageladine A analogs

Synthesis of the 37 ageladine A analogs was accomplished by employing the total synthetic route of natural ageladine A previously explored by us. From the matrix metalloproteinase-12 (MMP-12) inhibitory activity assay carried out using the novel analogs, it appeared evident that the halogen atom at the 2-position of pyrrole ring was essential for the inhibitory activity and that the introduction of a bromine atom into the 4-position of pyrrole ring is very effective for producing potent activity. In addition, exchange of the pyrrole ring to an imidazole ring was extremely effective in increasing activity, and the analog 29 thus obtained was found to show approximately 4 times more potent activity than natural ageladine A.     

URB602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices

The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.     

Clorobiocin biosynthesis in Streptomyces: identification of the halogenase and generation of structural analogs

Clorobiocin (clo) and novobiocin (nov) are potent inhibitors of bacterial DNA gyrase. The two substances differ in the substitution pattern at C-8' of the aminocoumarin ring, carrying a chlorine atom or a methyl group, respectively. By gene inactivation, clo-hal was identified as the gene of the halogenase responsible for the introduction of the chlorine atom of clorobiocin. Inactivation of cloZ did not affect clorobiocin formation, showing that this ORF is not essential for clorobiocin biosynthesis. Expression of the methyltransferase gene novO in the clo-hal(-) mutant led to the very efficient formation of a hybrid antibiotic containing a methyl group instead of a chlorine atom at C-8'. Comparison of the antibacterial activity of clorobiocin analogs with -Cl, -H, or -CH(3) at C-8' showed that chlorine leads to 8-fold higher activity than hydrogen and to 2-fold higher activity than a methyl group.     

Synthesis and biological evaluation of substituted indazolyl amide derivatives as S-adenosyl-L-homocysteine hydrolase inhibitors

A series of novel amide derivatives bearing an indazole moiety were synthesized and evaluated for their in vitro S-adenosyl-L-homocysteine hydrolase (SAHase) inhibitory activity. Among these compounds, 8b, 8m, 8r and 8w showed better or similar inhibitory effects compared to the positive control aristeromycin. These results provide a novel lead for the discovery of more potent non-adenosine analogs as SAHase inhibitors.     

Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis

The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.     

Anti-Inflammatory Effect of Homo- and Heterodimers of Natural Enkephalinase Inhibitors in Experimental Colitis in Mice

Background: the pharmacological treatment and/or maintenance of remission in inflammatory bowel diseases (IBDs) is currently one of the biggest challenges in the field of gastroenterology. Method: our aim was the synthesis of homo- and heterodimers of natural enkephalinase inhibitors (opiorphin; sialorphin; spinorphin) and the in vitro characterization of their effect on the degradation of enkephalin by neutral endopeptidase (NEP) and stability in human plasma. We investigated the in vivo heterodimer of Cys containing analogs of sialorphin and spinorphin (peptide X) in a mouse model of colitis. The extent of inflammation was evaluated based on the microscopic score; macroscopic score; ulcer score, colonic wall thickness, colon length and quantification of myeloperoxidase activity. Results: we showed that the homo- and heterodimerization of analogs of sialorphin, spinorphin and opiorphin containing Cys residue at the N-terminal position resulted in dimeric forms which in vitro exhibited higher inhibitory activity against NEP than their parent and monomeric forms. We showed that peptide X was more stable in human plasma than sialorphin and spinorphin. Peptide X exerts potent anti-inflammatory effect in the mouse model of colitis. Conclusion: we suggest that peptide X has the potential to become a valuable template for anti-inflammatory therapeutics for the treatment of gastrointestinal (GI) tract inflammation.     

Studies on analgesic oligopeptides. VII. Solid phase synthesis and biological properties of Tyr-D-Arg-Phe-beta Ala-NH2 and its fluorinated aromatic amino acid derivatives.

Tyr-D-Arg-Phe-beta Ala-NH2 (I) and its six fluorinated analogs were synthesized. Their opioid receptor binding properties were examined in vitro and their analgesic activity in vivo using the mouse writhing test. It was found that I was one of the most selective and potent mu-receptor agonists reported to date. [Tyr(2F)1](VI) and [Tyr(3F)1](V) derivatives of I showed similar biological properties to those of I. Since these peptides resist enzymatic degradation, it is expected that they are excellent reagents for the studies of function of mu-receptor-mediated biological properties in vivo and in vitro.