Inhibition of monoamine oxidase by selected C5- and C6-substituted isatin analogues

Previous studies have shown that (E)-5-styrylisatin and (E)-6-styrylisatin are reversible inhibitors of human monoamine oxidase (MAO) A and B. Both homologues are reported to exhibit selective binding to the MAO-B isoform with (E)-5-styrylisatin being the most potent inhibitor. To further investigate these structure-activity relationships (SAR), in the present study, additional C5- and C6-substituted isatin analogues were synthesized and evaluated as inhibitors of recombinant human MAO-A and MAO-B. With the exception of 5-phenylisatin, all of the analogues examined were selective MAO-B inhibitors. The C5-substituted isatins exhibited higher binding affinities to MAO-B than the corresponding C6-substituted homologues. The most potent MAO-B inhibitor, 5-(4-phenylbutyl)isatin, exhibited an IC₅₀ value of 0.66 nM, approximately 13-fold more potent than (E)-5-styrylisatin and 18,500-fold more potent than isatin. The most potent MAO-A inhibitor was found to be 5-phenylisatin with an IC₅₀ value of 562 nM. The results document that substitution at C5 with a variety of substituents is a general strategy for enhancing the MAO-B inhibition potency of isatin. Possible binding orientations of selected isatin analogues within the active site cavities of MAO-A and MAO-B are proposed.     

4- and 6-(p-Sulphamoylphenyl)androstenediones: Studies of aromatase inhibitor-based oestrone sulphatase inhibition

4-(p-Sulphamoylphenyl)androstenedione (3) and 6α-p-sulphamoylphenyl analogues 12-14 were synthesised and tested as aromatase inhibitors as well as oestrone sulphatase inhibitors in human placental microsomes. All of the p-sulphamoylphenyl compounds synthesised were powerful inhibitors of aromatase with apparent K_i values ranging between 30 and 97 nM. In addition, the aromatase inhibitory activities of 6α-p-hydroxyphenyl compounds 9-11, which may be produced from their respective sulphamoylphenyl compounds by action of oestrone sulphatase, were also high in a range of 23 and 75 nM of the K_i values. On the other hand, all of the sulphamoylphenyl compounds were poor inhibitors of oestrone sulphatase with more than about 200 μM of IC₂₅ values. Although the present findings of the oestrone sulphatase inhibition are disappointing, such attempts may be valuable to develop a new class of drugs having a dual function, aromatase inhibitor and oestrone sulphatase inhibitor, for the treatment of oestrogen-dependent breast cancer.     

Isolation and characterization of a bacterial dipeptidyl carboxypeptidase inhibitor from Bacillus subtilis 3-16-20

A bacterial dipeptidyl carboxypeptidase inhibitor was isolated from the culture broth of a bacterium identified as Bacillus subtilis. The inhibitor was purified 33-fold from the culture supernatant of B. subtillis 3-16-20 strain by Q-, and S-Sepharose fast flow, C₁₈ column chromatography, ethanol treatment, and ODS column chromatography. The purified inhibitor has an amino acid sequence of glycyl-prolyl-phenylalanyl-prolylisoleucine. IC₅₀ values of the inhibitor were 177 μM (rabbit lung ACE) and 35 μM (bacterial DCP).     

Natural Inhibitor for Volatile C6-Aldehyde Formation from C18-Unsaturated Fatty Acids

The homogenate of tea seed cotyledons contained an inhibitor for C₆-aldehyde formation from linoleic acid and linolenic acid by isolated tea chloroplasts. Seed homogenates of other plants, such as soybean, kidney bean, cucumber, Japanese radish and rice, also contained the inhibitor for C₆-aldehyde formation. The inhibitor from tea seed and cucumber seed inhibited C₆-aldehyde formation by the homogenate of cucumber hypocotyl. Hydroperoxides of linoleic acid detected were reduced when the tea seed inhibitor was added to the reaction mixture, but the enzyme activities of lipoxygenase and hydroperoxide lyase were not inhibited. This means that the inhibitor is a decomposer of fatty acid hydroperoxides as an intermediate of C₆-aldehyde formation. The tea seed inhibitor was formed during the seed ripening and it was stable during the seed germination. These findings obtained here suggest that the inhibitor is widely present in plant seeds and inhibits C₆-aldehyde formation by a variety of plant tissues.     

Palladium-Catalyzed Animation of 6-Chloropurine. Synthesis of N6-Substituted Adenosine Analogues

Abstract

Room-temperature treatment of persilylated 6-chloro-9-β-D-ribofuranosyl-purine with a variety of aliphatic and aromatic amines, in the presence of Pd₂(dba)₃, BINAP and base, leads to N⁶-substituted adenosine analogues in fair to good yields. Coupling of chloropurine with a chiral aziridinyl diester is applied in the synthesis of a potential adenylosuccinate lyase inhibitor.     

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone: A potent inhibitor of the blood platelet cyclo-oxygenase

Previous studies have demonstrated that 13-azaprostanoic acid (13-APA) is a potent and specific antagonist of thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) at the platelet receptor level. In the present study we evaluated the effects of a new azaprostanoid, 2-(6-carboxyhexyl) cyclopentanone hexylhydrazone (CPH), on human platelet function. This hydrazone was found to completely inhibit arachidonic acid (AA)-induced platelet aggregation at 1 uM CPH. On the other hand, CPH was not an effective inhibitor of PGH₂-induced aggregation. Furthermore, 100 uM CPH was completely ineffective in blocking platelet aggregation stimulated by adenosine diphosphate (ADP) or the stable prostaglandin endoperoxide analog U46619 (which presumably acts at the TXA₂/PGH₂ receptor). Measurement of platelet thromboxane B₂ (TXB₂) production demonstrated that the primary site-of-action of CPH is at the cyclo-oxygenase level. Thus, CPH inhibited TXB₂ formation from AA in a dose-dependent manner (0.1 uM–100 uM CPH)². In contrast, CPH blocked TXB₂ production from PGH₂ only at the highest CPH concentration tested, i.e., 100 uM. These results indicate that relative to 13-APA, addition of a second nitrogen at C₁₄ and a double bond between the 12- and 13- positions results in a loss of receptor activity but produces a high affinity for the platelet cyclo-oxygenase.     

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (K_d = 0.52 nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC₅₀ = 0.098 μM, NCI-N87 IC₅₀ = 0.066 μM) and also displayed high oral bioavailability in mice (F = 44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition = 136%).     

Synthesis and evaluation of non-hydrolyzable d-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-d-mannose as a new strong inhibitor of phosphomannose isomerases

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs) from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-d-mannose and 6-deoxy-6-carboxymethyl-d-mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxymethyl-d-mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E. coli PMIs with K_m/K_i ratios of 28 and 8, respectively. We thus report the first malonate-based inhibitor of an aldose–ketose isomerase to date. Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy intermediate postulated for the isomerization reaction catalyzed by PMIs were also synthesized but behave as poor inhibitors of PMIs. A polarizable molecular mechanics (SIBFA) study was performed on the complexes of d-mannose 6-phosphate and two of its analogues with PMI from Candida albicans, an enzyme involved in yeast infection homologous to S. cerevisiae and E. coli PMIs. It shows that effective binding to the catalytic site occurs with retention of the Zn(II)-bound water molecule. Thus the binding of the hydroxyl group on C1 of the ligand to Zn(II) should be water-mediated. The kinetic study reported here also suggests the dianionic character of the phosphate surrogate as a likely essential parameter for strong binding of the inhibitor to the enzyme active site.     

Rat plasma 25-hydroxyvitamin D3 binding protein: An inhibitor of the 25-hydroxyvitamin D3-1 α-hydroxylase

An antibody was prepared from serum of rabbits injected with a pure inhibitor protein obtained from rat serum for chick renal 25-hydroxyvitamin D₃-1α-hydroxylase. The antibody was separated from the endogenous inhibitor in rabbit serum. The antibody shows a single precipitin line with the rat serum antigen and with crude calf serum. Furthermore, the antibody removes the 4.0 S 25-hydroxyvitamin D₃ binding protein from rat serum. The removal of the 25-hydroxyvitamin D₃ binding protein from rat serum with antibody brings about a proportionate removal of inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase. The pure inhibitor binds 25-hydroxyvitamin D₃, as demonstrated by sucrose density gradient sedimentation, and shows specificity of binding identical to the serum transport globulin for 25-hydroxyvitamin D₃. Thus, the previously reported inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase in rat preparations is the serum 25-hydroxyvitamin D₃ transport protein or some derivative thereof. The antibody added to rat renal mitochondrial preparations does increase the activity of the 1- and 24-hydroxylases slightly but not markedly.     

Stimulation of human neutrophil degranulation with 1-0-octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine: Modulation by inhibitors of arachidonic acid metabolism

1-0-octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (C₁₈-AGEPC) stimulated a concentration (10-¹⁰-10-⁶M)-dependent release of granule-associated enzymes from human neutrophils. Cells which were not preincubated with cytochalasin B prior to exposure to C₁₈-AGEPC did not degranulate. C₁₈-AGEPC-elicited enzyme release was significantly reduced, but not abolished, in the absence of extracellular calcium. The lipoxygenase inhibitor, nordihydroguaiaretic acid and the lipoxygenase/cyclo-oxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid, an acetylenic analog of arachidonic acid, caused a concentration-dependent suppression of enzyme discharge from neutrophils exposed to C₁₈-AGEPC. However, the cyclo-oxygenase inhibitors, indomethacin, ibuprofen and flurbiprofen had no effect on C₁₈-AGEPC-induced enzyme extrusion.     

Purification and Some Catalytic Properties of Glucose-6-Phosphate Dehydrogenase Isoforms from Barley Leaves

The cytosolic and chloroplastic isoforms of glucose-6-phosphate dehydrogenase (G₆PDH) were separated and purified from barley leaves (Hordeum vulgare L.). In etiolated leaves, only the cytosolic isoform was expressed. The molecular mass of the cytosolic enzyme, G₆PDH₁, was 112±8 kDa and that of the chloroplast enzyme, G₆PDH₂, was 136±7 kDa. The K_m values for glucose-6-phosphate and NADP were 0.133 and 0.041 mM for G₆PDH₁, and 0.275 and 0.062 mM for G₆PDH₂, respectively. The pH optimum was 8.2 for G₆PDH₁ and 7.8 for G₆PDH₂. The enzyme is absolutely specific for NADP. NADPH is a competitive inhibitor of the G₆PDH₁ in respect to glucose-6-phosphate (G₆P) and NADP (K_i = 0.050 and 0.025 mM, respectively). NADPH is a competitive inhibitor of the G₆PDH₂ in respect to NADP (K_i = 0.010 mM), but a non-competitive inhibitor in respect to the G₆P. ADP, AMP, UTP, NAD, and NADH had no effect on the activity of G₆PDH. ATP inhibited the G₆PDH₂ activity.     

Merging of ruxolitinib and vorinostat leads to highly potent inhibitors of JAK2 and histone deacetylase 6 (HDAC6)

Inhibition of more than one pathway in a cancer cell with a single molecule could result in better therapies with less complex dosing regimens. In this work multi-component ligands have been prepared by joining together key pharmacophores of two different enzyme inhibitors in a way which increases potency against the individual pathways. Selective JAK1/2 inhibitor, ruxolitinib (3), and pan-HDAC inhibitor vorinostat (4) were linked together by a single nitrogen atom to create a new series of compounds with very potent JAK2 and HDAC6 inhibition with selectivity against HDAC1. A preferred compound, 13b, had unprecedented sub-nanomolar JAK2 potency with an IC₅₀ of 41?pM and a sub-nanomolar IC₅₀ against HDAC6 of 200?pM. Binding models show a good fit into both JAK2 and HDAC6.     

Potentiation of lipopolysaccharide-induced IL-6 release by uridine triphosphate in macrophages: Cross-interaction with cyclooxygenase-2-dependent prostaglandin E2 production

Our previous study has demonstrated the potentiation by uridine triphosphate (UTP) of nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in lipopolysaccharide (LPS)-stimulated murine J774 macrophages. In this study, we found that the amount of interleukin-6 (IL-6) release in response to LPS stimulation was greatly enhanced in the presence of UTP. This enhancement exhibited concentration dependence and occurred after 8 h of treatment with LPS. RT-PCR analysis indicated that the steady-state level of IL-6 mRNA induced by LPS was apparently increased upon co-addition of UTP. The potentiation by UTP was inhibited by the treatment with U73122 (a phosphatidylinositol-phospholipase C inhibitor), BAPTA/AM (an intracellular Ca²⁺ chelator), KN-93 (a selective inhibitor of calmodulin-dependent protein kinase) or PDTC (a nuclear factor B inhibitor). To understand the cross-regulation among NO, PGE₂ and IL-6, all of which are dramatically induced after LPS stimulation, the effects of L-NAME (a nitric oxide synthase inhibitor), indomethacin (a cyclooxygenase inhibitor), NS-398 (a cycloxygenase-2 inhibitor) and IL-6 antibody were tested. The results revealed the positive regulation between PGE₂ and IL-6 synthesis because NS-398 and indomethacin inhibited LPS plus UTP-induced IL-6 release, and IL-6 antibody attenuated LPS plus UTP-induced PGE₂ release. Taken together these results reinforce the role of UTP as a regulatory element in inflamed sites by demonstrating the capacity of this nucleotide to potentiate LPS-induced release of inflammatory mediators.     

Incorporation of Streptidine-C6-phosphate into Phosphorylated Streptomycin by Resting Cell of Streptomyces griseus

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities.

The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M_r 19,242 and M_r 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45%) with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 μM. Kinetic analyses revealed that at a concentration below 0.5 μM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (K_a) and stoichiometry (r) for the enzyme-dye complex.     

An active-site-directed irreversible inhibitor of delta 5-3-ketosteroid isomerase

The α and β isomers of spiro-3-oxiranyl-5α-androstan-17β-ol were tested as possible inhibitors of Δ⁵-3-ketosteroid isomerase of $\text{Pseudomonas}\text{testosteroni}$. The β-oxirane causes a first-order irreversible inactivation of the enzyme and shows saturation kinetics (K_I, 17 μM). Protection against inactivation is exhibited by 19-nortestosterone, a competitive inhibitor of the isomerase. Although the α-oxirane was found to be a good reversible inhibitor (K_i, 21 μM), prolonged incubation with it failed to produce any inactivation of the isomerase. The results obtained are consistent with the presence of a nucleophilic group situated near the 3-keto group of the substrate in the enzyme-steroid complex.     

Irreversible Inhibition of Thymidylate Synthase by Pyridoxine (B6) Analogues

Abstract

Three vitamin B₆ analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower K_i) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B₆ analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.     

Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.     

Isolation and Characterization of a Spore Germination Inhibitor from Streptomyces sp. CB-1-1, a Phytopathogen Causing Root Tumor of Melon

The germination rate and activation conditions of spores were examined for four strains of Streptomyces sp., a phytopathogen causing root tumor of melon. An inhibitor was isolated from the agar-cultured material of strain CB-1-1 and then characterized. The inhibitor selectively acted on spore germination and did not affect hyphal growth, and inhibition was abolished by washing the spores in water. The inhibitor was produced by an agar culture, and most of the inhibitor existed in the spores. The IC₅₀ value for the inhibitor was approximately 0.25 μg/ml.