GPR91 antagonist and TGF-ß inhibitor suppressed collagen production of high glucose and succinate induced HSC activation.

Activated hepatic stellate cells (HSCs) play a central role in fibrillary collagen production, the primary cause of liver fibrosis. Although it is known that primary cultured HSCs are activated by plastic culture dish stiffness, HSC activation and quiescent-state-reversion mechanisms are still unclear. In this study, we used cultured normal rat HSCs on 3.2 kPa collagen normal liver stiffness equivalent gel, to determine whether high glucose or high succinate conditions induce HSC activation, and examined whether activated HSCs reverted to a quiescent state when suppressed by GPR91 antagonists or TGF-ß1 receptor inhibitors. We measured the gene expression levels of α-SMA and type I collagen HSC activation markers using real-time PCR. Our data indicated that high glucose conditions induced HSC activation, and showed that under continuous high glucose exposure HSC activation progressed. A GPR91 antagonist, 2 d, and a TGF-ß1 receptor inhibitor, SB525334, suppressed the Col1α mRNA expression level of these activated HSCs. Similarly, under extended high succinate exposure, 2 d and SB525334 reduced Col1α mRNA expression levels of activated HSCs. From the above, we determined that even though HSCs had already been activated by high glucose or succinate conditions which persisted after activation, the GPR91 antagonist and the TGF-ß1 receptor inhibitor were able to reduce the production of type I collagen from activated HSCs.

Discovery of new low-molecular-weight p53-Mdmx disruptors and their anti-cancer activities.

Although several p53-Mdm2-binding disruptors have been identified to date, few studies have been published on p53-Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200-300 selectively inhibited the p53-Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53-Mdmx interaction over the p53-Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53-Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.

Discovery of dihydroquinazolinone derivatives as potent, selective, and CNS-penetrant M(1) and M(4) muscarinic acetylcholine receptors agonists.

We designed and synthesized a series of dihydroquinazolinone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Introduction of the N-carbethoxy piperidine unit into a HTS hit compound followed by optimization of the amine linker and the carbamoyl moiety led to the identification of compound 1 as a potential candidate. The identified compound 1 showed high selectivity for M1 and M4 muscarinic acetylcholine receptors with M4 partial agonistic activity. In addition, compound 1 showed good brain penetration and reversed methamphetamine-induced hyperlocomotion in rats (ED50=3.0 mg/kg, sc).

Identification of 2,3-disubstituted pyridines as potent, non-emetic PDE4 inhibitors.

A series of 2,3-disubstituted pyridines were synthesized as potential non-emetic PDE4 inhibitors. To decrease brain exposure and minimize emesis, we modified the lipophilic moiety of a series of emetic PDE4 inhibitors and found that introduction of a hydroxy group into the pyridine moiety of the side chain led to non-emetic compounds with preserved PDE4 inhibitory activity. Following optimization at the phenoxy group, we identified compound 1 as a potent non-emetic PDE4 inhibitor. Compound 1 showed significant efficacy in an animal model of asthma without inducing emesis.

Discovery of N-sulfonyl-7-azaindoline derivatives as potent, orally available and selective M(4) muscarinic acetylcholine receptor agonists.

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M4 muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M4 muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M4 mAChR agonist. Compound 1 showed a highly selective M4 mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1-10 mg/kg, po).

Discovery of N-substituted 7-azaindoline derivatives as potent, orally available M1 and M4 muscarinic acetylcholine receptors selective agonists.

We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M1 and M4 muscarinic acetylcholine receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M1 and M4 mAChRs agonistic activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.

Syntheses of [14C]-labeled 2-(3-chlorophenyloxy)-3-[3-(3-hydroxy) pyridin-4-yl propoxy]pyridine, a phosphodiesterase 4 inhibitor and its metabolites.

We describe here the synthesis of [(14)C]-2-(3-chlorophenyloxy)-3-[3-(3-hydroxy)pyridin-4-yl propoxy]pyridine (1), a phosphodiesterase 4 inhibitor. [(14)C]-Labeled 1 was prepared in three steps from [(14)C]-2-bromopyridin-3-ol in an overall yield of 32%. Preparation of [(14)C]-labeled 2 and 3, two metabolites of 1, is also described.

Acquired curved hair is caused by fusion of multiple hair matrix cells.

BACKGROUND: "Curved hair" caused by acquired factors is considered to have adverse cosmetic effects, but the detailed mechanism behind curved hair remains obscure. OBJECTIVE: We attempted to clarify the causes of curved hair that appeared to have occurred via acquired factors. METHODS: Outer root sheath cells (ORSC) isolated from plucked human hair follicles were used to evaluate the expression of type IV collagen. Straight and curved hairs with hair follicle tissue attached were also collected from the same individuals and subjected to morphological, immunohistochemical, and gene expression analyses. RESULTS: The amount of type IV collagen increased upon inducing endoplasmic reticulum stress in ORSC. Meanwhile, in curved hair follicle tissue, the gene expression of type IV collagen decreased. In addition, the curved hair follicle tissue obtained from participants in their 30 s to 50 s had distorted shapes compared with that of straight hair from the same individuals. It was also observed that hair matrix cells based on multiple hair germs fused to eventually form a single hair follicle and hair shaft. In curved hair follicle tissue, KRT71 protein, a marker of inner root sheath differentiation, was unevenly distributed and there was elevated expression of Dickkopf-1 (DKK1) protein, an inhibitor of the Wnt signaling pathway. CONCLUSION: Our study revealed the fusion of hair matrix cells during hair follicle regeneration as a cause of acquired curved hair. We consider that such fusion causes hair follicle tissue to abnormally differentiate, resulting in asymmetric hair follicle shapes and curved hair.

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M(1) and M(4) muscarinic acetylcholine receptors agonists.

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M1 and M4 agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.

Identification of 2,3-disubstituted pyridines as potent, orally active PDE4 inhibitors.

A series of 2,3-disubstituted pyridines were synthesized and evaluated for their PDE4 inhibitory activity. We successfully modified undesirable cyano group of initial lead compound 2 to 4-pyridyl group with improvement of in vitro efficacy and optimized the position of nitrogen atoms in pyridine moiety and alkylene linker. The most potent compound showed significant efficacy in animal models of asthma and inflammation.

Cloning of two gene clusters involved in the catabolism of 2,4-dinitrophenol by Paraburkholderia sp. strain KU-46 and characterization of the initial DnpAB enzymes and a two-component monooxygenases DnpC1C2.

Little is currently known about the metabolism of the industrial pollutant 2,4-dinitrophenol (DNP), particularly among gram-negative bacteria. In this study, we identified two non-contiguous genetic loci spanning 22 kb of Paraburkholderia (formerly Burkholderia) sp. strain KU-46. Additionally, we characterized four key initial genes (dnpA, dnpB, and dnpC1C2) responsible for DNP degradation, providing molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription polymerase chain reaction (PCR) analysis indicated that dnpA, which encodes the initial hydride transferase, and dnpB which encodes a nitrite-eliminating enzyme, were induced by DNP and organized in an operon. Moreover, we purified DnpA and DnpB from recombinant Escherichia coli to demonstrate their effect on the transformation of DNP to NP through the formation of a hydride-Meisenheimer complex of DNP, designated as H--DNP. The function of DnpB appears new since all homologs of the DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER, and DnpC1 and DnpC2 were functionally characterized as the FAD reductase and oxygenase components of the two-component DNP monooxygenase, respectively. By elucidating the hqdA1A2BCD gene cluster, we are now able to delineate the final degradation pathway of hydroquinone to ß-ketoadipate before it enters the tricarboxylic acid cycle.

Asymmetric synthesis of the carbon-14-labeled selective glucocorticoid receptor modulator using cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate.

We describe in this study the asymmetric synthesis of radioisotope (RI)-labeled selective glucocorticoid receptor modulator. This synthesis is based on optimization of the cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate and Negishi coupling of zinc cyanide to the 6-bromoindole moiety. [¹4C] Labeled (-)-{4-[(1-{2-[6-cyano-1-(cyclohexylmethyl)-1H-indol-3-yl]-3,3,3-trifluoro-2-hydroxypropyl}piperidin-4-yl)oxy]-3-methoxyphenyl}acetic acid (-)-1 was synthesized successfully with high enantioselectivity (>99% ee) and sufficient radiochemical purity.

Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood-Brain Barrier.

A proton-coupled organic cation (H+/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.

MDMX elevation by a novel Mdmx-p53 interaction inhibitor mitigates neuronal damage after ischemic stroke.

Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein-Protein Interaction (PPI) inhibitors, K-181, on Mdmx-p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.

Discovery of Benzylpiperazine Derivatives as CNS-Penetrant and Selective Histone Deacetylase 6 Inhibitors.

Inhibition of histone deacetylase 6 (HDAC6) in the brain is a highly attractive therapeutic target for the treatment of neurodegenerative diseases. The low blood-brain barrier permeability of most known HDAC6 inhibitors, however, prevents their application as central nervous system (CNS) drugs. To overcome this problem, we designed and synthesized benzylpiperazine derivatives using a hybrid strategy of combining HDAC6 inhibitors and brain-penetrant histamine H1 receptor antagonists. Introducing the benzylpiperazine units to the cap region of hydroxamate-type HDAC6 inhibitors led us to identify isozyme-selective and CNS-penetrant HDAC6 inhibitor KH-259 (1) with the appropriate pharmacokinetic and safety properties. Intraperitoneal administration of KH-259 (10 mg/kg) had antidepressant activity and increased acetylated α-tubulin in the brain without promoting acetylated histone H3K9. These findings indicate that our hybrid strategy of combining HDAC6 inhibitors and histamine H1 receptor antagonists is an effective methodology for designing CNS-penetrant HDAC6 inhibitors.

A novel piperidine degradation mechanism in a newly isolated piperidine degrader Pseudomonas sp. strain KU43P.

The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg2+. These results clearly show that pipA encodes γ-glutamylpiperidide synthetase and that piperidine is first glutamylated and then hydroxylated in the piperidine degradation pathway of Pseudomonas sp. strain KU43P. This study has filled a void in the general knowledge of the microbial degradation of amine compounds.

Design, synthesis and evaluations of spiro-fused benzoxaborin derivatives as novel boron-containing compounds.

Drug design using boron-containing heterocycles has attracted a great deal of attention because these compounds are believed to possess high biological activity. However, information on the synthetic methodology and pharmacokinetic profiling of boron-containing compounds is limited. In this study, we provide a new synthetic route for preparation of spiro-fused benzoxaborin derivatives and investigate their in vitro pharmacokinetic properties. Our efforts led to the successful construction of a chemical library of spiro-fused benzoxaborin derivatives with appropriate physicochemical and in vitro pharmacokinetic properties for oral drugs. These results indicate that the synthesized boron-containing compounds are therefore eligible for classification in a novel chemical library.

Design, Synthesis, and Blood-Brain Barrier Transport Study of Pyrilamine Derivatives as Histone Deacetylase Inhibitors.

We designed and synthesized a pyrilamine derivative 1 as a selective class I HDAC inhibitor that targets pyrilamine-sensitive proton-coupled organic cation antiporter (PYSOCA) at the blood-brain barrier (BBB). Introduction of pyrilamine moiety to benzamide type HDAC inhibitors kept selective class I HDAC inhibitory activity and increased BBB permeability. Our BBB transport study showed that compound 1 is a substrate of PYSOCA. Thus, our findings suggest that the hybrid method of HDAC inhibitor and substrate of PYSOCA such as pyrilamine is useful for development of HDAC inhibitors with increased BBB permeability.

Discovery of novel N-substituted oxindoles as selective m1 and m4 muscarinic acetylcholine receptors partial agonists.

Activation of the M1 and M4 muscarinic acetylcholine receptors is thought to play an important role in improving the symptoms of schizophrenia. However, discovery of selective agonists for these receptors has been a challenge, considering the high sequence homology and conservation of the orthosteric acetylcholine binding site among muscarinic acetylcholine receptor subtypes. We report in this study the discovery of novel N-substituted oxindoles as potent muscarinic acetylcholine receptor partial agonists selective for M1 and M4 over M2, M3, and M5. Among these oxindoles, compound 1 showed high selectivity for the M1 and M4 receptors with remarkable penetration into the central nervous system. Compound 1 reversed methamphetamine- and apomorphine-induced psychosis-like behaviors with low potency to extrapyramidical and peripheral side effects.