Discovery of new ERRγ agonists regulating dopaminergic neuronal phenotype in SH-SY5Y cells.

The discovery of small molecules that regulate specific neuronal phenotypes is important for the development of new therapeutic candidates for neurological diseases. Estrogen-related receptor γ (ERRγ), an orphan nuclear receptor widely expressed in the central nervous system (CNS), is closely related to the regulation of neuronal metabolism and differentiation. We previously reported that upregulation of ERRγ could enhance dopaminergic neuronal phenotypes in the neuroblastoma cell line, SH-SY5Y. In this study, we designed and synthesized a series of new ERRγ agonists using the X-ray crystal structure of the GSK4716-bound ERRγ complex and known synthetic ligands. Our new ERRγ agonists exhibited increased transcriptional activities of ERRγ. In addition, our molecular docking results supported the experimental findings for ERRγ agonistic activity of the potent analogue, 5d. Importantly, 5d not only enhanced the expression of dopaminergic neuronal-specific molecules, TH and DAT but also activated the relevant signaling events, such as the CREB-mediated signaling pathway. The results of the present study may provide useful clues for the development of novel ERRγ agonists for neurological diseases related to the dopaminergic nervous system.

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of L,D-Carboxypeptidase.

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.

Resveratrol-salicylate derivatives as selective DNMT3 inhibitors and anticancer agents.

Resveratrol is a natural polyphenol with plethora of biological activities. Resveratrol has previously shown to decrease DNA-methyltransferase (DNMT) enzymes expression and to reactivate silenced tumor suppressor genes. Currently, it seems that no resveratrol analogs have been developed as DNMT inhibitors. Recently, we reported the synthesis of resveratrol-salicylate derivatives and by examining the chemical structure of these analogs, we proposed that these compounds could exhibit DNMT inhibition especially that they resembled NSC 14778, a compound we previously identified as a DNMT inhibitor by virtual screening. Indeed, using in vitro DNMT inhibition assay, some of the resveratrol-salicylate analogs we screened in this work that showed selective inhibition against DNMT3 enzymes which were greater than resveratrol. A molecular docking study revealed key binding interactions with DNMT3A and DNMT3B enzymes. In addition, the most active analog, 10 showed considerable cytotoxicity against three human cancer cells; HT-29, HepG2 and SK-BR-3, which was greater than resveratrol. Further studies are needed to understand the anticancer mechanisms of these derivatives.

Rationalization of activity cliffs of a sulfonamide inhibitor of DNA methyltransferases with induced-fit docking.

Inhibitors of human DNA methyltransferases (DNMT) are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure-activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of 'activity cliffs', e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

Docking of a novel DNA methyltransferase inhibitor identified from high-throughput screening: insights to unveil inhibitors in chemical databases.

Inhibitors of DNA methyltransferase (DNMT) are attractive compounds not only as potential therapeutic agents for the treatment of cancer and other diseases, but also as research tools to investigate the role of DNMTs in epigenetic events. Recent advances in high-throughput screening (HTS) for epigenetic targets and the availability of the first crystallographic structure of human DNMT1 encourage the integration of research strategies to uncover and optimize the activity of DNMT inhibitors. Herein, we present a binding model of a novel small-molecule DNMT1 inhibitor obtained by HTS, recently released in a public database. The docking model is in agreement with key interactions previously identified for established inhibitors using extensive computational studies including molecular dynamics and structure-based pharmacophore modeling. Based on the chemical structure of the novel inhibitor, a sequential computational screening of five chemical databases was performed to identify candidate compounds for testing. Similarity searching followed by molecular docking of chemical databases such as approved drugs, natural products, a DNMT-focused library, and a general screening collection, identified at least 108 molecules with promising DNMT inhibitory activity. The chemical structures of all hit compounds are disclosed to encourage the research community working on epigenetics to test experimentally the enzymatic and demethylating activity in vivo. Five candidate hits are drugs approved for other indications and represent potential starting points of a drug repurposing strategy.

Antiviral activity of novel 2'-fluoro-6'-methylene-carbocyclic adenosine against wild-type and drug-resistant hepatitis B virus mutants.

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (9) was synthesized and evaluated its anti-HBV activity. The titled compound demonstrated significant antiviral activity against wild-type as well as lamivudine, adefovir and double lamivudine/entecavir resistant mutants. Molecular modeling study indicate that the 2'-fluoro moiety by a hydrogen bond, as well as the van der Waals interaction of the carbocyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the drug resistant mutants.

Homology modeling, docking and structure-based pharmacophore of inhibitors of DNA methyltransferase.

DNA methyltransferase 1 (DNMT1) is an emerging epigenetic target for the treatment of cancer and other diseases. To date, several inhibitors from different structural classes have been published. In this work, we report a comprehensive molecular modeling study of 14 established DNTM1 inhibitors with a herein developed homology model of the catalytic domain of human DNTM1. The geometry of the homology model was in agreement with the proposed mechanism of DNA methylation. Docking results revealed that all inhibitors studied in this work have hydrogen bond interactions with a glutamic acid and arginine residues that play a central role in the mechanism of cytosine DNA methylation. The binding models of compounds such as curcumin and parthenolide suggest that these natural products are covalent blockers of the catalytic site. A pharmacophore model was also developed for all DNMT1 inhibitors considered in this work using the most favorable binding conformations and energetic terms of the docked poses. To the best of our knowledge, this is the first pharmacophore model proposed for compounds with inhibitory activity of DNMT1. The results presented in this work represent a conceptual advance for understanding the protein-ligand interactions and mechanism of action of DNMT1 inhibitors. The insights obtained in this work can be used for the structure-based design and virtual screening for novel inhibitors targeting DNMT1.

Discovery of a novel CDK7 inhibitor YPN-005 in small cell lung cancer.

In contrast to non-small cell lung cancer, there has been no significant progress in the development of therapies for the small cell lung cancer (SCLC) in recent decades. Although various targeted agents, including immunotherapies, have recently been developed for testing in clinical trials, novel therapeutic agents are still needed for SCLC. We developed a potent inhibitor of cyclin-dependent kinase 7 (CDK7), designated YPN-005, and sought to determine whether it showed any anticancer effects in SCLC cells, cisplatin or etoposide-resistant cells, or organoids derived from SCLC patients. In a panel of kinases assay, YPN-005 was highly selective for CDK7 and showed antiproliferative effects in SCLC and cells with acquired resistance to conventional anticancer drugs. Similar to other CDK7 inhibitors, YPN-005 treatment significantly decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Consistent with the in vitro results, YPN-005 treatment showed a significant inhibition of tumor growth through the suppression of RNA polymerase II phosphorylation. Finally, YPN-005 showed potent anticancer effects in organoids derived from SCLC patients compared to another CDK7 inhibitor, THZ1. Therapeutic targeting of CDK7 in SCLC might be suitable for clinical investigation, and YPN-005 may be a promising therapeutic option for primary SCLC and SCLC with acquired resistance to conventional therapy.

Synthesis and biological evaluation of 4(5)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles as transforming growth factor-beta type 1 receptor kinase inhibitors.

A series of 4(5)-(6-methylpyridin-2-yl)imidazoles 16-19 and -pyrazoles 22-29, 33, and 34 have been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in cell-based luciferase reporter assays. The 6-quinolinyl imidazole analogs 16 and 18 inhibited ALK5 phosphorylation with IC(50) values of 0.026 and 0.034 microM, respectively. In a luciferase reporter assay using HaCaT cells transiently transfected with p3TP-luc reporter construct, 18 displayed 66% inhibition at 0.05 microM, while competitor compounds 2 and 3 showed 44% inhibition. The binding mode of 18 generated by flexible docking studies with ALK5:18 complex shows that it fits well into the active site cavity of ALK5 by forming broad and tight interactions.

Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase.

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity.

Concise syntheses of (+)-macrosphelides A and B: studies on the macro-ring closure strategy.

Highly concise syntheses of (+)-macrosphelides A and B were accomplished in this study. The key feature of our synthetic route involved the direct three-carbon homologation of the readily available Weinreb amide 6 by the addition of a trans-vinylogous ester anion equivalent and facile construction of the 16-membered macrolide skeleton of macrosphelides via an intramolecular nitrile oxide-olefin cycloaddition. The syntheses of macrosphelides A and B were completed with a 30 and 20% overall yield, respectively. This paper describes the details of our syntheses.

Analysis of Saccharomyces cerevisiae histone H3 mutants reveals the role of the alphaN helix in nucleosome function.

To understand the role of histone H3 sub-domains in chromatin function, 35 histone H3 tandem alanine mutants were generated and tested for their viability and sensitivity to DNA damaging agents. Among 13 non-viable H3 mutants, 6 were mapped around the alphaN helix and preceding tail region. Mutants with individual alanine substitutions in this region were viable but developed multiple sensitivities to DNA damaging agents. The only viable triple mutant, REI49-50A, in the alphaN helix region could not grow when combined with histone chaperone mutations, such as asf1Delta, cac1Delta, or hir1Delta, suggesting that this particular region is important when the histone assembly/disassembly pathway is compromised. In addition, further analysis showed that T45, E50, or F54 of the alphaN helix genetically interacted with a histone chaperone (Asf1) and transcription elongation factors (Paf1 and Hpr1). These results suggest a specific role of the H3 alphaN helix in histone dynamics mediated by histone chaperones, which might be important during transcription elongation.

Synthesis and biological evaluation of 4(5)-(6-alkylpyridin-2-yl)imidazoles as transforming growth factor-beta type 1 receptor kinase inhibitors.

A series of 4(5)-(6-alkylpyridin-2-yl)imidazoles 13a-p, 17a, and 17b have been synthesized and evaluated for ALK5 inhibitory activity in an enzyme assay and in cell-based luciferase reporter assays. The quinoxalinyl analogue 13e inhibited ALK5 phosphorylation with an IC50 of 0.012 muM and showed more than 90% inhibition at 0.05 muM in a luciferase reporter assay using HaCaT cells transiently transfected with p3TP-luc reporter construct. The binding mode of 13e generated by flexible docking studies shows that 13e fits well into the active site cavity of ALK5 by forming several tight interactions.

Computer-aided identification of new histone deacetylase 6 selective inhibitor with anti-sepsis activity.

Histone deacetylase (HDAC) inhibitors have been recognized as promising approaches to the treatment of various human diseases including cancer, inflammation, neurodegenerative diseases, and metabolic disorders. Several pan-HDAC inhibitors are currently approved only as anticancer drugs. Interestingly, SAHA (vorinostat), one of clinically available pan-HDAC inhibitors, shows an anti-inflammatory effect at concentrations lower than those required for inhibition of tumor cell growth. It was also reported that HDAC6 selective inhibitor tubastatin A has anti-inflammatory and anti-rheumatic effect. In our efforts to develop novel HDAC inhibitors, we rationally designed various HDAC inhibitors based on the structures of two hit compounds identified by virtual screening of chemical database. Among them, 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) was identified as a HDAC6 selective inhibitor (IC50 values of 0.199 µM for HDAC6 versus 13.8 µM for HDAC1), and it did not show significant cytotoxicity against HeLa cells. In vivo biological evaluation of 9a was conducted on a lipopolysaccharide (LPS)-induced mouse model of sepsis. The compound 9a significantly improved 40% survival rate (P = 0.0483), and suppressed the LPS-induced increase of TNF-α and IL-6 mRNA expression in the liver of mice. Our study identified novel HDAC6 selective inhibitor 9a, which may serve as a potential lead for the development of anti-inflammatory or anti-sepsis agents.

Dehydroevodiamine•HCl Protects Against Memory Impairment and Cerebral Amyloid-β Production in Tg2576 Mice by Acting as a β-Secretase Inhibitor.

We previously demonstrated that dehydroevodiamine•HCl (DHED), which was purified from Evodia rutaecarpa Bentham (Rutaceae), has beneficial effects on memory impairment and neuronal damage in three disease models. To investigate the preventive action of DHED in Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by memory decline, amyloid-ß (Aß) protein-containing neuritic plaques and neurofibrillary tangles, in this study, we proposed that DHED may be therapeutically effective against the memory impairment and disease-related neurochemical changes that occur in Tg2576 (Tg) mice. DHED (0.5 mg/kg) was intraperitoneally administered to 7-month-old Tg and wild type mice for 4 months. In passive avoidance and water maze tests, DHED improved memory impairment of Tg mice after 4 months of administration. DHED also reduced cortical levels of soluble Aß40, soluble Aß42 and total Aß peptides in the Tg mice. Additionally, we investigated whether DHED may be a ß-secretase inhibitor that affects the production of Aß related to the formation of neuritic plaques. DHED directly inhibited ß-secretase activity in a concentrationdependent manner. The concentration required for 50 % enzyme inhibition (IC50) was 40.96 µM, and DHED may act as a competitive inhibitor of ß-secretase. Moreover, DHED interacted strongly with BACE1 (ß-secretase 2QP8), as demonstrated in the analysis of the binding mode of DHED in the active site of human BACE1. In conclusion, DHED may exhibit therapeutic effects for AD as a ß-secretase inhibitor.

Discovery and development of DNA methyltransferase inhibitors using in silico approaches.

Multiple strategies have evolved during the past few years to advance epigenetic compounds targeting DNA methyltransferases (DNMTs). Significant progress has been made in HTS, lead optimization and determination of 3D structures of DNMTs. In light of the emerging concept of epi-informatics, computational approaches are employed to accelerate the development of DNMT inhibitors helping to screen chemical databases, mine the DNMT-relevant chemical space, uncover SAR and design focused libraries. Computational methods also synergize with natural-product-based drug discovery and drug repurposing. Herein, we survey the latest developments of in silico approaches to advance epigenetic drug and probe discovery targeting DNMTs.

Structure-Activity Relationship of Indole-Tethered Pyrimidine Derivatives that Concurrently Inhibit Epidermal Growth Factor Receptor and Other Angiokinases.

Antiangiogenic agents have been widely investigated in combination with standard chemotherapy or targeted cancer agents for better management of advanced cancers. Therapeutic agents that concurrently inhibit epidermal growth factor receptor and other angiokinases could be useful alternatives to combination therapies for epidermal growth factor receptor-dependent cancers. Here, we report the synthesis of an indole derivative of pazopanib using a bioisosteric replacement strategy, which was designated MKP101. MKP101 inhibited not only the epidermal growth factor receptor with an IC50 value of 43 nM but also inhibited angiokinases as potently as pazopanib. In addition, MKP101 effectively inhibited vascular endothelial growth factor-induced endothelial proliferation, tube formation, migration of human umbilical vein endothelial cells and proliferation of HCC827, an epidermal growth factor receptor-addicted cancer cell line. A docking model of MKP101 and the kinase domain of the epidermal growth factor receptor was generated to predict its binding mode, and validated by synthesizing and evaluating MKP101 derivatives. Additionally, a study of structure-activity relationships of indolylamino or indolyloxy pyrimidine analogues derived from MKP101 demonstrated that selectivity for epidermal growth factor receptor and other angiokinases, especially vascular endothelial growth factor receptor 2 depends on the position of substituents on pyrimidine and the type of link between pyrimidine and the indole moiety. We believe that this study could provide a basis for developing angiokinase inhibitors having high affinity for the epidermal growth factor receptor, from the pyrimidine scaffold.

Rational design of an indolebutanoic acid derivative as a novel aldose reductase inhibitor based on docking and 3D QSAR studies of phenethylamine derivatives.

A series of 45 phenethylamine derivatives were synthesized and evaluated for their inhibitory activity against pig kidney aldose reductase (ALR2, EC 1.1.1.21). Their IC(50) values ranged from 400 microM to 24 microM. The binding modes of compounds at the active site of ALR2 were examined using flexible docking. The results indicated that phenethylamine derivatives nicely fit into the active pocket of ALR2 by forming various hydrogen bonding and hydrophobic interactions. 3D-QSAR analysis was also conducted using FlexX-docked alignment of the compounds. The best prediction was obtained by CoMSIA combined with hydrophobic and hydrogen bond donor/acceptor field (q(2) = 0.557, r(2) = 0.934). A new derivative, 4-oxo-4-(4-hydroxyindole)butanoic acid, was designed, taking into account the CoMSIA field and the binding mode derived by FlexX docking. This rationally designed compound exhibits an ALR2 inhibition with an IC(50) value of 7.4 microM, which compares favorably to that of a well-known ALR2 inhibitor, tolrestat (IC(50) = 16 microM) and represents a potency approximately 240-fold higher than that of an original phenethylamine lead compound, YUA001.

Synthesis and biological evaluation of 3-(4-substituted-phenyl)-N-hydroxy-2-propenamides, a new class of histone deacetylase inhibitors.

Inhibitors of histone deacetylases (HDACs) have been shown to induce differentiation and/or apoptosis of human tumor cells. Novel 3-(4-substituted-phenyl)-N-hydroxy-2-propenamides have been prepared as a new class of HDAC inhibitors and evaluated for their antiproliferative activity and HDAC inhibitory activity. Incorporation of a 1,4-phenylene carboxamide linker, shown by 5, and a 4-(dimethylamino)phenyl or 4-(pyrrolidin-1-yl)phenyl group as a cap substructure generated highly potent hydroxamic acid-based HDAC inhibitors 5a and 5b.

Synthesis and anti-renal fibrosis activity of conformationally locked truncated 2-hexynyl-N(6)-substituted-(N)-methanocarba-nucleosides as A3 adenosine receptor antagonists and partial agonists.

Truncated N(6)-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4'-thioadenosines. Hydrophobic N(6) and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N(6)-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with Ki values of 7.8-16.0 nM. N(6)-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-ß1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-ß1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 µM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.