The Stat3 inhibitor F0648-0027 is a potential therapeutic against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease characterized by chronic joint inflammation, pain and joint destruction, leading to alteration in activities of daily living, yet pathological mechanisms underlying the condition are not fully clarified. To date, various therapeutic agents have been developed as RA therapy including DMARDs and/or biological agents that target inflammatory cytokines or inhibit JAK. Here we asked whether inhibiting signal transducer and activator of transcription 3 (Stat3) activity would antagonize RA. Stat3 forms dimers when activated and undergoes nuclear translocalization; thus we screened approximately 4.9 million small compounds as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening. We identified 15 as strong candidates as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening from those compounds. Four of the 15 significantly inhibited expression of IL-6 and RANKL, both of which are direct targets of Stat3, induced by IL-6. Among four, one compound, F0648-0027, significantly inhibited arthritis development without apparent adverse effects in vivo in collagen-induced arthritis model mice. F0648-0027 also significantly blocked Stat3 phosphorylation and nuclear localization following IL-6 stimulation of fibroblasts. These data suggest that Stat3 is a target for collagen-induced arthritis in mice, and that F0648-0027 could serve as a therapeutic reagent against comparable conditions in humans.

Identification of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.

Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is overexpressed in many cancers. Numerous EZH2 inhibitors have been developed as anticancer agents, but recent studies have also focused on protein-protein interaction (PPI) between embryonic ectoderm development (EED) and EZH2 as a novel drug discovery target. Because EED indirectly enhances EZH2 enzymatic activity, EED-EZH2 PPI inhibitors suppress the methyltransferase activity and inhibit cancer growth. By contrast to the numerous promising EZH2 inhibitors, there are a paucity of EED-EZH2 PPI inhibitors reported in the literature. Here, we aimed to discover novel EED-EZH2 PPI inhibitors by first identifying possible binders of EED using an in-house knowledge-based in silico fragment mapping method. Next, 3D pharmacophore models were constructed from the arrangement pattern of the potential binders mapped onto the EED surface. In all, 16 compounds were selected by 3D pharmacophore-based virtual screening followed by docking-based virtual screening. In vitro evaluation revealed that five of these compounds exhibited inhibitory activities. This study has provided structural insights into the discovery and the molecular design of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

[Identifying Inhibitors of USP7-HDM2 Protein-Protein Interaction (PPI) by the in Silico Fragment-mapping Method].

Proteolysis mediated by the ubiquitin-proteome system plays an important role in cancer. Recently, a deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7) has attracted attention as a key regulator of the p53-human double minute 2 (HDM2) pathway in cancer cells. Although some USP7 enzyme inhibitors have been identified, issues related to activity and selectivity prevent their therapeutic application. In this study, we aimed to search for novel USP7-HDM2 protein-protein interaction (PPI) inhibitors that do not affect the USP7 enzyme activity. Using the fragment-mapping program Fsubsite and the canonical subsite-fragment database (CSFDB) developed in our laboratory, we mapped a variety of fragments onto USP7 protein and constructed 3D-pharmacophore models based on the arrangement patterns of the mapped fragments. Finally, we performed 3D pharmacophore-based virtual screening of a commercial compound database and successfully selected promising USP7-HDM2 PPI inhibitor candidates.

Validation of molecular force field parameters for peptides including isomerized amino acids.

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age-related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l-α-amino acids, the accuracy of the computational results for d-amino acids and ß-amino acids was comparable to those for l-α-amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.

Exploration of DPP-IV inhibitors with a novel scaffold by multistep in silico screening.

Dipeptidyl peptidase-IV (DPP-IV), an enzyme that degrades incretins-hormones that promote insulin secretion-is a therapeutic target for type 2 diabetes, with a number of its inhibitors having been launched as therapies for diabetes. Since adverse effects of these inhibitors have recently been reported, the development of novel DPP-IV inhibitors with higher efficacy and safety is required. We, therefore, screened for novel DPP-IV inhibitors using the combination of an in silico drug discovery technique and a DPP-IV assay system. We initially selected seven candidate compounds as DPP-IV inhibitors from a database consisting of four million compounds by a multistep in silico screening procedure combining pharmacophore-based screening, docking calculation and the analysis of three-dimensional quantitative structure-activity relationship. We then measured the inhibitory activity of the selected compounds and identified a hit compound. In addition, we discuss the structure-activity relationship between the binding mode model and inhibitory activity of the hit compound.

Multi-step virtual screening to develop selective DYRK1A inhibitors.

Developing selective inhibitors for a particular kinase remains a major challenge in kinase-targeted drug discovery. Here we performed a multi-step virtual screening for dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) inhibitors by focusing on the selectivity for DYRK1A over cyclin-dependent kinase 5 (CDK5). To examine the key factors contributing to the selectivity, we constructed logistic regression models to discriminate between actives and inactives for DYRK1A and CDK5, respectively, using residue-based binding free energies. The residue-based parameters were calculated by molecular mechanics-generalized Born surface area (MM-GBSA) decomposition methods for kinase-ligand complexes modeled by computer ligand docking. Based on the findings from the logistic regression models, we built a three-dimensional (3D) pharmacophore model and chose filter criteria for the multi-step virtual screening. The virtual hit compounds obtained from the screening were assessed for their inhibitory activities against DYRK1A and CDK5 by in vitro assay. Our screening identified two novel selective DYRK1A inhibitors with IC50 values of several µM for DYRK1A and >100µM for CDK5, which can be further optimized to develop more potent selective DYRK1A inhibitors.

Prediction of three-dimensional structures and structural flexibilities of wild-type and mutant cytochrome P450 1A2 using molecular dynamics simulations.

In this study, we investigated the effect of genetic polymorphism on the three-dimensional (3D) conformation of cytochrome P450 1A2 (CYP1A2) using molecular dynamics (MD) simulations. CYP1A2, a major drug-metabolizing enzyme among cytochrome P450 enzymes (CYPs), is known to have many variant alleles. The genetic polymorphism of CYP1A2 may cause individual differences in the pharmacokinetics of medicines. By performing 100ns or longer MD simulations, we investigated the influence of amino acid mutation on the 3D structures and the dynamic properties of proteins. The results show that the static structures were changed by the mutations of amino acid residues, not only near the mutated residues but also in distant portions of the proteins. Moreover, the mutation of only one amino acid was shown to change the structural flexibility of proteins, which may influence the substrate recognition and enzymatic activity. Our results clearly suggest that it is necessary to investigate the dynamic property as well as the static 3D structure for understanding the change of the enzymatic activity of mutant CYP1A2.

Inhibition of adenosine deaminase (ADA)-mediated metabolism of cordycepin by natural substances.

Cordycepin, which is an analogue of a nucleoside adenosine, exhibits a wide variety of pharmacological activities including anticancer effects. In this study, ADA1- and ADA2-expressing HEK293 cells were established to determine the major ADA isoform responsible for the deamination of cordycepin. While the metabolic rate of cordycepin deamination was similar between ADA2-expressing and Mock cells, extensive metabolism of cordycepin was observed in the ADA1-expressing cells with K m and V max values of 54.9 µmol/L and 45.8 nmole/min/mg protein. Among five natural substances tested in this study (kaempferol, quercetin, myricetin, naringenin, and naringin), naringin strongly inhibited the deamination of cordycepin with K i values of 58.8 µmol/L in mouse erythrocytes and 168.3 µmol/L in human erythrocytes. A treatment of Jurkat cells with a combination of cordycepin and naringin showed significant cytotoxicity. Our in silico study suggests that not only small molecules such as adenosine derivatives but also bulky molecules like naringin can be a potent ADA1 inhibitor for the clinical usage.

Genistein and daidzein, typical soy isoflavones, inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes.

SCOPE: Previous reports suggest that soy isoflavones have multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about whether soy isoflavones can inhibit the downregulation of adiponectin and their molecular mechanisms. In the present study, we demonstrate that genistein (Gen) or daidzein (Dai) can significantly inhibit the downregulation of adiponectin expression via unique and different molecular mechanisms. METHODS AND RESULTS: Pretreatment with Gen or Dai significantly inhibited the tumor necrosis factor-alpha (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Gen inhibited the TNF-α-induced c-Jun-NH(2) -terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on JNK1 with Gen or Dai clearly supported our experimental results. However, Dai did not significantly inhibit JNK signaling. Dai did, however, inhibit the TNF-α-induced downregulation of forkhead box-containg protein O1, which is also involved in adiponectin expression. CONCLUSION: These data demonstrate that: (i) both Gen and Dai significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes; (ii) Gen is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin; and (iii) Dai can inhibit the downregulation of adiponectin by restoring the TNF-α-mediated reduction of forkhead box-containg protein O1 protein expression.

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy.

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

Brazilian propolis-derived components inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes.

BACKGROUND: Previous reports suggest that Brazilian propolis has multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about the molecular mechanisms by which these compounds inhibit the downregulation of adiponectin. METHODS: The effect of various Brazilian propolis-derived components on inhibition of tumor necrosis factor-α (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes and molecular mechanism was investigated. RESULTS AND CONCLUSIONS: Pretreatment with either artepillin C (C3) or its derivative (C4) significantly inhibited TNF-α-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Interestingly, C3 strongly activated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. Treatment of adipocytes with C3 resulted in the upregulation of adiponectin and fatty acid-binding protein 4 expression, but C4 did not significantly induce PPARγ transactivation. C4 did, however, inhibit the TNF-α-induced c-Jun-NH(2)-terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on hPPARγ with C3 and JNK1 with C4 clearly supported our experimental results. These data demonstrate that 1) both C3 and C4 significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes, 2) C3 functions as a PPARγ agonist, and its inhibition of the effect of TNF-α is due to this PPARγ transactivation, and 3) C4 is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin. GENERAL SIGNIFICANCE: Brazilian propolis-derived components (C3 and C4) can significantly inhibit TNF-α-mediated downregulation of adiponectin in adipocytes, although they do so via different mechanisms.

Novel 12-membered non-antibiotic macrolides from erythromycin A; EM900 series as novel leads for anti-inflammatory and/or immunomodulatory agents.

Herein, we report the design and synthesis of the novel 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900), which was found to be a potent anti-inflammatory and/or immunomodulatory agent, capable of promoting monocyte to macrophage differentiation. This molecule shows improved acid stability, does not exhibit any anti-bacterial activity and has relatively low cytotoxicity against THP-1 cells. In addition, one of its analogues, (8R,9S)-4″,13-O-diacetyl-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM911), was found to be twice as effective as EM900.

Treatment of arthritis with a selective inhibitor of c-Fos/activator protein-1.

To inhibit arthritis upstream of inflammatory cytokine release and matrix metalloproteinase (MMP) action, we designed de novo a small-molecule inhibitor of c-Fos/activator protein-1 (AP-1) using three-dimensional (3D) pharmacophore modeling. This model was based on the 3D structure of the basic region-leucine zipper domain of AP-1-DNA complex. Administration of this inhibitor prevented type II collagen-induced arthritis from day 21, before the onset of arthritis, or from day 27, resolved arthritis after its onset. Suppression of disease was accomplished by reducing the amounts of inflammatory cytokines and MMPs in vivo in sera and joints and in vitro in synovial cell and chondrocyte cultures. The primary action of this molecule was the inhibition of matrix-degrading MMPs and inflammatory cytokines including interleukin 1beta; this molecule also synergized with anti-tumor necrosis factor alpha to inhibit arthritis. Thus, selective inhibition of c-Fos/AP-1 resolves arthritis in a preclinical model of the disease.

A possible role of multidrug resistance-associated protein 2 (Mrp2) in hepatic excretion of PCB126, an environmental contaminant: PBPK/PD modeling.

3,3',4,4',5'-Pentachlorobiphenyl (PCB126) is a carcinogenic environmental pollutant and its toxicity is mediated through binding with aryl hydrocarbon receptor (AhR). Earlier, we found that PCB126 treated F344 rats had 110-400 times higher PCB126 concentration in the liver than in the fat. Protein binding was suspected to be a major factor for the high liver concentration of PCB126 despite its high lipophilicity. In this research, we conducted a combined pharmacokinetic/pharmacodynamic study in male F344 rats. In addition to blood and tissue pharmacokinetics, we use the development of hepatic preneoplastic foci (glutathione-S-transferase placental form [GSTP]) as a pharmacodynamic endpoint. Experimental data were utilized for building a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model. PBPK/PD modeling was consistent with the experimental PK and PD data. Salient features of this model include: (1) bindings between PCB126 and hepatic proteins, particularly the multidrug resistance-associated protein (Mrp2), a protein transporter; (2) Mrp2-mediated excretion; and (3) a relationship between area under the curve of PCB126 in the livers and % volume of GSTP foci. Mrp2 involvement in PCB126 pharmacokinetics is supported by computational chemistry calculation using a three-dimensional quantitative structure-activity relationship model of Mrp2 developed by S. Hirono et al. (2005, Pharm. Res. 22, 260-269). This work, for the first time, provided a plausible role of a versatile hepatic transporter for drugs, Mrp2, in the disposition of an important environmental pollutant, PCB126.

Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor.

BACKGROUND: We previously synthesized a novel s-triazine derivative, ZSTK474 [2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine], that strongly inhibited the growth of tumor cells. We identified its molecular target, investigated its effects on cellular signaling pathways, and examined its antitumor efficacy and toxicity in vivo. METHODS: We used COMPARE analysis of chemosensitivity measurements from 39 human cancer cell lines and identified phosphatidylinositol 3-kinase (PI3K) as a molecular target for ZSTK474. PI3K was immunoprecipitated from A549 cell lysates, and its activity was measured by assessing the incorporation of 32P into phosphatidylinositol. We used the crystal structure of the PI3K-LY294002 complex to model the binding of ZSTK474 to PI3K (where LY294002 is a known PI3K inhibitor). PI3K downstream activity was analyzed by immunoblotting. Antitumor activity of ZSTK474 was examined against A549, PC-3, and WiDr xenografts in nude mice. Phosphorylation of Akt, a serine/threonine protein kinase and a major signaling component downstream of PI3K, was assessed in vivo by immunohistochemistry. RESULTS: PI3K was identified as a molecular target for ZSTK474 by COMPARE analysis. We confirmed that ZSTK474 directly inhibited PI3K activity more efficiently than the PI3K inhibitor LY294002. At concentrations of 1 microM, ZSTK474 and LY2194002 reduced PI3K activity to 4.7% (95% confidence interval [CI] = 3.2% to 6.1%) and 44.6% (95% CI = 38.9% to 50.3%), respectively, of the untreated control level. Molecular modeling of the PI3K-ZSTK474 complex indicated that ZSTK474 could bind to the ATP-binding pocket of PI3K. ZSTK474 inhibited phosphorylation of signaling components downstream from PI3K, such as Akt and glycogen synthase kinase 3beta, and mediated a decrease in cyclin D1 levels. ZSTK474 administered orally to mice had strong antitumor activity against human cancer xenografts without toxic effects in critical organs. Akt phosphorylation was reduced in xenograft tumors after oral administration of ZSTK474. CONCLUSION: ZSTK474 is a new PI3K inhibitor with strong antitumor activity against human cancer xenografts without toxic effects in critical organs. ZSTK474 merits further investigation as an anticancer drug.

Three-dimensional solution structure of EM703 with potent promoting activity of monocyte-to-macrophage differentiation.

EM703, which is an erythromycin derivative synthesized by our group, has a potent promoting activity of monocyte-to-macrophage differentiation in vitro. Its activity is approximately 300 times higher than that of erythromycin A (EM-A). In this study, we determined three-dimensional (3D) solution structures of EM703 and EM-A, and compared them using a superposition method, in order to investigate the 3D structure-activity relationship. We found a distinct difference between the 3D structures of these molecules, which might be an important factor in their divergent activities.

[Anti-AP-1 treatment].

This review emphasizes our first discovery on the contribution of over-activation of c-fos gene to the pathogenesis of rheumatoid joint destruction. In particular, c-Fos signalling was required for increased activity of synovial mesenchymal cells which finally leads to rheumatoid joint destruction and peri-articular osteoporosis. Over-activation of c-fos via Wee1 kinase is responsible for tumor-like synovial over-growth. Our team designed anti-c-Fos drugs that specifically inhibit action of c-Fos at the AP-1 consensus sequence by using a computer-assisted drug design, which was the front-runner work executed in Japan.

New AMBER force field parameters of heme iron for cytochrome P450s determined by quantum chemical calculations of simplified models.

The heme protein, cytochrome P450, is an oxidoreductase that plays an important role in drug metabolism. To model P450s using molecular mechanics methods and classical molecular dynamics simulations, force field parameters and atomic charges are required. Because these parameters are generally obtained by quantum chemical methods, an appropriate simplified model for the iron-porphyrin system was needed. In this study, two models with a five-coordinated Fe(III) mimicking the sextet spin state of P450s are proposed, which are optimized by semiempirical and ab initio unrestricted Hartree-Fock methods. The results produced using the simpler of the two models were similar to those of the more complex model; therefore, the more simplified model of P450 can be used without a loss of accuracy. Furthermore, several quantum chemical calculations were carried out on the simpler model to investigate which method was most suitable for iron-porphyrin systems. The results calculated by hybrid density functional theory (DFT), with the MIDI basis set for iron, reproduced the three-dimensional structures determined by X-ray diffraction and extended X-ray absorption fine-structure experiments. From these results, atomic charges and force-field parameters for molecular mechanics and molecular dynamics calculations were obtained.

Use of photoaffinity labeling and site-directed mutagenesis for identification of the key residue responsible for extraordinarily high affinity binding of UCN-01 in human alpha1-acid glycoprotein.

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor anticancer drug currently undergoing a phase II clinical trial. The low distribution volumes and systemic clearance of UCN-01 in human patients have been found to be caused in part by its extraordinarily high affinity binding to human alpha1-acid glycoprotein (hAGP). In the present study, we photolabeled hAGP with [3H]UCN-01 without further chemical modification. The photolabeling specificity of [3H]UCN-01 was confirmed by findings in which other hAGP binding ligands inhibited formation of covalent bonds between hAGP and [3H]UCN-01. The amino acid sequence of the photolabeled peptide was concluded to be SDVVYTDXK, corresponding to residues Ser-153 to Lys-161 of hAGP. No PTH derivatives were detected at the 8th cycle, which corresponded to the 160th Trp residue. This strongly implies that Trp-160 was photolabeled by [3H]UCN-01. Three recombinant hAGP mutants (W25A, W122A, and W160A) and wild-type recombinant hAGP were photolabeled by [3H]UCN-01. Only mutant W160A showed a marked decrease in the extent of photoincorporation. These results strongly suggest that Trp-160 plays a prominent role in the high affinity binding of [3H]UCN-01 to hAGP. A docking model of UCN-01 and hAGP around Trp-160 provided further details of the binding site topology.