Small-Scale Panel Comprising Diverse Gene Family Targets To Evaluate Compound Promiscuity.

Despite the recent advances in the life sciences and the remarkable investment in drug discovery research, the success rate of small-molecule drug development remains low. Safety is the second most influential factor of drug attrition in clinical studies; thus, the selection of compounds with fewer toxicity concerns is crucial to increase the success rate of drug discovery. Compounds that promiscuously bind to multiple targets are likely to cause unexpected pharmacological activity that may lead to adverse effects. Therefore, avoiding such compounds during early research stages would contribute to identifying compounds with a higher chance of success in the clinic. To evaluate the interaction profile against a wide variety of targets, we constructed a small-scale promiscuity panel (PP) consisting of eight targets (ROCK1, PDE4D2, GR, PPARγ, 5-HT2B, adenosine A3, M1, and GABAA) that were selected from diverse gene families. The validity of this panel was confirmed by comparison with the promiscuity index evaluated from larger-scale panels. Analysis of data from the PP revealed that both lipophilicity and basicity are likely to increase promiscuity, while the molecular weight does not significantly contribute. Additionally, the promiscuity assessed using our PP correlated with the occurrence of both in vitro cytotoxicity and in vivo toxicity, suggesting that the PP is useful to identify compounds with fewer toxicity concerns. In summary, this small-scale and cost-effective PP can contribute to the identification of safer compounds that would lead to a reduction in drug attrition due to safety issues.

Discovery and characterization of a small-molecule enteropeptidase inhibitor, SCO-792.

Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated Kinact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated koff rate of 0.047/hour, which suggested that SCO-792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against enteropeptidase. SCO-792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting enteropeptidase on biological actions.

Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application.

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.

Discovery of Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1) Inhibitors as Anticancer Agents.

A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a poor pharmacokinetic (PK) profile and limited chemical accessibility, making it suboptimal for use in anticancer research. To identify potent SCD1 inhibitors with more promising PK profiles, we newly designed a series of 'non-spiro' 4, 4-disubstituted piperidine derivatives based on molecular modeling studies. As a result, we discovered compound 1a, which retained moderate SCD1 binding affinity. Optimization around 1a was accelerated by analyzing Hansch-Fujita and Hammett constants to obtain 4-phenyl-4-(trifluoromethyl)piperidine derivative 1n. Fine-tuning of the azole moiety of 1n led to compound 1o (T-3764518), which retained nanomolar affinity and exhibited an excellent PK profile. Reflecting the good potency and PK profile, orally administrated compound 1o showed significant pharmacodynamic (PD) marker reduction (at 0.3mg/kg, bid) in HCT116 mouse xenograft model and tumor growth suppression (at 1mg/kg, bid) in 786-O mouse xenograft model. In conclusion, we identified a new series of SCD1 inhibitors, represented by compound 1o, which represents a promising new chemical tool suitable for the study of SCD1 biology as well as the potential development of novel anticancer therapies.

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells.

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as "enzyme bags" and incubated at 30 degrees C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 microg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26 degrees C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.