Zenyu Shiokawa

Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine derivative, as a highly potent and orally active hedgehog signaling inhibitor: modification of the core skeleton for improved solubility.

We recently reported the discovery of the novel pyrrolo[3,2-c]quinoline-4-one derivative 1 as a potent inhibitor of Hedgehog (Hh) pathway signaling. However, the PK evaluation of 1 at high dosage (100 mg/kg) revealed the C(max) value 3.63 μg/mL, likely due to poor solubility of this compound. Efforts to improve solubility by reducing the aromatic ring count of the core system led to N-methylpyrrolo[3,2-c]pyridine derivative 11. Further optimization of the 3-alkoxy group led to compound 11d with acceptable solubility and potent Hh inhibitory activity. Compound 11d suppressed transcription factor Gli1 mRNA expression in tumor-associated stromal tissue and inhibited tumor growth (treatment/control ratio, 3%) in a mouse medulloblastoma allograft model owing to the improved PK profile based on increased solubility. Compound 11d (TAK-441) is currently in clinical trials for the treatment of advanced solid tumors.

Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog signaling inhibitors.

The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimization. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allograft model.

Design and Synthesis of Potent Inhibitor of Apoptosis (IAP) Proteins Antagonists Bearing an Octahydropyrrolo[1,2-a]pyrazine Scaffold as a Novel Proline Mimetic.

To develop novel inhibitor of apoptosis (IAP) proteins antagonists, we designed a bicyclic octahydropyrrolo[1,2-a]pyrazine scaffold as a novel proline bioisostere. This design was based on the X-ray co-crystal structure of four N-terminal amino acid residues (AVPI) of the second mitochondria-derived activator of caspase (Smac) with the X-chromosome-linked IAP (XIAP) protein. Lead optimization of this scaffold to improve oral absorption yielded compound 45, which showed potent cellular IAP1 (cIAP1 IC(50): 1.3 nM) and XIAP (IC(50): 200 nM) inhibitory activity, in addition to potent tumor growth inhibitory activity (GI(50): 1.8 nM) in MDA-MB-231 breast cancer cells. X-ray crystallographic analysis of compound 45 bound to XIAP and to cIAP1 was achieved, revealing the various key interactions that contribute to the higher cIAPI affinity of compound 45 over XIAP. Because of its potent IAP inhibitory activities, compound 45 (T-3256336) caused tumor regression in a MDA-MB-231 tumor xenograft model (T/C: -53% at 30 mg/kg).

Design, synthesis, and biological activities of novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists with improved membrane permeability across MDR1 expressing cells.

We previously reported octahydropyrrolo[1,2-a]pyrazine derivative 2 (T-3256336) as a potent antagonist for inhibitors of apoptosis (IAP) proteins. Because compound 2 was susceptible to MDR1 mediated efflux, we developed another scaffold, hexahydropyrazino[1,2-a]indole, using structure-based drug design. The fused benzene ring of this scaffold was aimed at increasing the lipophilicity and decreasing the basicity of the scaffold to improve the membrane permeability across MDR1 expressing cells. We established a chiral pool synthetic route to yield the desired tricyclic chiral isomers. Chemical modification of the core scaffold led to a representative compound 50, which showed strong inhibition of IAP binding (X chromosome-linked IAP [XIAP]: IC50 23 nM and cellular IAP [cIAP]: IC50 1.1 nM) and cell growth inhibition (MDA-MB-231 cells: GI50 2.8 nM) with high permeability and low potential of MDR1 substrate.

Synthesis and evaluation of hedgehog signaling inhibitor with novel core system.

As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next focused on modification of the lower part including core skeletons to discover new Hh signaling inhibitors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure analysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1 expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441) because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical candidate with good structural and pharmacokinetic advantages.

Gene Signature–Based Approach Identified MEK1/2 as a Potential Target Associated With Relapse After Anti-TNFα Treatment for Crohn’s Disease

Abstract Background Anti–tumor necrosis factor alpha (anti-TNFα) therapy has become the mainstay of therapy for Crohn’s disease (CD). However, post-therapy, the recurrence rate is still high. The aim of this study was to dissect the molecular mechanism for recurrence of CD treated with anti-TNFα therapy and investigate novel therapeutic options that could induce complete remission. Methods We re-analyzed publicly available mucosal gene expression data from CD patients pre– and post–infliximab therapy to extract the transcriptional differences between responders and healthy controls. We used a systematic computational approach based on identified differences to discover novel therapies and validated this prediction through in vitro and in vivo experimentation. Results We identified a set of 3545 anti-TNFα therapy-untreatable genes (TUGs) that are significantly regulated in intestinal epithelial cells, which remain altered during remission. Pathway enrichment analysis of these genes clearly showed excessive growth state and suppressed terminal differentiation, whereas immune components were clearly resolved. Through in silico screening strategy, we observed that MEK inhibitors were predicted to revert expression of genes dysregulated in infliximab responders. In vitro transcriptome analysis demonstrated that selective MEK1/2 inhibitor significantly normalized reference genes from TUGs. In addition, in vitro functional study proved that MEK1/2 inhibitor facilitated intestinal epithelial differentiation. Finally, using murine colitis model, administration of MEK1/2 inhibitor significantly improved diarrhea and histological score. Conclusions Our data revealed the abnormalities in anti-TNFα responders’ CD colons that would be cause of recurrence of CD. Also, we provided evidence regarding MEK1/2 inhibitor as a potential treatment against CD to achieve sustainable remission.