Corrigendum to "Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor" [Biochem. Biophys. Rep. 21 (2020) 100726].

[This corrects the article DOI: 10.1016/j.bbrep.2020.100726.].

Synergistic apoptotic effects in cancer cells by the combination of CLK and Bcl-2 family inhibitors.

Emerging evidence indicates that alternative splicing plays a critical role in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have recently attracted considerable attention as a novel class of cancer therapeutics. CDC-like kinases (CLKs) are central to exon recognition in mRNA splicing and CLK inhibitors exhibit anti-tumour activities. Most importantly, molecular mechanism-based combination strategies for cancer therapy must be considered. However, it remains unclear whether CLK inhibitors modulate expression and splicing of apoptosis-related genes, and whether CLK inhibitors enhance cytotoxicity in combination with apoptosis inducers. Here we report an appropriate mechanism-based drug combination approach. Unexpectedly, we found that the CLK inhibitor T3 rapidly induced apoptosis in A2780 cells and G2/M cell cycle arrest in HCT116 cells. Regardless of the different phenotypes of the two cancer cell types, T3 decreased the levels of anti-apoptotic proteins (cIAP1, cIAP2, XIAP, cFLIP and Mcl-1) for a short period of exposure and altered the splicing of the anti-apoptotic MCL1L and CFLAR isoform in A2780 and HCT116 cells. In contrast, other members of the Bcl-2 family (i.e., Bcl-xL and Bcl-2) were resistant to T3-induced expression and splicing modulation. T3 and a Bcl-xL/Bcl-2 inhibitor synergistically induced apoptosis. Taken together, the use of a CLK inhibitor is a novel therapeutic approach to sensitise cancer cells to Bcl-xL/Bcl-2 inhibitors.

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor.

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling. The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor. Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.

Discovery of a Novel, Highly Potent, and Selective Thieno[3,2-d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent.

In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.

Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor.

Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.

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.

2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase.

In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50=0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.

Identification of a new class of potent Cdc7 inhibitors designed by putative pharmacophore model: Synthesis and biological evaluation of 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-ones.

Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays important roles in the regulation of DNA replication process. A genetic study indicates that Cdc7 inhibition can induce selective tumor-cell death in a p53-dependent manner, suggesting that Cdc7 is an attractive target for the treatment of cancers. In order to identify a new class of potent Cdc7 inhibitors, we generated a putative pharmacophore model based on in silico docking analysis of a known inhibitor with Cdc7 homology model. The pharmacophore model provided a minimum structural motif of Cdc7 inhibitor, by which preliminary medicinal chemistry efforts identified a dihydrothieno[3,2-d]-pyrimidin-4(1H)-one scaffold having a heteroaromatic hinge-binding moiety. The structure-activity relationship (SAR) studies resulted in the discovery of new, potent, and selective Cdc7 inhibitors 14a, c, e. Furthermore, the high selectivity of 14c, e for Cdc7 over Rho-associated protein kinase 1 (ROCK1) is discussed by utilizing a docking study with Cdc7 and ROCK2 crystal structures.

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 3. Evaluation of 5-amino-linked thiazolo[5,4-d]pyrimidine and thiazolo[5,4-b]pyridine derivatives.

Our aim was to discover RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors that possess strong activity and sufficient oral absorption, and thus, we selected a 5-amino-linked thiazolo[5,4-d]pyrimidine derivative as the lead compound because of its potential kinase inhibitory activities and its desired solubility. The novel tertiary 1-cyano-1-methylethoxy substituent was designed to occupy the hydrophobic region of 'back pocket' of BRAF on the basis of the X-ray co-crystal structure data of BRAF. In addition, we found that N-methylation of the amine linker could control the twisted molecular conformation leading to improved solubility. These approaches produced N-methyl thiazolo[5,4-b]pyridine-5-amine derivative 5. To maximize the in vivo efficacy, we attempted salt formation of 5. Our result indicated that the besylate monohydrate salt form (5c) showed significant improvement of both solubility and oral absorption. Owing to the improved physicochemical properties, compound 5c demonstrated regressive antitumor efficacy in a HT-29 xenograft model.

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 2. Synthesis and characterization of a novel imide-type prodrug for improving oral absorption.

As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorption of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubility (19µg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy (T/C=-6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising candidate and are performing further studies. Herein, we report the design, synthesis, and characterization of novel imide-type prodrugs.

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. 1. Exploration of [5,6]-fused bicyclic scaffolds.

To develop RAF/VEGFR2 inhibitors that bind to the inactive DFG-out conformation, we conducted structure-based drug design using the X-ray cocrystal structures of BRAF, starting from an imidazo[1,2-b]pyridazine derivative. We designed various [5,6]-fused bicyclic scaffolds (ring A, 1-6) possessing an anilide group that forms two hydrogen bond interactions with Cys532. Stabilizing the planarity of this anilide and the nitrogen atom on the six-membered ring of the scaffold was critical for enhancing BRAF inhibition. The selected [1,3]thiazolo[5,4-b]pyridine derivative 6d showed potent inhibitory activity in both BRAF and VEGFR2. Solid dispersion formulation of 6d (6d-SD) maximized its oral absorption in rats and showed significant suppression of ERK1/2 phosphorylation in an A375 melanoma xenograft model in rats by single administration. Tumor regression (T/C = -7.0%) in twice-daily repetitive studies at a dose of 50 mg/kg in rats confirmed that 6d is a promising RAF/VEGFR2 inhibitor showing potent anticancer activity.