Kentaro Yoshimatsu

E7070, a novel sulphonamide agent with potent antitumour activity in vitro and in vivo

E7070 (N-(3-Chloro-7-indolyl)-1,4-benzenedisulphonamide) was selected from our sulphonamide compound collections via antitumour screening and flow cytometric analysis. Following treatment with E7070, the cell cycle progression of P388 murine leukaemia cells was disturbed in the G1 phase. The cell-killing effect on human colon cancer HCT116 cells was found to be time-dependent. In the panel of 42 human tumour cell lines, E7070 showed an antitumour spectrum that was distinct from those of other anticancer drugs used in clinic. Animal tests using human tumour xenograft models demonstrated that E7070 could cause not only tumour growth suppression, but also tumour regression in three of five colorectal and two of two lung cancers. In the HCT116 xenograft model, E7070 was shown to be superior to 5-FU, MMC and CPT-11 (irinotecan). Furthermore, complete regression of advanced LX-1 tumours was observed in 80% of E7070-treated mice. All of these observations have promoted this drug to clinical evaluation.

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage

BackgroundLenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.MethodWe evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.ResultLenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.ConclusionThese results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

In Vitro Activity of E1210, a Novel Antifungal, against Clinically Important Yeasts and Molds

ABSTRACT E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC90 of ≤0.008 to 0.06 μg/ml), except for Candida krusei (MICs of 2 to >32 μg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC90 of 0.13 μg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 μg/ml), Scedosporium prolificans (MIC of 0.03 μg/ml), and Paecilomyces lilacinus (MICs of 0.06 μg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.

Efficacy of Oral E1210, a New Broad-Spectrum Antifungal with a Novel Mechanism of Action, in Murine Models of Candidiasis, Aspergillosis, and Fusariosis

ABSTRACT E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action—inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210s potential for the treatment of disseminated fungal infections are indicated.

Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly

Glycosylphosphatidylinositol (GPI)‐anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small molecular inhibitors of the cell surface presentation of GPI‐anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI‐anchored cell wall mannoproteins. By means of a yeast cell‐based screening procedure, we discovered a compound, 1‐[4‐butylbenzyl]isoquinoline (BIQ), that inhibits cell wall localization of GPI‐anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compound resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage‐dependent suppressor of the BIQ‐induced phenotypes. GWT1 knock‐out cells showed similar phenotypes to BIQ‐treated wild‐type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to BIQ each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compound.

An Angiogenesis Inhibitor E7820 Shows Broad-Spectrum Tumor Growth Inhibition in a Xenograft Model: Possible Value of Integrin α2 on Platelets as a Biological Marker

We reported previously that an angiogenesis inhibitor, E7820, inhibits in vitro tube formation of human umbilical vein endothelial cell through the suppression of integrin α2 expression. Here we describe the antiangiogenic and antitumor effects of E7820 in mice and discuss the feasibility of using platelet integrin α2 expression on platelets as a biological marker of the efficacy of E7820. Oral administration of E7820 significantly inhibited basic fibroblast growth factor-induced angiogenesis in Matrigel implants and human colon WiDr tumor-induced angiogenesis in a dorsal air sac model. Twice-daily treatment with E7820 clearly inhibited the s.c. tumor growth of seven tumor cell lines derived from human colon, breast, pancreas, and kidney, and completely suppressed the growth of human pancreatic KP-1 and human colon LoVo cell lines. Moreover, E7820 significantly inhibited the growth of KP-1 and human colon tumor Colo320DM cells orthotopically implanted in the pancreas and cecum, respectively. The efficacy of E7820 was comparable in the s.c. and orthotopic transplantation models. Immunohistochemical analyses using anti-CD31 antibody showed that E7820 significantly reduced microvessel density in orthotopically implanted KP-1 tumor. E7820 reduced integrin α2 expression on a megakaryocytic cell line, Dami cells, induced by phorbol 12-myristate 13-acetate treatment. It also decreased the expression level of integrin α2 on platelets withdrawn from mice bearing s.c. KP-1 tumor at a dosage close to that affording antitumor activity. These data demonstrate that E7820 showed a broad-spectrum antitumor effect in mice through inhibition of angiogenesis and indicate that the decrease of integrin α2 on platelets might serve as a biological marker for the antitumor efficacy of E7820.

Schizosaccharomyces pombe och1+ encodes α-1,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides

The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N‐linked oligosaccharides on many of its glycoproteins. We identified an S. pombe och1 mutant that did not synthesize the outer chains on acid phosphatase. The S. pombe och1 + gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated α‐1,6‐linked mannose into pyridylaminated Man9GlcNAc2, indicating that och1 + encodes an α‐1,6‐mannosyltransferase. Our results indicate that SpOch1p is a key enzyme of outer chain elongation. The substrate specificity of SpOch1p was different from that of S. cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p.

A focused compound library of novel N-(7-indolyl)benzenesulfonamides for the discovery of potent cell cycle inhibitors

A series of compounds containing an N-(7-indolyl)benzenesulfonamide pharmacophore was synthesized and evaluated as a potential antitumor agent. Cell cycle analysis with P388 murine leukemia cells revealed that there were two different classes of potent cell cycle inhibitors; one disrupted mitosis and the other caused G1 accumulation. Herein described is the SAR summary of the substituent patterns on this pharmacophore template.

Preferential binding of E7010 to murine β3-tubulin and decreased β3-tubulin in E7010-resistant cell lines

N‐[2‐[(4‐Hydroxyphenyl)amino]‐3‐pyridyl]‐4‐methoxybenzenesulfonamide (E7010) is a novel sulfonamide antimitotic agent, which is active against mouse and human tumors. E7010 binds to β‐tubulin and inhibits polymerization of microtubules. In order to clarify the mechanisms of E7010‐resistance, two murine leukemic P388 subclones resistant to E7010, 0.5r‐D and 4.0r‐M, were characterized. The two clones showed approximately 10‐ and 100‐fold resistance to E7010‐induced growth‐inhibitory effects, respectively, compared with the parental cells in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. These cell lines showed no cross‐resistance to other anticancer agents such as taxanes, vinca alkaloids, mitomycin C, cisplatin and irinotecan hydrochloride (CPT‐11). Increased α‐ and β‐tubulin protein and mRNA levels were observed in 0.5r‐D and 4.0r‐M cells as compared with the parental cells. We examined the isotype‐specific expression of β‐tubulin in these E7010‐resistant cells by a competitive reverse transcription‐polymerase chain reaction method. Although a 50% increase in β5 isotype mRNA levels was observed in 4.0r‐M cells, the levels of β3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with β‐tubulin isotypes, we prepared isotype‐specific fusion proteins of β‐tubulins. Direct photoaffinity labelling of the isotype‐specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the β3 isotype rather than β2, β4, and β5 isotype proteins. Therefore, altered expression of β‐tubulin isotypes, especially β3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.

Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.