Yoshiharu Mizui

Pladienolides, New Substances from Culture of Streptomyces platensis Mer-11107 : II. Physico-chemical properties and Structure Elucidation

In the course of our screening using fermented broth from soil microorganisms, novel metabolites (pladienolides), possessing inhibitory activity against vascular endothelial growth factor (VEGF) expression and cancer cell proliferation, were isolated from Streptomyces platensis Mer-11107. Pladienolides A (1), B (2), C (3), D (4), E (5), F (6), and G (7) were found to be novel 12-membered macrolides by spectroscopic studies including 1H, 13C NMR, HMQC, HMBC, and NOE experiments. Pladienolides are unusual 12-membered macrolides having a long side chain at the carbon that bears a lactone oxygen.

Biological validation that SF3b is a target of the antitumor macrolide pladienolide

Pladienolide is a naturally occurring macrolide that binds to the SF3b complex to inhibit mRNA splicing. It has not been fully validated whether the splicing impairment is a relevant mechanism for the potent antitumor activity of pladienolide. We established pladienolide‐resistant clones from WiDr and DLD1 colorectal cancer cells that were insensitive to the inhibitory action of pladienolide on cell proliferation and splicing. An mRNA‐Seq differential analysis revealed that these two cell lines have an identical mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex. Reverse expression of the mutant protein transferred pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation analysis using a radiolabeled probe showed that the mutation impaired the binding affinity of paldienolide to its target. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

Phase I Pharmacokinetic and Pharmacodynamic Study of the First-in-Class Spliceosome Inhibitor E7107 in Patients with Advanced Solid Tumors

Purpose: To assess the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of E7107 administered as 5-minute bolus infusions on days 1, 8, and 15 in a 28-day schedule. Experimental Design: Patients with solid tumors refractory to standard therapies or with no standard treatment available were enrolled. Dose levels of 0.6 to 4.5 mg/m2 were explored. Results: Forty patients [24M/16F, median age 61 years (45–79)] were enrolled. At 4.5 mg/m2, dose-limiting toxicity (DLT) consisted of grade 3 diarrhea, nausea, and vomiting and grade 4 diarrhea, respectively, in two patients. At 4.0 mg/m2, DLT (grade 3 nausea, vomiting, and abdominal cramps) was observed in one patient. Frequently occurring side effects were mainly gastrointestinal. After drug discontinuation at 4.0 mg/m2, one patient experienced reversible grade 4 blurred vision. The maximum tolerated dose (MTD) is 4.0 mg/m2. No complete or partial responses during treatment were observed; one patient at 4.0 mg/m2 had a confirmed partial response after drug discontinuation. Pharmacokinetic analysis revealed a large volume of distribution, high systemic clearance, and a plasma elimination half-life of 5.3 to 15.1 hours. Overall drug exposure increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes monitored in peripheral blood mononuclear cells showed a reversible 15- to 25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a reversible 10- to 25-fold increase. Conclusion: The MTD for E7107 using this schedule is 4.0 mg/m2. Pharmacokinetics is dose-dependent and reproducible within patients. Pharmacodynamic analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107. Clin Cancer Res; 19(22); 6296–304. ©2013 AACR.

Cloning and chromosomal mapping of mouse ADAM11, ADAM22 and ADAM23.

A cellular disintegrin, also called MDC and ADAM is a recently discovered gene family that encodes protein with disintegrin-like and metalloprotease-like domains. We have reported the identification of human cDNAs encoding novel ADAM family proteins that we named MDC2 and MDC3 because of their structural similarity to the MDC (Sagane, K. et al., 1998. Biochem. J. 334, 93-98). The Human Gene Nomenclature Committee assigned the gene symbols ADAM11 for the MDC, ADAM22 for the MDC2 and ADAM23 for the MDC3. Here we report the isolation of three novel murine cDNAs encoding the proteins closely related to the human ADAM11, ADAM22 and ADAM23. Their chromosomal locations in the mouse were identified by interspecies backcross mapping. The loci of these murine ADAM genes were in good accordance with the location of each human ortholog, ADAM11, ADAM22 and ADAM23. These findings suggest that three murine cDNAs that we have isolated are the murine ADAM11, ADAM22 and ADAM23 cDNAs. Northern blot analysis shows that all of these three murine ADAMs were highly expressed in the mouse brain. The structures of these ADAM proteins strongly suggest that they could function as integrin receptors. The implications of the cellular disintegrins in neural development are discussed.

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8, 9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione], a Novel Kinase Inhibitor of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase (MEK)-1 and MEK Kinase-1: In Vitro Characterization of Its Anti-Inflammatory and Antihyperproliferative Activities

The goal of this study is to identify a novel inhibitor with anti-inflammatory and antiproliferative properties for the treatment of psoriasis. Compound f152A1 [(3S,5Z,8S,11E)-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,9,10-tetrahydro-1H-benzo[c][1]oxacyclotetradecine1,7(8H)-dione] was identified as the main active metabolite with strong inhibitory activity against tumor necrosis factor-α (TNFα) transcription in a fraction originated from the fermentation broth of the fungus Curvularia verruculosa. Although active in cell-based assays, f152A1 was unstable in plasma and liver microsome preparations, thus limiting its pharmaceutical utilization. To improve the metabolic properties of f152A1, a medicinal chemistry program was undertaken, resulting in the generation of over 400 analogs of f152A1. Eventually, E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] was identified as a promising analog in this series. In the present study, we characterized the in vitro activities of E6201 and discovered that the compound inhibits lipopolysaccharide-activated TNFα reporter activity in THP-1-33 cells with an IC50 value of 50 nM and selectively inhibits mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1 and MEK kinase-1 in cell-free biochemical assays. In addition, E6201 showed inhibitory activity in several other cell-based systems: 1) phosphorylation of c-jun N-terminal kinase and p38 MAPKs; 2) nuclear factor-κB and activated protein-1 activation in various cell types; 3) interleukin (IL)-2 production from human lymphocytes; 4) hyperproliferation of human keratinocytes; 5) IL-8 production from human keratinocytes; and 6) proinflammatory cytokine production from human peripheral blood mononuclear cells. Based on the data presented here, E6201 may be beneficial for treatment of inflammatory and hyperproliferative diseases such as psoriasis through its anti-inflammatory activities on immune cells and antihyperproliferative activities on keratinocytes.

Discovery of a potent, metabolically stabilized resorcylic lactone as an anti-inflammatory lead

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor–encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.

Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A–SF3b complex

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A–SF3B1 forms a central node for binding to these splicing modulators.

Total Synthesis of 6-Deoxypladienolide D and Assessment of Splicing Inhibitory Activity in a Mutant SF3B1 Cancer Cell Line

A total synthesis of the natural product 6-deoxypladienolide D (1) has been achieved. Two noteworthy attributes of the synthesis are (1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and (2) the development of a scalable and cost-effective synthetic route to support drug discovery efforts. 6-Deoxypladienolide D (1) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing.

Microregional antitumor activity of a small-molecule hypoxia-inducible factor 1 inhibitor.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that play crucial roles in the adaptation of cancer cells to hypoxia. HIF-1α overexpression has been associated with poor prognosis in patients with various types of cancer. Here, we describe ER-400583-00 as a novel HIF-1 inhibitor. ER-400583-00 suppressed the production of HIF-1α protein in response to hypoxia, with a half-maximal inhibitory concentration value of 3.7 nM in human U251 glioma cells. The oral administration of 100 mg/kg ER-400583-00 to mice bearing U251 tumor xenografts resulted in a rapid suppression of HIF-1α that persisted for 24 h. Immunohistochemical analysis revealed that ER-400583-00 suppressed the proliferation of cancer cells most prominently in areas distal to the region of blood perfusion, where HIF-1α-expressing hypoxic cancer cells were located. These hypoxic cancer cells were resistant to radiation therapy. ER-400583-00 showed a synergistic interaction with radiation therapy in terms of antitumor activity. These data suggest that HIF-1 blockade by small compounds may have therapeutic value in cancer, especially in combination with radiation therapy.