Yoshinori Yamashita

Halohydrin and oxime derivatives of radicicol: synthesis and antitumor activities.

Novel halohydrin and oxime derivatives of radicicol (1) were prepared and evaluated for their v-src tyrosine kinase inhibitory, antiproliferative, and antitumor activities. Some of the resulting derivatives showed significantly improved antitumor activities than those of 1 in vitro as tested in a cell proliferation assay and in vivo using sc-inoculated human breast carcinoma and epidermoid tumor models. Design and synthesis of radicicol-based novel affinity probes are also described.

TELOMERASE INHIBITORS – OLIGONUCLEOTIDE PHOSPHORAMIDATES AS POTENTIAL THERAPEUTIC AGENTS

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3′ → P5′ phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2′-deoxy, 2′-hydroxy, 2′-methoxy, 2′-ribo-fluoro, and 2′-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3′ → P5′ thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells.

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G(1) phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cell-based assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic.

UCS15A, a non-kinase inhibitor of Src signal transduction.

Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activated-src over-expression system. In the present study, we describe the identification of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src specific tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identified as bona-fide src substrates, cortactin and Sam68. UCS15A differed from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to differ from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory effects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway.

Oligonucleotide N3′ → P5′ Thio-phosphoramidate Telomerase Template Antagonists as Potential Anticancer Agents

Abstract Human telomerase is a reverse transcriptase that is expressed in essentially all cancer cells, but not in the vast majority of normal somatic cells. Therefore, the specific inhibition of telomerase activity in tumors might have significant beneficial therapeutic effects. We have designed and evaluated oligonucleotide N3′ → P5′ thio-phosphoramidates as telomerase template antagonists. In biochemical cell-free assays 11-13-mer thio-phosphoramidate oligonucleotides demonstrated sequence specific and dose dependent inhibition of telomerase with pico-molar IC50 values. Optimization of the oligonucleotide sequence and length resulted in the identification of a 13-mer-oligonucleotide thio-phosphoramidate GRN163 as a drug development candidate. In cell cultures GRN163 was able to inhibit telomerase activity in the absence of cationic lipid with ∼1 µM IC50 values. Telomerase inhibition by GRN163 produced gradual telomere shortening, followed by cellular senescence and/or apoptosis of cancer derived cell lines.

Novel and orally active 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives as selective FLT3 inhibitors

5-(1,3,4-Oxadiazol-2-yl)pyrimidine derivative 1 was identified as a new class of FLT3 inhibitor from our compound library. With the aim of enhancement of antitumor activity of 2 prepared by minor modification of 1, structure optimization of side chains at the 2-, 4-, and 5-positions of the pyrimidine ring of 2 was performed to improve the metabolic stability. Introduction of polar substituents on the 1,3,4-oxadiazolyl group contributed to a significant increase in the metabolic stability. As a result, a series of compounds showed increased efficacy against MOLM-13 xenograft model in mice by oral administration.

Conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity.

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1-A4 also adds to their potential as lead compounds for anti-tumor drugs.

Reconstitution of telomerase activity utilizing human catalytic subunit expressed in insect cells.

Telomerase is a specialized reverse transcriptase responsible for maintaining the termini of linear chromosomes. The human enzyme is a ribonucleoprotein complex minimally comprising a catalytic protein moiety (hTERT) and an RNA subunit (hTR) which acts as the template for the reverse transcriptase reaction. Here we report expression of recombinant hTERT protein in insect cells utilizing a baculovirus expression system. The recombinant hTERT protein reconstitutes telomerase activity in the presence of hTR, either when co-expressed in insect cells or when added in vitro. Reconstitution of telomerase activity using this system will facilitate further analysis of the biochemical and biophysical properties of this enzyme.