SummaryThe HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min−1 and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-012-9912-9) contains supplementary material, which is available to authorized users.

Aureobasidin A (LY295337) is a cyclic depsipeptide antifungal agent with activity against Candida spp. The mechanism of action of LY295337 remains unknown. LY295337 also shows activity against the yeast Saccharomyces cerevisiae. Generation of a mutant of S. cerevisiae resistant to LY295337 is reported. Resistance was found to reside in a dominant mutation of a single gene which has been named AUR1 (for aureobasidin resistance). This gene was cloned and sequenced. A search for homologous sequences in GenBank and by BLAST did not elucidate the function of this gene, although sequence homology to an open reading frame from the Saccharomyces genome sequencing project and several other adjacent loci was noted. Deletion of aur1 was accomplished in a diploid S. cerevisiae strain. Subsequent sporulation and dissection of the aur1/aur1⌬ diploid resulted in tetrads demonstrating 2:2 segregation of viable and nonviable spores, indicating that deletion of aur1 is lethal. As LY295337 is fungicidal and deletion of aur1 is lethal, aur1 represents a potential candidate for the target of LY295337.Aureobasidins are antifungal cyclic depsipeptides isolated from Aureobasidium pullulans R106 (15, 25). Aureobasidin A (LY295337) is the major factor isolated from the fermentation broth of A. pullulans R106 (25). This compound has potent in vitro antifungal activity against Candida albicans, other species of Candida, and Cryptococcus neoformans (9,26).The model yeast Saccharomyces cerevisiae is susceptible to LY295337. The study of mutants of S. cerevisiae resistant to the action of LY295337 could lead to an understanding of its mechanism of action. In this study, generation of a dominant mutant of S. cerevisiae which is resistant to LY295337 has allowed the cloning of a gene, ABR1, which encodes resistance to LY295337 (12, 13). Hashida-Okado and coworkers and Okado et al. have recently isolated the same gene as ABR1 through resistance to aureobasidin A (named AUR1) from S. cerevisiae (11,16). The gene encoding resistance to aureobasidin A will hereafter be referred to as AUR1. AUR1 is unique and essential for viability of S. cerevisiae. AUR1 may encode the target for LY295337. MATERIALS AND METHODSStrains, media, plasmids, and transformations. S. cerevisiae strains used in this study are presented in Table 1. Cultivation, storage, and genetic manipulation of S. cerevisiae were carried out as previously described (19). The yeast shuttle plasmid pRS416 was used for construction of the yeast library, and pRS406 was used for construction of a deletion plasmid (24). Transformation of yeast strains was carried out by the method of Reddy and Maley (18). Transformants bearing the URA3-marked plasmids were cured by the use of 5-fluoroorotic acid (5-FOA) (4).Selective medium for drug resistance was standard yeast-peptone-dextrose (YPD) agar with 5 g of LY295337 per ml and 1% ethanol (19).Library construction and propagation of plasmids were performed with Escherichia coli DH5␣, grown and selected for by standard methods (21).Determination of ant...

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target “agnostic” fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD2), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD2 assay panel. Analysis of PD2 submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD2 have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD2, may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.

Cloned cDNAs representing the entire, homologous (80%) translated sequences of human phosphoribosylpyrophosphate synthetase (PRS) 1 and PRS 2 cDNAs were utilized as probes to localize the corresponding human PRPS1 and PRPS2 genes, previously reported to be X chromosome linked. PRPS1 and PRPS2 loci mapped to the intervals Xq22-q24 and Xp22.2-p22.3, respectively, using a combination of in situ chromosomal hybridization and human x rodent somatic cell panel genomic DNA hybridization analyses. A PRPS1-related gene or pseudogene (PRPS1L2) was also identified using in situ chromosomal hybridization at 9q33-q34. Human HPRT and PRPS1 loci are not closely linked. Despite marked cDNA and deduced amino acid sequence homology, human PRS 1 and PRS 2 isoforms are encoded by genes widely separated on the X chromosome.