Karen L. Cox

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

SummaryA gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage λ Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

Open Innovation for Phenotypic Drug Discovery: The PD2 Assay Panel

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.

Highly transformable mutants of Streptomyces fradiae defective in several restriction systems

SummaryStreptomyces fradiae JS85 is a mutant defective in tylosin production and an efficient recipient for conjugal transfer of tylosin genes. JS85 was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and derivatives defective in restriction were isolated by sequential selection for increased transformability by several plasmid DNAs. From the number of mutation and selection cycles required to eliminate most restriction, it was estimated that wild type S. fradiae expressed at least five restriction systems. From the patterns of restriction enzyme digestion of chromosomal DNA observed in the series of mutants that became progressively less restricting, it was suggested that wild type S. fradiae normally expresses modification (and presumably restriction) systems similar or analogous to PstI, XhoI, ScaI and EcoRI. The least restricting mutant of S. fradiae was readily transformable by many plasmids, including a bifunctional cosmid vector containing a large insert of Streptomyces DNA.

Effect of growth hormone secretagogue LY444711 on IGF-1, growth hormone, and cortisol levels in beagle dogs after one and seven daily oral doses

Growth hormone (GH) release involves interaction of somatostatin and an endogenous GH secretagogue (GHS) on the hypothalamus. GH causes release of IGF‐1, which acts by negative feedback to restrain subsequent GH release. GH secretagogues produce increases in cortisol. In this study, we determined if compound LY444711 produces sustained elevation of GH and IGF‐1 in beagle dogs without sustained alteration of baseline cortisol secretions after one and seven daily doses. Adult male beagle dogs received oral doses of LY444711 at 1 mg/kg/day, or vehicle (10% hydroxypropyl beta‐cyclodextrin). Jugular vein blood was collected periodically after one and seven doses, and plasma levels of IGF‐1, GH, and cortisol were determined. LY444711 increased IGF‐1 levels by approximately 60% over controls after one and seven daily doses. IGF‐1 was elevated within 6 h of dosing on Day 1 and remained elevated 24 h postdose. GH levels (AUC) increased approximately 50‐fold above controls following a single dose of LY444711. With repeated dosing, GH levels rose to approximately 8‐fold over controls. Regardless of the reduction in GH AUC with repeat dosing, sufficient GH was produced to cause sustained IGF‐1 elevation after seven doses. LY444711 produced little or no effect on cortisol AUC level after one or seven doses. These data demonstrate that LY444711 functions as a GH secretagogue in dogs, with associated increases in IGF‐1 levels and an absence of meaningful increases in cortisol levels. Drug Dev. Res. 49:260–265, 2000.

Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays

Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.

Abstract B248: Characterization and preclinical development of LCI‐1, a selective and potent Chk1 inhibitor in phase 1 clinical trials

Interference of DNA damage checkpoints has been demonstrated to be a highly effective means of increasing the cytotoxicity of a wide number of anti‐cancer therapies. Ionizing radiation, DNA cross‐linkers, topoisomerase inhibitors and anti‐metabolites all cause severe cellular DNA damage and activation of multiple DNA damage checkpoints. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until the DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA proceed into premature mitosis followed by rapid apoptotic death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 leads to abrogation of the DNA damage checkpoints and has been shown to enhance the preclinical activity of many standard of care chemotherapeutic agents. LCI‐1 is a novel small molecule inhibitor of Chk1 (IC50 = 7 nM). In cell‐based experiments LCI‐1 inhibited doxorubicin‐dependent autophosphorylation of Chk1 (IC50 = 52 nM). Treatment of cells with LCI‐1 alone caused a phenotype identical to that reported for cells depleted of Chk1, demonstrating the mechanism‐dependent activity of the compound. Treated with 100 nM doxorubicin, HeLa cells arrested at the G2M checkpoint. When treated 24 hours later with LCI‐ 1, the arrested cells traversed the G2M checkpoint, allowing cells to proceed into mitosis with unresolved replicated chromosomes. Consistent with abrogation of the Chk1‐dependent G2M checkpoint, HT‐29 cells (mutant p53) treated with LCI‐1 were more sensitive to killing by gemcitabine then were LCI‐1 treated HCT116 cells (wild‐type p53). In vivo, LCI‐1 effectively inhibited Chk1 autophosphorylation, as well as released the S and G2M block induced by gemcitabine treatment. When used preclinically in combination with DNA damaging agents, LCI‐1 increased DNA damage and cell death over gemcitabine alone. These results indicate that LCI‐1 may prove to be an effective potentiator of DNA damaging therapies in the clinic. LCI‐1 is currently undergoing clinical testing in combination with gemcitabine and pemetrexed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B248.

Synthesis of a radioiodinated park nucleotide analog: a new tool for antibacterial screen development

The Park nucleotide is an important biological building block used in the construction of bacterial cell walls. Herein, we describe the synthesis of a radiolabeled Park nucleotide analog, p-iodophenoxyacyl-Ala-(d)-iso-Glu-Lys-(d)-Ala-(d)-Ala-OH-[123I], using electrophilic destannylation. Anti-Park nucleotide antibody binding assays using a scintillation proximity assay (SPA) system showed good recognition of the radiolabeled surrogate. This methodology could be used for establishing a screen to identify inhibitors of peptidoglycan biosynthesis.