Emerging evidence demonstrates that stromal cell-derived factor 1 (SDF-1) and CXCR4, a chemokine and chemokine receptor pair, play important roles in tumorigenesis. In this report, we describe a small cyclic peptide, LY2510924, which is a potent and selective CXCR4 antagonist currently in phase II clinical studies for cancer. LY2510924 specifically blocked SDF-1 binding to CXCR4 with IC 50 value of 0.079 nmol/L, and inhibited SDF-1-induced GTP binding with Kb value of 0.38 nmol/L. In human lymphoma U937 cells expressing endogenous CXCR4, LY2510924 inhibited SDF-1-induced cell migration with IC 50 value of 0.26 nmol/L and inhibited SDF-1/ CXCR4-mediated intracellular signaling. LY2510924 exhibited a concentration-dependent inhibition of SDF-1-stimulated phospho-ERK and phospho-Akt in tumor cells. Biochemical and cellular analyses revealed that LY2510924 had no apparent agonist activity. Pharmacokinetic analyses suggested that LY2510924 had acceptable in vivo stability and a pharmacokinetic profile similar to a typical small-molecular inhibitor in preclinical species. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non-Hodgkin lymphoma, renal cell carcinoma, lung, and colon cancer cells that express functional CXCR4. In MDA-MB-231, a breast cancer metastatic model, LY2510924 inhibited tumor metastasis by blocking migration/homing process of tumor cells to the lung and by inhibiting cell proliferation after tumor cell homing. Collectively, the preclinical data support further investigation of LY2510924 in clinical studies for cancer. Mol Cancer Ther; 14(2); 480-90. Ó2014 AACR.

We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/ reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat alltrans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (<73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3␣-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD ؉ and NADH are the preferred cofactors, with apparent K m values 250 -1500 times lower than those for NADP ؉ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent K i values of 5.8 and 3.5 M, respectively. All-transretinol bound to cellular retinol-binding protein (type I) exhibits a similar K i value of 3.6 M. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD ؉ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3␣-androstane-diol.Short chain alcohol dehydrogenases/reductases are either cytosolic or membrane-bound enzymes with a subunit molecular mass of 25-35 kDa that utilize a vast variety of substrates, including steroids and prostaglandins (1). Recently, this family of enzymes has expanded to include the retinol-oxidizing dehydrogenases (2-6). Retinol dehydrogenases are involved in the biosynthesis of all-trans-retinoic acid, the activating ligand for a family of nuclear receptors (7). All-trans-retinoic acid is produced from all-trans-retinol in two oxidative steps: all-transretinol is oxidized to all-trans-retinal and then further to alltrans-retinoic acid. Retinol dehydrogenases catalyze the ratelimiting step: the oxidation of retinol to retinaldehyde (8). Although the effects of retinoic acid on gene transcription and regulation have been intensively studied during the last decade, the exact enzymes that synthesize this morphogen and the mechanisms that regulate its production in tissues are not fully understood. Enzymatic activity capable of oxidizing retinol to retinaldehyde is readily detected in the cytosolic and microsomal fractions of total cell homogenates (9). The cytosol...

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD ؉ and NADH as the preferred cofactors. The enzyme is most efficient as a 3␣-hydroxysteroid dehydrogenase, converting 3␣-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3␣-androstanediol to dihydrotestosterone with similar catalytic efficiency (V max values of 13-14 nmol/min/mg microsomal protein and K m values of 5-7 M). Despite ϳ44 -47% sequence identity with retinol/ 3␣-hydroxysterol dehydrogenases, the enzyme is not active toward retinols. The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal NAD ؉

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.