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The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.

The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3

Preclinical experiments and clinical observations suggest the potential effectiveness of selective 5-HT1F receptor agonists in migraine. Identifying compounds with enhanced selectivity is crucial to assess its therapeutic value. Replacement of the indole nucleus in 2 (LY334370) with a monocyclic phenyl ketone moiety generated potent and more selective 5-HT1F receptor agonists. Focused SAR studies around this central phenyl ring demonstrated that the electrostatic and steric interactions of the substituent with both the amide CONH group and the ketone CO group play pivotal roles in affecting the adopted conformation and thus the 5-HT1F receptor selectivity. Computational studies confirmed the observed results and provide a useful tool in the understanding of the conformational requirements for 5-HT1F receptor agonist activity and selectivity. Through this effort, the 2-F-phenyl and N-2-pyridyl series were also identified as potent and selective 5-HT1F receptor agonists.

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 (PD(2)), 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 PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) 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 PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

Preclinical experiments and clinical observations suggest the potential effectiveness of selective 5-HT1F receptor agonists in migraine. Identifying compounds with enhanced selectivity is crucial to assess its therapeutic value. Replacement of the indole nucleus in 2 (LY334370) with a monocyclic phenyl ketone moiety generated potent and more selective 5-HT1F receptor agonists. Focused SAR studies around this central phenyl ring demonstrated that the electrostatic and steric interactions of the substituent with both the amide CONH group and the ketone CO group play pivotal roles in affecting the adopted conformation and thus the 5-HT1F receptor selectivity. Computational studies confirmed the observed results and provide a useful tool in the understanding of the conformational requirements for 5-HT1F receptor agonist activity and selectivity. Through this effort, the 2-F-phenyl and N-2-pyridyl series were also identified as potent and selective 5-HT1F receptor agonists.

Synthesis and evaluation of a series of 2,3,5- and 3,5-substituted furo[3,2- b ]pyridines were undertaken in order to investigate their utility as bioisosteres of 5-HT 1F receptor agonist indole analogues, 1 – 3 . The replacement proved to be effective, providing compounds with similar 5-HT 1F receptor affinity and improved selectivity when compared with the indole analogues. Through these studies we identified 4-fluoro- N -[3-(1-methyl-piperidin-4-yl)-furo[3,2- b ]pyridin-5-yl]-benzamide ( 5 ), a potent and selective 5-HT 1F receptor agonist with the potential to treat acute migraine.

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 IC50 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 IC50 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. ©2014 AACR.

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 3α-hydroxysteroid dehydrogenase, converting 3α-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3α-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 μm). Despite ∼44–47% sequence identity with retinol/3α-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+-dependent 3α-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.

All-trans retinoic acid (atRA) is a powerful morphogen synthesized in a variety of tissues. Oxidation of all-trans retinol to all-trans retinal determines the overall rate of atRA biosynthesis. This reaction is catalyzed by multiple dehydrogenases in vitro. In the cells, most all-trans retinol is bound to cellular retinol binding protein (CRBP). Whether retinoic acid is produced from the free or CRBP-bound retinol in vivo is not known. The current study investigated whether human medium-chain alcohol/retinol dehydrogenases (ADH) can oxidize the CRBP-bound retinol. The results of this study suggest that retinol bound to CRBP cannot be channeled to the active site of ADH. Thus, the contribution of ADH isozymes to retinoic acid biosynthesis will depend on the amount of free retinol in each cell. Physiological levels of ethanol will substantially inhibit the oxidation of free retinol by human ADHs: class I, alpha alpha and beta 2 beta 2; class II, pi pi; and class IV, sigma sigma.

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 all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (

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.

Several fused bicyclic systems have been investigated to serve as the core structure of potent and selective 5-HT1F receptor agonists. Replacement of the indole nucleus in 2 with indazole and 'inverted' indazole provided more potent and selective 5-HT1F receptor ligands. Indoline and 1,2-benzisoxazole systems also provided potent 5-HT1F receptor agonists, and the 5-HT1A receptor selectivity of the indoline- and 1,2-benzisoxazole-based 5-HT1F receptor agonists could be improved with modification of the benzoyl moiety of the benzamides. Through these studies, we found that the inherent geometries of the templates, not the nature of hybridization of the linking atom, were important for the 5-HT1F receptor recognition.

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.

This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed using the Baculovirus System localized in the microsomal fraction and catalyzed oxidation and reduction of the functional groups on steroids at carbons 3 and 17. Autoradiography assays revealed that the enzyme was most efficient as a 3alpha-hydroxysteroid oxidoreductase. High affinity of the enzyme for NADH (Km of 0.18 microM), lack of stereospecificity in the reductive direction, and poor efficiency for 3beta- versus 3alpha-hydroxyl oxidation could account for the observed transient accumulation of 3beta-stereoisomers in the oxidative reaction. Consistent with the 65% sequence identity with RoDH dehydrogenases, the enzyme oxidized all-trans-retinol with the Km value of 3.2 microM and Vmax value of 1.2 nmol/min per milligram microsomes. 13-cis-Retinol and all-trans-retinol bound to the cellular retinol-binding protein were not substrates. Neurosteroid allopregnanolone was a better substrate than all-trans-retinol with the Km and Vmax values of 0.24 microM and 14.7 nmol/min per milligram microsomes. Northern blot analysis revealed that the corresponding mRNA was present in adult human brain (caudate nucleus, amygdala, hippocampus, substantia nigra, thalamus) and spinal cord in addition to other tissues. The message was also detected in fetal lung, liver, and brain. Antibodies against the enzyme recognized a protein of approximately 35 kDa in the particulate fraction of human tissues. This study presents new information about enzymatic properties, substrate specificity, and tissue distribution of this enzyme, and provides a better insight into its possible physiological function(s).

We have previously characterized the first human NAD(+)-dependent short chain dehydrogenase capable of oxidizing all-trans-retinol and androgens, and found only in the liver and skin. In a search for related human enzymes, we identified a partial open reading frame, which exhibited >60% sequence identity to human RoDH-4. The full-length cDNA for this enzyme was determined in our laboratory by 5'-RACE PCR and was found to be identical to the recently reported novel type of oxidative human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Analysis of the genomic structure revealed that the gene for RoDH-like 3alpha-HSD has four translated exons and, possibly, a fifth exon that codes for the 5'-untranslated region. The gene for RoDH-4 appears to have only four exons. The positions of exon-intron boundaries and the sizes of the protein coding regions are identical in 3alpha-HSD and RoDH-4. Moreover, both genes are mapped to chromosome 12q13, and are located in a close proximity to each other. Both genes appear to have satellite pseudogenes. Thus, RoDH-4 and 3alpha-HSD genes share similar structural organization and cluster on human chromosome 12, near the gene for 11-cis retinol dehydrogenase.

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.