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Nicotinamide (2) is a potent and selective inhibitor of the PDE4D isozyme and as a chemical tool selectively blocks eosinophil mediator release and chemotaxis thus linking the role of PDE4D to eosinophil function.

Abstract: The M. tuberculosis rpoA gene, encoding the a subunit of RNA polymerase, was isolated by a polymerase chain reaction-based strategy using primers directed against regions of a that are highly conserved among Gram-positive and Gram-negative bacteria. The amplified DNA was utilized as a hybridization probe to recover the entire rpoA gene from a cosmid library of genomic DNA from virulent M. tuberculosis strain H37Rv. Nucleotide sequencing indicated that the 1044 bp M. tuberculosis rpoA open reading frame (ORF) encodes a protein of 347 amino acids which shows significant structural similarity to the a subunits of diverse bacterial species. Recombinant M. tuberculosis a subunit was overproduced in soluble form in E. coli as a hexahistidine-tagged fusion protein and purified to homogeneity by affinity chromatography.

Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smo's actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Products of a number of mutant p53 genes bind with high affinity to members of the hsp70 family of chaperonin proteins, whereas wild type p53 lacks this type of association. Examination of the sequences of p53 genes from five different species enabled us to predict domains on p53 which may be involved in the association with hsp70 family members. A synthetic polypeptide (Pro-17-Gly) corresponding to the candidate hsp70 binding domain bound to in vitro translated hsp70 as determined by affinity chromatography and nondenaturing gel mobility shift assays. In addition, the Pro-17-Gly peptide competitively inhibited association between hsp70 and p53, an activity which was determined by immunoprecipitation with anti-p53 monoclonal antibody PAb240. The data indicate that p53 contains a hsp70 binding domain, which is located in a highly conserved region at the amino terminus of the protein, and may participate in the cellular function of wild-type p53 or in the transforming capacity of p53 mutants.

Oxindole (2) is a potent and selective PDE2 inhibitor with a favorable ADME, physiochemical and pharmacokinetic profile to allow for use as a chemical tool in elucidating the physiological role of PDE2.

The RNA polymerase holoenzyme (RNAP) of Enterobacter cloacae was purified by gel filtration and heparin affinity chromatography and shown to consist of four subunits (beta', beta, alpha and sigma) of 156, 151, 45 and 82 kDa, as measured by SDS-PAGE. The 82 kDa protein was shown to be related to the Escherichia coli primary sigma factor by western blot analysis with polyclonal antisera raised against purified E. coli sigma 70. Functional reconstitution of E. cloacae core enzyme with purified E. coli sigma 70 showed that E. cloacae and E. coli sigma factors are closely related. The RNAP of E. cloacae initiated transcription from the tac promoter with an efficiency similar to that of E. coli. Measuring promoter-specific transcription, the dependence of holoenzyme activity on salt, divalent cation and temperature was also similar to that of the E. coli RNAP. We also showed that the transcriptional inhibitor, rifampicin, inhibits the enzyme activity of the purified RNAP of E. cloacae and E. coli at similar concentrations. We conclude, based on these data, that the RNAP of E. cloacae and E. coli, both enterobacteria, are closely related.

Vascular endothelial growth factor (VEGF) is potent mitogen for endothelial cells that signals through VEGF Receptor-type2 (VEGFR-2)/Flk-1/KDR. Binding of VEGF to VEGFR-2, a membrane bound receptor tyrosine kinase of the VEGF-receptor family, leads to receptor dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling pathways stimulating angiogenesis. In adults, angiogenesis normally occurs during wound healing to restore blood flow post injury and during menstruation and pregnancy. Angiogenesis is also involved in several pathological disease states such as rheumatoid arthritis, psoriasis, diabetic retinopathy and cancer. While physiological angiogenesis involves interplay between pro- and anti-angiogenic factors, the ability to inhibit pathological angiogenesis has therapeutic potential. Inhibitors of VEGFR-2 provide targeted treatments for tumor growth as well as abnormal neovascularization related diseases. The caveat of traditional biochemical VEGFR-2 assays is that only the catalytic domain of the receptor is used to identify hits. One strategy to identify true novel inhibitors against membrane associated proteins is to screen these targets in a more physiological environment, where lipid-protein and protein-protein interactions are included in the assay. We achieved this goal by docking the entire cytoplasmic domain of the receptor on a liposome. Template Directed Assembly (TDA) is a screening technology designed to accomplish this objective. TDA technology utilizes recombinant VEGFR-2 cytoplasmic domain tagged with polyhistidine at the juxtamembrane position and docked to the liposome via the nickel residues. The lipids in TDA are able to diffuse within the surface of the liposome and, therefore, proteins can diffuse to form oligomeric complexes such as homo- or hetero-dimers formation. To compliment the TDA biochemical screen, we are implementing an image based high content screen (HCS) utilizing human umbilical vein endothelial cells (HUVECs) that express native VEGFR-2. The HCS screen is designed to identify potent and specific inhibitors of VEGFR-2 as assessed by a reduction in the proliferation marker Ki67. We are currently (1) validating the TDA assay with known VEGFR-2 inhibitors (2) screening a focus small molecule library to identify novel hits (3) testing the primary hits against a kinase selectivity panel and (4) designing and developing a VEGFR2 cell-based HCS assay to confirm the primary hits. The hit to lead program will be guided by using both physiologically relevant biochemical-based and cell-based assays. The combination of our novel TDA screening platform and the HCS assay technology permits us to identify unique VEGFR-2 inhibitors. Citation Format: Kelvin Lam, Edward Esposito, Deanna Navaroli, Ryan Clinton, Kerrin Beovich, Norman Garceau. Novel TDA technology and high content imaging as complementary assay platforms to identify VEGFR-2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2013-4544

Tropomyosin-related kinases (Trks A, B, and C) are a family of membrane-associated receptor tyrosine kinases (RTKs) activated by neurotrophins. RTKs are shown to be implicated in Alzheimer's disease, depression, pain sensation and various cancers including tumor cell growth and survival signaling. Inhibitors of Trk receptor kinases might provide targeted treatments for neurodegenerative diseases, pain and cancer. One strategy to indentify Trk isoform-specific inhibitors is to develop an assay where compound libraries can be screened in a more-native membrane associated lipid environment. Template-Directed Self Assembly (TDA) is a biomimetic technology designed to restore membrane-associated proteins to more relevant physiological conditions. This technology utilizes recombinant histidine-tagged proteins bound to the surface of nickel-chelated liposomes. Recombinant HIS-tagged proteins are engineered on the correct terminus of the protein to reflect the polarity of the enzyme with respect to the membrane. This allows the HIS-tagged proteins to bind to the liposome creating an environment much like a cellular membrane. The lipids in TDA are fully fluid within the surface of the liposome and associated proteins can rotate freely. Furthermore it promotes the formation of high-order structures such as homo- or hetro-dimers and shown to recruit accessory factors. Coupling of membrane associsted kinases to liposomes reconstitutes an environment that confers proper polarity, stereochemistry, topology and other critical properties, thereby creating a more relevant biochemical assay. To date, we have shown that several RTKs have enhanced biochemical specific activity and substrate specificity that resembles physiological conditions when coupled to TDA. The TDA approach restores TrkA activity to a more-native environment; therefore, we hypothesize that we will be able to identify compounds that are more specific and selective. To address the need of an enzyme assay near a lipid membrane environment, we successfully developed a robust TrkA assay shown to exhibit up to 20-fold increase in specific activity and a reduction in Km for ATP relative to TrkA in a conventional assay. Furthermore, TDA-associated TrkA exhibited a rapid and robust trans-activation, obviating the need for a pre-activation step. Thus, assays of TrkA coupled to TDA have the potential to identify more chemically diverse and specific inhibitors. We are currently (1) validating the TDA assay with known inhibitors (2) screening a focus library to identify novel hits (3) testing the primary hits against a small kinase selectivity panel and (4) designing and developing a TrkA specific reporter cell line to confirm the primary hits. The advantages of the TDA technology will be also discussed against other therapeutically relevant RTKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2842. doi:1538-7445.AM2012-2842

SummaryThe Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog-responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wild-type Smo, and mutant forms of Smo, SmoM2, and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.