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Green fluorescent protein (GFP) is useful as an intracellular scaffold for the display of random peptide libraries in yeast. GFPs with a different sequence from Aequorea victoria have recently been identified from Renilla mulleri and Ptilosarcus gurneyi. To examine these proteins as intracellular scaffolds for peptide display in human cells, we have determined the expression level of retrovirally delivered human codon-optimized versions in Jurkat-E acute lymphoblastic leukemia cells using fluorescence activated cell sorting and Western blots. Each wild type protein is expressed at 40% higher levels than A. victoria mutants optimized for maximum fluorescence. We have compared the secondary structure and stability of these GFPs with A. victoria GFP using circular dichroism (CD). All three GFPs essentially showed a perfect beta-strand conformation and their melting temperatures (Tm) are very similar, giving an experimental evidence of a similar overall structure. Folded Renilla GFP allows display of an influenza hemagglutinin epitope tag in several internal insertion sites, including one which is not permissive for such display in Aequorea GFP, giving greater flexibility in peptide display options. To test display of a functional peptide, we show that the SV-40 derived nuclear localization sequence PPKKKRKV, when inserted into two different potential loops, results in the complete localization of Renilla GFP to the nucleus of human A549 cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Background: Few examples exist of peptides of < 35 residues that form a stable tertiary structure without disulfide bonds. A method for stabilization and noncovalent constraint of relatively short peptides may allow the construction and use of intracellular peptide libraries containing protein minidomains.Results: We have examined a novel method for the noncovalent constraint of peptides by attaching the peptide EFLIVKS (single-letter amino acid code), which forms dimers, to the amino and carboxyl termini of different peptide inserts. An 18 residue random coil taken from the inhibitor loop of barley chymotrypsin inhibitor 2 was inserted between the peptides to produce a 32-mer minidomain that is attacked only slowly by elastase, has numerous slowly exchanging protons, contains a high β-structure content and has a Tm above 37°C. A point mutation disrupting the hydrophobic interior in both dimerizing peptides causes a loss of all slowly exchanging protons and of secondary structure. Adding specific charged residues to each terminus substantially increased the Tm, as did point mutants designed to add interdimerizer ion pairs. Three flexible epitope tag inserts and a nonamer insert do not appear to be folded in a stable structure by EFLIVKS. The properties of two peptides selected for expression in HeLa cells suggest they do form a stable tertiary structure.Conclusions: Attaching short dimerizing peptides to both the amino and carboxyl termini of several 18-mer peptides appears to create stable monomeric tertiary structures. Mutations in the dimerizers can either destabilize or significantly stabilize a standard 18-mer insert. Dimerizing peptides flanking random insert sequences could be used as a strategy to generate heterogeneous peptide libraries with both extended and folded members.

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.

Germinal center kinases (GCKs) compose a subgroup of the Ste20 family of kinases. Here we describe the cloning and characterization of a novel GCK family kinase, Traf2- and Nck-interacting kinase (TNIK) that interacts with both Traf2 and Nck. TNIK encodes a polypeptide of 1360 amino acids with eight spliced isoforms. It has 90% amino acid identity to the Nck-interacting kinase in both the N-terminal kinase domain and the C-terminal germinal center kinase homology region. The homology drops to 53% in the intermediate region. TNIK specifically activates the c-Jun N-terminal kinase pathway when transfected into Phoenix-A cells (derivatives of 293 cells), similar to many GCKs. However, in contrast to other GCKs, this activation is mediated solely by the GCK homology region of TNIK. In addition, in Phoenix-A, NIH-3T3, and Hela cells, overexpression of wild type TNIK, but not the kinase mutant form of TNIK, results in the disruption of F-actin structure and the inhibition of cell spreading. Furthermore, TNIK can phosphorylate Gelsolin in vitro. This is the first time that a GCK family kinase is shown to be potentially involved in the regulation of cytoskeleton.

Background: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-α, and leukotrienes. Methods: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme β-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. Results: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP–green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and β-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. Conclusions: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis. Cytometry 36:340–348, 1999. © 1999 Wiley-Liss, Inc.

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E 2 and F 1α from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E 2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.

Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3' untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.

Dipeptides containing fluorescein or biotin have been incorporated into proteolytic substrate cleavage products of bovine serum albumin generated by human cathepsin S or neutrophil elastase and into a fragment of the 31-kDa interleukin 1beta precursor by human interleukin 1beta-converting enzyme. Incorporation of the nucleophile is blocked by prior inhibition of the enzymes, and is not seen when proteolysis occurs in the absence of label, and the protease is then inhibited before the addition of label. Labeling is dependent on the pH, the time of reaction, and the concentrations of the nucleophile and substrate. Labeling of proteins can be readily detected by SDS-polyacrylamide gel electrophoresis. The pattern of elastase-labeled bovine serum albumin bands differs among P1' Phe, Ala, and Gly, suggesting that nucleophilic attack on acyl enzyme intermediates derived from a large protein may differ from attack on small intermediates. The only observed labeled fragment catalyzed by interleukin 1beta-converting enzyme is fragment 28-116 from the interleukin 1beta precursor, suggesting that the cleavage between residues 27 and 28 is at least as efficient as between residues 116 and 117. This labeling method does not require organic solvent or nonphysiological pH values and thus may be useful for the discovery of novel protease substrates in cells or other in vivo systems or for diagnostic applications.

The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid-based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation.

We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.

We have identified a novel human cyclophilin (hCyP-60) which interacts with the proteinase inhibitor eglin c using the yeast two-hybrid system. A cDNA isolated from a Raji B lymphocyte library reveals a domain showing sequence similarity to known cyclophilins flanked by unique N- and C-terminal residues. In addition, hCyP-60 contains a tyrosine residue (Tyr389) instead of a tryptophan residue found in most eukaryotic cyclophilins at a position important for cyclosporin binding. Northern and Western analysis reveal widespread expression with considerable tissue-specific variation. Specifically, the highest levels of mRNA are detected in the thymus, pancreas, testis, and K-562 cell line, while the most protein is detected in the kidney. Immunohistochemistry indicates a nuclear-specific localization both in transfected cells and tissue sections. hCyP-60's specific subcellular localization and conserved amino acid sequence suggest that it may play a specific role in the nucleus.

Introduction: In normal tissue homeostasis, interaction of phosphatidylserine externalized on apoptotic cells (ACs) with the TAM (Tyro3, Axl MerTK) family RTK MerTK, via its ligands Gas6 and Protein S, leads to AC phagocytosis (efferocytosis). The resulting clearance of AC antigens, immunosuppressive M2 macrophage polarization, and suppression of pro-inflammatory cytokine production promotes tolerance to AC-derived self-antigens. This homeostatic response is coopted in tumors, which are abundant in both ACs and TAM ligands, leading to a blunted anti-tumor immune response. Syngeneic tumors implanted in MerTK -/- mice exhibit poor growth and metastasis, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10 compared with those implanted in WT mice. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens and promoting an anti-tumor immune response, or by directly blocking tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell number, apoptosis and proliferation. Effects on immune function were tested in human primary dendritic cells (LPS-induced IL-23 production) and human primary T cells (anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5). Efferocytosis of apoptotic Jurkat cells by human primary macrophages was detected by flow cytometry. For the PD assay, MerTK expressing tumors were grown in nude mice. 30-60 minutes post-compound dosing, MerTK was stimulated in vivo for 1hr. Tumors were snap frozen and tumor lysates were blotted with anti-phospho-MerTK antibodies. Anti-tumor efficacy was studied in syngeneic models. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both classes of compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Note: This abstract was not presented at the conference. Citation Format: Sacha J. Holland, Alexander M. Owyang, Sylvia Braselmann, Chrystelle Lamagna, Sothy Yi, Chi Young, Roy Frances, Arthur Bagos, Meagan Chan, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Jiaxin Yu, Ihab Darwish, Somasekhar Bhamidipati, Donald G. Payan, Esteban Masuda. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B060.

Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies. Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 346.

5’-AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and is critical for maintaining energy homeostasis under conditions of nutrient stress. During cellular transformation, metabolic reprogramming enables the aberrant growth and proliferation of tumor cells. Both positive and negative roles for AMPK in tumor cell proliferation and survival have been reported. However, only a limited number of studies addressed this question with potent direct AMPK activators. AMPK exists as heterotrimers composed of the catalytic subunit α and reguratory subunits β and γ. We expressed the full length of all three human AMPK subunits in insect cells, purified the heterotrimer complexes, and used them for biochemical screening and characterization of AMPK activators. The purified complexes displayed basal activity, which was further enhanced by AMP. The compounds we identified potently activated the complexes in vitro at AC(2X)s (the concentration that gives a twofold activation) of 0.001-0.3 μM. Importantly, the compounds up-regulated substrate phosphorylation (pS79 Acetyl-CoA Carboxylase) and/or auto-phosphorylation (pT172 AMPKα) in multiple cancer cell lines including HepG2 hepatoma cells, A549 liver kinase B1 (LKB1) null lung cancer cells, and MOLM14 myeloid leukemia cells, indicating activation was irrespective of functional status of LKB1, which is a key AMPK-activation kinase. Activation of AMPK by the compounds was also confirmed using native AMPK isolated from normal tissues and tumor cells. We further investigated anti-proliferative effects of the compounds and found that up-regulation of AMPK kinase activity was correlated with anti-proliferative effects in A549 and MOLM14, but not in HepG2, suggesting that positive effects of direct AMPK activators could be cell-type dependent. Interestingly, we identified compounds that display comparable AMPK activation in HepG2 and A549 yet possessed divergent activities on proliferation across a panel of tumor lines. Analysis of cellular signaling across several of these tumor lines with this set of the compounds revealed dose-dependent effects on mTORC1 substrates, feedback signaling to PI3K and mTORC2, and inhibition of kinases downstream of RAF. Direct activation of AMPK could be a good therapeutic strategy for the treatment of subsets of cancers. Citation Format: Yasumichi Hitoshi, Yonchu Jenkins, Yingwu Li, Elmer Sampang, Xiang Xu, Guodong Dong, Jianing Huang, Nan Lin, Dane Goff, Simon Shaw, Luke Boralsky, Rajinder Singh, Sarkiz D. Issakani, Donald G. Payan. Development of small molecule direct AMPK activators for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3021.

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4869.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.

MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands (Gas6 or Protein S) bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK −/− mice exhibit impaired growth and metastasis compared with those implanted in WT mice. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor-induced immunosuppression, or blocking tumor cell survival. Here we describe novel and potent MerTK-selective and Mer-Axl small molecule inhibitors that block both MerTK in vitro kinase activity and MerTK autophosphorylation and downstream signaling in cells. Moreover, compounds were able to inhibit phagocytosis of ACs by MerTK-expressing human primary macrophages, and they block activation of MerTK in vivo. The MerTK inhibitors were not overtly cytotoxic or antiproliferative, and did not block the activity of TLR or T cell immune effector pathways. Limited off-target activity was observed in an in vitro kinase panel. Activity of these novel MerTK inhibitors in tumor models is currently under investigation.

IRAK4 is a key protein kinase in immune cells. It is responsible for initiating MyD88-dependent signaling from most Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R), resulting in downstream production of pro-inflammatory cytokines. Hence, inhibitors of IRAK4 kinase activity represent valuable therapeutic tools to treat cytokine-driven autoimmune and inflammatory diseases. Through cell-based screening, we identified potent small molecule IRAK4 kinase inhibitors that block TLR4- and IL-1R-induced cytokine production with potencies less than 100nM. Our inhibitors exhibit good selectivity against a broad panel of kinases. In vivo, our lead compound decreases serum IL-6 in an acute mouse model of IL-1beta-induced cytokine release, demonstrating excellent pharmacokinetics properties. In addition, our IRAK4 inhibitor blocks monosodium urate crystals-induced peritonitis in mice, reduces paw swelling in the chronic rat model of collagen-induced arthritis, as well as the clinical score and overall inflammatory phenotype of mice in the imiquimod-induced psoriasis model. The in vitro and in vivo characterization of our lead candidate is promising and confirms IRAK4 as an attracting therapeutic target for the management of cytokine-driven diseases.

The transcription factor Nrf2 (nuclear factor erythroid-derived-2-like 2) is a key mediator of the anti-oxidant response. Nrf2 can be activated by alkylating its chaperone, Keap-1, resulting in a retardation of Nrf2 degradation, and increased Nrf2 in the cytoplasm. Upon translocation to the nucleus, Nrf2 binds an Antioxidant Response Element (ARE), which leads to the transcription of antioxidant gene products, many of which may be cytoprotective. Activation of the Nrf2 pathway may be useful against a wide-range of disorders, such as multiple sclerosis, psoriasis, and Huntington’s Disease, among others. For example, monomethylfumarate (MMF), the active component of the multiple sclerosis drug, dimethylfumarate (DMF), has been shown to activate the Nrf2-pathway at therapeutic concentrations. However, MMF also serves as an agonist of the niacin receptor, which is likely responsible for the flushing side-effect seen in patients taking DMF. We have discovered a novel class of molecules that are potent activators of Nrf2 and are inactive at the niacin receptor, yet are efficacious in vivo in a model of multiple sclerosis. Lead candidate R970 is an orally-bioavailable small molecule activator of Nrf2 (EC50 = 1 uM; MMF EC50 = 127 uM), which is inactive at the niacin receptor (0% activity at 500 uM; MMF EC50 ca. 1 uM). Additionally, R970 is selective against a range of kinase, G-protein-coupled receptor, ion-channel, and transporter targets. When dosed orally in a murine model of multiple sclerosis, R970 delayed the onset and also suppressed clinical disease. Further exploration with R970 and other selective activators of Nrf2 is ongoing and may yield a next-generation Nrf2-therapeutic, with an improved side-effect profile.

We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.

The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. Cancer Res; 70(4); 1544–54

1. A protein A-rat substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed in Escherichia coli driven by the heat-inducible lambda promoter (lambda Pr). 2. The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR). 3. Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked). 4. The Ca2+ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not induce intracellular Ca2+ mobilization normally observed when cells were incubated with SP. 5. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.

1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with aKd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10−11–10−6M SP and was maximal at 10−7M SP, with an EC50 of 0.3–0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl− current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will now permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.

In order to analyze the mechanisms by which a single biogenic amine like histamine is capable of inducing a wide variety of both physiologic and pathologic functions in various tissues/cells, histamine responses were dissected in detail from a biochemical and pharmacologic point of view. Histamine is synthesized by multiple isozymes of histidine decarboxylase, and catabolized by either diamine oxidase or histamine-N-methyltransferase. Synthesized intracellular histamine may play a role in cell proliferation, whereas released histamine binds to at least three different histamine-specific receptors, then activates various intracellular components, such as Ca++, cAMP, protein kinase, and ion channels. These second messenger pathways interact differentially with each other in various tissues/cells. Moreover, histamine not only activates its own receptors, but also activates other related receptors such as the serotonin 1c receptor. Therefore, to understand the complex actions of histamine, new approaches should be established, in which multiple phenomena can be monitored simultaneously.

Janus kinases (JAKs) are critical components of cytokine signaling pathways which regulate immunity, inflammation, hematopoiesis, growth, and development. The recent discovery of JAK2-activating mutations as a causal event in the majority of patients with Philadelphia chromosome negative (Ph−) myeloproliferative disorders (MPDs) prompted many pharmaceutical companies to develop JAK2-selective inhibitors for the treatment of MPDs. JAK2 inhibitors effectively reduce JAK2-driven phosphorylation of signal transducer and activator of transcription 5, and cell proliferation and cell survival in JAK2-activated cells in vitro and in vivo. Most inhibitors are currently being evaluated in patients with one form of MPD, myelofibrosis. Patients treated with these inhibitors experienced a rapid reduction of splenomegaly, significant improvement of constitutional symptoms, and increased daily activity with few adverse events. A partial reduction of JAK2V617F disease burden during the treatment with JAK2 inhibitors was also observed. The inhibitors appear to have a therapeutic benefit in the treatment of these disorders. The results of ongoing clinical trials will allow further evaluation of clinical benefits and safety of these compounds. In this review, the authors summarize the status of JAK2 inhibitors in development and discuss their benefits and challenges.

JAK3, a member of the Janus kinase family, is predominantly expressed in hemopoietic cells and binds specifically to the common γ chain of a subfamily of cytokine receptors that includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies suggest that this tyrosine kinase plays key roles in mediating T cell functions, and inhibition of JAK3 has been shown to prevent graft rejection and decrease the severity of arthritis in rodent models. However, the functions of JAK3 in the development of skin immune responses and diseases such as psoriasis have not been determined. CD18 mutant PL/J mice develop spontaneous T cell-dependent psoriasiform skin disease with several similarities to human psoriasis. In this study, we treated mice with established skin disease with R348, a small molecule inhibitor of JAK3, and observed a marked attenuation of skin lesions following 6 wk of treatment. Histological analyses revealed major reductions of both epidermal and dermal lesion severity scores in R348-treated CD18-deficient PL/J mice compared with vehicle controls, which was associated with decreased CD4+ T cell infiltration. In addition, systemic levels of IL-17, IL-22, IL-23, and TNF-α were significantly lower in mice receiving the compound, and T cells isolated from R348-treated mice also showed reduced phosphorylation of Stat5 after stimulation with IL-2. These findings suggest that small-molecule inhibitors of JAK3 may be useful in the treatment of inflammatory skin diseases such as psoriasis and strongly implicate JAK signaling events as important in the pathogenesis of this disease.

Measurement of antiproliferative capacity of a compound is central to early oncology drug discovery, with information about the precise mechanism of compound action typically being acquired during later downstream assays. Here we describe the development and validation of an in vitro image-based assay that simultaneously measures tumor cell count, late apoptotic morphology, and nuclear DNA content (termed the proliferation, apoptosis, and DNA content [PAD] assay) by using a DNA binding fluorescent dye. The PAD assay determines whether a compound's antiproliferative effect occurs via cell cycle arrest or induction of apoptosis, replacing downstream assays for 1/50(th) the cost. We used this assay to screen a kinase inhibitor-biased library and discovered an Aurora kinase inhibitor, and we also used it to drive structure-activity relationship to clinical candidate Investigational New Drug filing within 2 years. The simplicity of the PAD assay was critical to the rapid time frame within which this candidate was identified and progressed. This inhibitor is currently beginning Phase II clinical trials.

To analyze the cross-reactivity between serotonin (5-HT) and histamine, the in vitro transcribed RNA for the 5-HT1c receptor was functionally expressed in Xenopus oocytes. 5-HT significantly increased 45Ca2+ efflux in RNA-injected oocytes, but not in uninjected and water-injected control oocytes. Furthermore, histamine and the H1 receptor agonists, but not the H2 and H3 agonists, significantly induced 45Ca2+ efflux in 5-HT1c receptor RNA-injected oocytes, but not in uninjected and water-injected oocytes. However, the H1, H2, and H3 antagonists failed to inhibit histamine-induced 45Ca2+ efflux at 10(-6) M. This finding suggests that the 5-HT1c receptor can be activated by both 5-HT and histamine, although the action of histamine is different from classic histamine pharmacology.

The amyloid beta protein (ABP) has been shown to interact with the substance P (SP) receptor in a cell culture model that may mimic the pathogenesis of Alzheimer's disease. In the present study, however, 4 fragments of ABP (beta 1-42, beta 1-16, beta 17-28, and beta 25-35) failed to interact with SP-induced Ca2+ mobilization in SP receptor-expressing cultured cells. Therefore, the action of these ABP-related peptides in our cultured cells is unrelated to the SP receptor.

Agrin is a component of the basal lamina that causes the aggregation of acetylcholine receptors on cultured muscle fibers. An agrin cDNA clone isolated from electromotor neurons of a marine ray was used to characterize the corresponding cDNAs from a rat embryonic spinal cord library. Analysis of a set of clones predicts a 1940 amino acid protein containing 141 cysteine residues. The predicted protein has nine domains homologous to protease inhibitors, a region similar to domain III of laminin, and four epidermal growth factor repeats. The agrin gene is expressed in rat embryonic nervous system and muscle. The rat agrin protein is concentrated at synapses, where it may play a role in development and regeneration.

Objective To assess whether R788, an orally bioavailable small molecule inhibitor of spleen tyrosine kinase (Syk)–dependent signaling, could modulate disease in lupus-prone (NZB × NZW)F1 (NZB/NZW) mice via inhibition of Fc receptor (FcR) and B cell receptor signaling. Methods R788 was administered to NZB/NZW mice before and after disease onset. Proteinuria, blood urea nitrogen levels, and autoantibody titers were examined periodically, and overall survival and renal pathologic features were assessed following long-term treatment (24–34 weeks). The distribution and immunophenotype of various splenic T cell and B cell subpopulations were evaluated at the time of study termination. Arthus responses in NZB/NZW mice pretreated with R788 or Fc-blocking antibody (anti-CD16/32) were also examined. Results When R788 was administered prior to or after disease onset, it delayed the onset of proteinuria and azotemia, reduced renal pathology and kidney infiltrates, and significantly prolonged survival of lupus-prone NZB/NZW mice; autoantibody titers were minimally affected throughout the study. Dose-dependent reductions in the numbers of CD4+ activated T cells expressing high levels of CD44 or CD69 were apparent in spleens from R788-treated mice. Minimal effects on the numbers of naive T cells expressing CD62 ligand and total CD8+ T cells per spleen were observed following long-term drug treatment. R788 pretreatment resulted in reduced Arthus responses in NZB/NZW mice, similar to results obtained in mice pretreated with FcR-blocking antibody. Conclusion We demonstrate that a novel Syk-selective inhibitor prevents the development of renal disease and treats established murine lupus nephritis. These data suggest that Syk inhibitors may be of therapeutic benefit in human lupus and related disorders.

The ubiquitin-proteasome system (UPS) plays an essential role in a wide variety of cell regulatory signaling pathways. The clinical effectiveness of the proteasome inhibitor Velcade in the treatment of several human cancers underscores the importance of the UPS as a novel target area for pharmaceutical intervention. E3 ubiquitin ligases are key enzyme complexes that regulate and determine the ubiquitination of specific substrates, whose abnormal regulation has been implicated in multiple disease phenotypes. Targeting a selective E3 ligase may allow specific manipulation of distinct pathways and eventually lead to a better therapeutic index with reduced nonspecific side effects. Here, we aim to discuss the challenges of interfering with small molecules in this target class, as well as current strategies and progress in E3 ligase drug discovery.

The binding of viridans group streptococci with human platelets was analyzed by two-color flow cytometry. Binding was detected within 15 s of mixing bacteria and platelets. At ratios of bacteria to platelets of 1:1, 10:1, 100:1, and 1,000:1, the percentages of bound streptococci (mean +/- standard deviation) were 93.2% +/- 5.4%, 80.0% +/- 8.6%, 39.8% +/- 11.1%, and 12.5% +/- 2.0%, respectively. Binding of labeled bacteria was reversed by adding a 500-fold excess of unlabeled streptococci. These results demonstrate that streptococcus-platelet binding is rapid, reversible, and saturable, which suggests a specific receptor-ligand interaction.

Human IM-9 B-lymphoblasts have been shown to express the receptor for the neuropeptide substance P (SP). The present study was undertaken to evaluate the effect of sex hormones on this receptor. Testosterone inhibited [125I]SP binding in a dose-dependent manner with an IC50 of approximately 100 nM, while both estradiol and progesterone failed to inhibit SP binding even at concentrations as high as 1 microM. Furthermore, Scatchard analysis indicated that the dissociation constant (Kd) of the SP receptor was markedly increased from 0.25 nM to 2.2 nM when cells were incubated with testosterone. These data indicate a unique neuro-endocrine interaction on lymphocytes involving the substance P receptor.

Substance P (SP) is an 11-amino-acid neuropeptide found in sensory neurons in the peripheral nervous system. In addition to having well-characterized functions as a peptide neurotransmitter, it also plays a major role in modulating inflammatory and immune responses. SP can alter the proliferative and physiological responses of both lymphocytes and macrophages. These effects are mediated by specific high-affinity SP receptors which have been characterized both kinetically and biochemically. The principle SP binding protein present on human lymphocyte cell membranes is a 58,000-MW hydrophobic glycoprotein. Cellular responses subsequent to the binding of substance P to its receptor that have been identified in various cell populations include phosphatidyl inositol turnover, arachidonic acid metabolism, immunoglobulin synthesis, and enzyme production and secretion. Evidence also suggests that SP modulation of inflammation is a factor in the pathophysiology of certain diseases such as rheumatoid arthritis.

We have used electrospray mass spectrometry to examine the dimerization of EFLIVKS, a reversed sequence analog of part of the neuropeptide head activator, and other similar analogs. Observation of the EFLIVKS gas phase dimer is concentration-dependent, with a half-saturation concentration for relative dimer formation of 7.8 μM, similar to that of SKVILFE of 12 μM. The lowest energy conformers from quenched molecular dynamics simulations suggest EFLIVKS may dimerize in the gas phase by formation of multiple ion pairs across the dimer interface. Alanine-scan mutants also dimerize in the gas phase, with replacement of the interior residues FLIVK diminishing dimerization. The concentration-dependence of the EFLIVKS circular dichroism spectrum at pH 7.5 suggests the existence of different conformation states at different concentrations, but does not provide evidence supporting the saturable dimer formation in solution. Different analogs of EFLIVKS, when fused to each end of a 18mer unfolded peptide, induce solution structures with Tms of 42–50°C. These peptides and analogs may thus be useful for the noncovalent constraint of peptides and peptide library members used in cellular screens. © 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88:55–63, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [ N 4-(2,2-dimethyl-3-oxo-4 H -pyrid[1,4]oxazin-6-yl)-5-fluoro- N 2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC 50 for degranulation = 56–64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor ( K i = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

Purpose: The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments. Experimental Design: A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC50, 20 nmol/L) when examined in a spectrum of tumor cell lines. Results: R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G2-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes. Conclusion: Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.

Ubiquitin (Ub, 76aa) is a small highly conserved protein present universally in eukaryotic cells. Covalent attachment of (Ub)(n) to target proteins is a well-known posttranslational modification that has been implicated in a wide array of cellular processes including cell biogenesis. Ubiquitin polymerization by the Ub activation-conjugation-ligation cascade and the reverse disassembly process catalyzed by Ub isopeptidases largely regulate substrate protein targeting to the 26S proteasome. Ub chains of four or more subunits attached by K48 isopeptide linkages have been shown to be necessary for the 26S proteasome association and subsequent degradation of protein molecules. To better understand this protein degradation event, it is important to develop Ub polymerization and depolymerization assays that monitor every reaction step involved in Ub attachment to, or detachment from, substrate protein molecules. In this chapter, we describe homogeneous, easy-to-use, nonradioactive, complementary continuous fluorescence assays capable of monitoring the kinetics of Ub chain formation by E3 Ub ligases, and their hydrolysis by isopeptidases, which rely on mixing a 1:1 population of fluorophore-labeled Ub molecules containing a FRET pair. The proximity of fluorescein (donor) and tetramethylrhodamine (acceptor) in Ub polymers results in fluorescein quenching on ligase-induced Ub chain assembly. Conversely, a dramatic enhancement of fluorescein emission was observed on Ub chain disassembly because of isopeptidase activity. These assays thus provide a valuable tool for monitoring Ub ligase and isopeptidase activities using authentic Ub monomers and polymers as substrates. Screening of a large number of small molecule compound libraries in a high-throughput fashion is achievable, warranting further optimization of these assays.

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the securin protein and mitotic cyclins, resulting in the regulation of the onset of sister-chromatid separation and mitotic exit. In an effort to identify novel therapeutic compounds that modulate cell proliferation and, therefore, have potential applications in oncology, a plate-based in vitro ubiquitination assay that uses recombinant purified E1, E2 (UbcH5c), E3 (APC11/APC2), and Flag-ubiquitin has been established and used to screen for small molecule inhibitors of APC E3 ligase activity. In this assay, APC2/APC11 is immobilized on the plate, and its E3 ligase activity (i.e., the incorporation of Flag-tagged polyubiquitin chain onto APC2/APC11 as a result of auto-ubiquitination) is detected with anti-Flag-horseradish peroxidase-conjugated antibody by monitoring the luminescence signal from the plate. Here we describe in detail the protocol for high-throughput screening of APC, including expression and purification of the individual proteins, assay development, and optimization. This assay has been validated in a 96-well plate format and successfully implemented to identify novel small molecule compounds that potently inhibit APC2/APC11 ligase activity.