Free fatty acid-2 (FFA2) receptor is a G-protein coupled receptor of interest in the development of therapeutics in metabolic and inflammatory disease areas. The discovery and optimization of an N-thiazolylamide carboxylic acid FFA2 agonist scaffold is described. Dual key objectives were to i) evaluate the potential of this scaffold for lead optimization in particular with respect to safety de-risking physicochemical properties, i.e. lipophilicity and aromatic content, and ii) to demonstrate the utility of selected lead analogues from this scaffold in a pertinent in vivo model such as oral glucose tolerance test (OGTT). As such, a concomitant improvement in bioactivity together with lipophilic ligand efficiency (LLE) and fraction sp3 content (Fsp3) parameters guided these efforts. Compound 10 was advanced into studies in mice on the basis of its optimized profile vs initial lead 1 (ΔLLE = 0.3, ΔFsp3 = 0.24). Although active in OGTT, 10 also displayed similar activity in the FFA2-knockout mice. Given this off-target OGTT effect, we discontinued development of this FFA2 agonist scaffold.

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

The chemokine G protein-coupled receptor CC chemokine receptor 5 (CCR5) is used as an entry gate by CCR5-tropic and dual- or CCR5/CXC chemokine receptor 4-tropic strains of HIV to enter the human host cells. Thus, CCR5 antagonists (i.e., maraviroc) have been proven to be clinically effective by preventing the interaction between viral glycoprotein 120 and CCR5 and thus impeding viral entry into host cells. However, the emergence of HIV strains resistant to CCR5 antagonists has been reported in vitro and in vivo, where the virus has adapted to enter the cells via antagonist-bound CCR5. An alternative strategy that should obviate this mode of viral resistance would entail the ablation of the CCR5 portal for HIV entry from the cell surface through agonist-induced receptor internalization. Although this protective effect has been demonstrated clearly with natural CCR5 ligands, the chemoattractant properties of these chemokines have precluded them from further consideration in terms of drug development. Thus, we sought to explore the possibility of developing novel small molecules and selective CCR5 agonists devoid of eliciting chemotaxis. Indeed, the CCR5 agonists described herein were found to induce profound down-modulation of CCR5 (and not CXC chemokine receptor 4) from the cell surface and its sustained sequestration in the intracellular compartment without inducing chemotaxis in vitro. The bioactivity profile of these novel CCR5 agonists is exemplified by the compound (R)-2-(4-cyanophenyl)-N-(1-(1-(N,1-diphenylmethylsulfonamido)propan-2-yl)piperidin-4-yl)acetamide (ESN-196) that potently inhibits HIV-1 infection in human peripheral blood mononuclear cells and macrophages in vitro with potencies comparable to that of maraviroc and moreover demonstrates full activity against a maraviroc-resistant HIV-1 RU570 strain.

IL-15 is critical for natural killer (NK)-cell development and function and for memory CD8(+) T-cell homeostasis. The IL-15 receptor consists of IL-15Ralpha, IL-2Rbeta, and the common cytokine receptor gamma chain (gamma(c)). IL-15Ralpha is known to "trans-present" IL-15 to an IL-2Rbeta/gamma(c) heterodimeric receptor on responding cells to initiate signaling. To investigate the importance of the IL-15Ralpha cytoplasmic domain, we generated a chimeric receptor consisting of the extracellular domain of IL-15Ralpha and intracellular domain of IL-2Ralpha (IL-15Ralpha(ext)/IL-2Ralpha(int)) and examined its function in 32D cells, in knock-in (KI) mice, and in adoptive-transfer experiments. The chimeric protein exhibited decreased cell-surface expression, and KI mice exhibited diminished NK, NKT, and CD8(+) T-cell development and defects in T-cell functional responses. However, 32D cells expressing the chimeric receptor had less IL-15-induced proliferation than wild-type (WT) transfectants with similar levels of IL-15Ralpha expression, indicating a signaling role for the IL-15Ralpha cytoplasmic domain beyond its effect on expression, and demonstrating that the IL-2Ralpha and IL-15Ralpha cytoplasmic domains are functionally distinct. Interestingly, adoptive-transfer experiments indicated that the chimeric IL-15Ralpha(ext)/IL-2Ralpha(int) receptor still supports trans-presentation. These experiments collectively indicate that IL-15Ralpha can act in cis in addition to acting in trans to present IL-15 to responding cells.

Free fatty acids (FFAs) fuel physiological processes in various tissues, such as skeletal muscle, the heart, liver and pancreas. Malfunction of FFA-mediated signaling pathways, as well as FFA metabolism, has been correlated to pathologies such as type 2 diabetes and dyslipidemia. The recent deorphanization of the FFA receptor family has drawn attention to these G-protein-coupled receptors as potential therapeutic targets for the treatment of metabolic diseases.

Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates lymphocyte proliferation and peripheral tolerance. IL-2 activates mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and signal transducer and activator of transcription (STAT) pathways and modulates expression of target genes. Systematic analysis of IL-2 target genes has revealed regulation of potential feedback inhibitors of IL-2 signaling, including several suppressor of cytokine signaling (SOCS) family members as well as MAPK pathway-regulating dual specificity phosphatases (DUSPs). Here we have evaluated the in vivo actions of DUSP5, an extracellular signal-regulated kinase 1/2 (ERK1/2)-specific phosphatase, by generating transgenic mice overexpressing DUSP5 within the lymphoid compartment. We show that transgenic DUSP5 expression results in a block in thymocyte development at the double positive stage. We also demonstrate that DUSP5-expressing mature T cells exhibit decreased IL-2-dependent proliferation and defective IL-2-mediated induction of genes. Finally, DUSP5 transgenic mice develop autoimmune symptoms, suggesting a role for the MAPK pathway in the regulation of tolerance. Thus, proper regulation of DUSP5 activity is critical for normal immune system development, IL-2 actions, and tolerance.

A structural model of the sushi domain of IL‐15Rα was first obtained by homology modeling to study its interactions with IL‐15 by means of molecular modeling, peptide scanning, and site‐directed mutagenesis. From these experimental data, a putative interacting surface of IL‐15Rα with a previously published IL‐15 model was inferred: Leu25, Leu44, and Glu46 of IL‐15 and Arg35 of IL‐15Rα were found to be key interfacial residues and were subsequently used as filters for the construction of docking solutions. Human IL‐15/IL‐15Rα complexes were constructed in two stages, with a preliminary docking procedure, treating the two partners as rigid bodies and using these filters. In this first stage, two classes of docking solutions were characterized. From a topological point of view, each solution could be derived from the other by reverse orientation of one partner in relation to the other. In a second stage, several further energy refinements clearly favored one solution. Moreover, this unique docking solution was confirmed by molecular modeling of IL‐15 mutants previously built and tested in our laboratory. Finally, this complex model, which is a useful tool to study the IL‐15/IL‐15Rα interface, was topologically compared to IL‐2/IL‐2Rα complexes (previous model in the literature and recent crystal structure). Proteins 2006. © 2006 Wiley‐Liss, Inc.

IL-15 and IL-2 are two structurally and functionally related cytokines whose high affinity receptors share the IL-2R beta-chain and gamma-chain in association with IL-15R a-chain (IL-15kalpha) or IL-211 alpha-chain, respectively. Whereas IL-2 action seems restricted to the adaptative T cells, IL-15 appears to be crucial for the function of the innate immune responses, and the pleiotropic expression of IL-15 and IL-15Ralpha hints at a much broader role for the IL-15 system in multiple cell types and tissues. In this report, using a highly sensitive radioimmunoassay, we show the existence of a soluble form of human IL-15Ralpha (sIL-15Ralpha) that arises from proteolytic shedding of the membrane-anchored receptor. This soluble receptor is spontaneously released from IL-15Ralpha-expressing human cell lines as well as from IL-15Ralpha transfected COS-7 cells. This release is strongly induced by PMA and ionomycin, and to a lesser extent by IL-1beta and TNF-alpha. The size of sIL-15Ralpha (42 kDa), together with the analysis of deletion mutants in the ectodomain of IL-15Ralpha, indicates the existence of cleavage sites that are proximal to the plasma membrane. Whereas shedding induced by PMA was abrogated by the synthetic matrix metalloproteinases inhibitor GM6001, the spontaneous shedding was not, indicating the occurrence of at least two distinct proteolytic mechanisms. The sIL-15Ralpha displayed high affinity for IL-15 and behaved as a potent and specific inhibitor of IL-15 binding to the membrane receptor, and of IL-15-induced cell proliferation (IC50 in the range from 3 to 20 pM). These results suggest that IL-15Ralpha shedding may play important immunoregulatory functions.

To identify the epitopes in human interleukin-15 (IL-15) that are responsible for binding to the interleukin-15 receptor α chain, antibody and receptor mapping by peptide scanning and site-directed mutagenesis was used. By using peptide scanning, we identified four regions in IL-15. The first region (85CKECEELEEKN95) is located in the C-D loop and is recognized by a set of non-inhibitory antibodies. The second region (102SFVHIVQMFIN112) is located in helix D and is recognized by two antibodies that are inhibitory of IL-15 bio-activity but not of IL-15 binding to IL-15Rα. The two remaining regions react with a recombinant soluble form of the IL-15Rα; the first (44LLELQVISL52, peptide 1) corresponds to a sequence located in the B-helix and the second (64ENLII68, peptide 2) to a sequence located in helix C. The latter is also contained in the epitope recognized by an antibody (monoclonal antibody B-E29) that prevents IL-15 binding to IL-15Rα. By site-directed mutagenesis, we confirmed that residues present in peptide 1 (Leu-45, Glu-46, Val-49, Ser-51, and Leu-52) and peptide 2 (Leu-66 and Ile-67) are involved in the binding of IL-15 to IL-15Rα. Furthermore, the results presented indicate that residues in the second peptide (Glu-64, Asn-65, and Ile-68) participate in IL-2Rβ recruitment. This finding could have implications for the dynamics of receptor assembly. These results also indicate that the modes of interaction of IL-15 and IL-2 with their respective α chains are not completely analogous. Finally, some of the IL-15 mutants generated in this study displayed agonist or antagonist properties and may be useful as therapeutic agents.

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15R alpha) transcripts resulting from exon-splicing mechanisms within the IL-15R alpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta 2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) art comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation stales, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15R alpha bound IL-15 with high affinity, Delta 2IL-15R alpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15R alpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15 IL-15R alpha complex at the nuclear level. In sharp contrast, Delta 2IL-15R alpha was found only in the non-nuclear membrane compartments, indicating that the exon alpha-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15R alpha is a natural process that might play regulatory roles at different levels.