ABSTRACT
The discovery of a new series of selective S1P1 agonists is described. This series of piperazinyl-oxadiazole derivatives was rapidly optimized starting from high-throughput screening hit 1 to afford potent and selective lead compound 10d. Further SAR studies showed that 10d was converted to the active phosphate metabolite 29 in vivo. Oral administration of compound 10d to rats was shown to induce lymphopenia at 3 mg/kg.
Subject(s)
Oxadiazoles/pharmacology , Piperazines/pharmacology , Receptors, Lysosphingolipid/agonists , Administration, Oral , Animals , Dose-Response Relationship, Drug , Female , Lymphopenia/chemically induced , Lymphopenia/pathology , Molecular Structure , Oxadiazoles/administration & dosage , Oxadiazoles/chemistry , Piperazines/administration & dosage , Piperazines/chemistry , Rats , Rats, Inbred Lew , Sphingosine-1-Phosphate Receptors , Structure-Activity RelationshipABSTRACT
Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1-/- animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1-/- mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.
Subject(s)
Neutrophil Infiltration/immunology , Neutrophils/immunology , Pain/immunology , Receptors, CCR1/immunology , Acetic Acid , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Transplantation/methods , Cell Movement/genetics , Cell Movement/immunology , Flow Cytometry , Freund's Adjuvant , Hyperalgesia/chemically induced , Hyperalgesia/genetics , Hyperalgesia/immunology , Leukocytes/immunology , Leukocytes/metabolism , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/immunology , Myeloid Cells/metabolism , Neutrophil Infiltration/genetics , Neutrophils/metabolism , Pain/chemically induced , Pain/genetics , Pain Measurement/methods , Peritonitis/genetics , Peritonitis/immunology , Peritonitis/metabolism , Receptors, CCR1/antagonists & inhibitors , Receptors, CCR1/geneticsABSTRACT
Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.
Subject(s)
Benzimidazoles/chemical synthesis , Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/chemical synthesis , Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Benzimidazoles/pharmacology , CD3 Complex/biosynthesis , Drug Design , Enzyme Inhibitors/pharmacology , Female , Hepatocytes/metabolism , Humans , Inhibitory Concentration 50 , Mice , Mice, Inbred BALB C , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Interleukin-2 inducible T-cell kinase (ITK) is a member of the Tec kinase family and is involved with T-cell activation and proliferation. Due to its critical role in acting as a modulator of T-cells, ITK inhibitors could provide a novel route to anti-inflammatory therapy. This work describes the discovery of ITK inhibitors through structure-based design where high-resolution crystal structural information was used to optimize interactions within the kinase specificity pocket of the enzyme to improve both potency and selectivity.
Subject(s)
Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Amino Acid Motifs , Anti-Inflammatory Agents/pharmacology , Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Crystallography, X-Ray/methods , Drug Design , Enzyme Inhibitors/chemical synthesis , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , Pyridines/chemistry , Structure-Activity RelationshipABSTRACT
Previously, we reported a series of novel benzimidazole based Itk inhibitors that exhibited excellent enzymatic potency and selectivity but low microsomal stability. Employing a structure based approach a new series of inhibitors with comparable potency and selectivity to the original series and with a potential for improved microsome stability was identified.
Subject(s)
Benzimidazoles/chemistry , Benzimidazoles/chemical synthesis , Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/chemical synthesis , Microsomes, Liver/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Adenosine Triphosphate/chemistry , Administration, Oral , Binding Sites , CD3 Complex/chemistry , Crystallography, X-Ray/methods , Drug Design , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Microsomes, Liver/chemistry , Structure-Activity Relationship , T-Lymphocytes/metabolismABSTRACT
A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.
Subject(s)
Amides/chemistry , Benzimidazoles/chemistry , Carboxylic Acids/chemistry , Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/chemical synthesis , Microsomes, Liver/metabolism , Protein-Tyrosine Kinases/chemistry , Animals , Benzimidazoles/chemical synthesis , Carboxylic Acids/chemical synthesis , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Mice , Models, Chemical , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Biochemistry in the context of a living cell or organism is complicated by many variables such as supramolecular organization, cytoplasmic viscosity, and substrate heterogeneity. While these variables are easily excluded or avoided in reconstituted systems, they must be dealt with in cellular environments. New developments have allowed researchers to begin probing the inner workings of the cell to gain new insight into cell function and metabolism. Advances in cellular imaging and in small molecule-controlled gene expression, signal transduction and cell surface modification are discussed in this review. These techniques have permitted the study of molecular components within the context of living cells.
Subject(s)
Biochemistry/methods , Cell Physiological Phenomena , Cells/chemistry , Animals , Cytological Techniques , Diagnostic Imaging/methods , Gene Expression , Humans , Membrane Glycoproteins , Molecular Probes , Signal TransductionABSTRACT
Tyrosylprotein sulfotransferases (TPSTs) catalyze the sulfation of tyrosine residues within secreted and membrane-bound proteins. The modification modulates protein-protein interactions in the extracellular environment. Here we use combinatorial target-guided ligand assembly to discover the first known inhibitors of human TPST-2.
