ABSTRACT
Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. C1q is composed of A, B, and C chains that require post-translational prolyl 4-hydroxylation of their N-terminal collagen-like domain to enable the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized prolyl 4-hydroxylases include collagen prolyl-4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small-molecule prolyl hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. In vitro studies showed that a C1q A chain peptide is not a substrate for PHD2 but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild-type mice or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.
Subject(s)
Complement C1q/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Hypoxia/metabolism , Macrophages/metabolism , Procollagen-Proline Dioxygenase/metabolism , Prolyl-Hydroxylase Inhibitors/pharmacology , Anemia/drug therapy , Anemia/etiology , Animals , Cell Line , Complement C1q/analysis , Complement Pathway, Classical , Female , Humans , Hydroxylation , Kidney Diseases/blood , Kidney Diseases/drug therapy , Kidney Diseases/pathology , Macrophages/drug effects , Male , Mice , Mice, Inbred C57BL , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Prolyl-Hydroxylase Inhibitors/therapeutic use , Protein Processing, Post-TranslationalABSTRACT
Intracellular levels of the hypoxia-inducible transcription factor (HIF) are regulated under normoxic conditions by prolyl hydroxylases (PHD1, 2, and 3). Treatment of cells with PHD inhibitors stabilizes HIF-1α, eliciting an artificial hypoxic response that includes the transcription of genes involved in erythropoiesis, angiogenesis, and glycolysis. The different in vivo roles of the three PHD isoforms are not yet known, making a PHD-selective inhibitor useful as a biological tool. Although several chemical series of PHD inhibitors have been described, significant isoform selectivity has not been reported. Here we report the synthesis and activity of dipeptidyl analogues derived from a potent but non-selective quinolone scaffold. The compounds were prepared by Pd-catalyzed reductive carbonylation of the 6-iodoquinolone derivative to form the aldehyde directly, which was then attached to a solid support via reductive amination. Amino acids were coupled, and the resulting dipeptidyl-quinolone derivatives were screened, revealing retention of PHD inhibitory activity but an altered PHD1, 2, and 3 selectivity profile. The compounds were found to be â¼10-fold more potent against PHD1 and PHD3 than against PHD2, whereas the specific parent compound had shown no appreciable selectivity among the different PHD isoforms.
Subject(s)
Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Quinolones/pharmacology , Combinatorial Chemistry Techniques , Dipeptides/chemical synthesis , Dipeptides/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Molecular Structure , Procollagen-Proline Dioxygenase/chemistry , Procollagen-Proline Dioxygenase/metabolism , Quinolones/chemical synthesis , Quinolones/chemistry , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The bradykinin B1 receptor has been shown to mediate pain response and is rapidly induced upon injury. Blocking this receptor may provide a promising treatment for inflammation and pain. We previously reported tetralin benzyl amines as potent B1 antagonists. Here we describe the synthesis and SAR of B1 receptor antagonists with homobenzylic amines. The SAR of different linkers led to the discovery of tetralin allylic amines as potent and selective B1 receptor antagonists (hB1 IC(50)=1.3 nM for compound 16). Some of these compounds showed modest oral bioavailability in rats.
Subject(s)
Benzylamines/chemistry , Bradykinin B1 Receptor Antagonists , Sulfonamides/chemistry , Tetrahydronaphthalenes/chemistry , Administration, Oral , Animals , Pain/drug therapy , Rats , Receptor, Bradykinin B1/metabolism , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic useABSTRACT
We report the structure-based design and synthesis of a novel series of aza-benzimidazoles as PHD2 inhibitors. These efforts resulted in compound 22, which displayed highly potent inhibition of PHD2 function in vitro.
Subject(s)
Aza Compounds/chemical synthesis , Aza Compounds/pharmacology , Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Aza Compounds/chemistry , Basic Helix-Loop-Helix Transcription Factors , Benzimidazoles/chemistry , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
We report the development of aryl sulfones as Bradykinin B1 receptor antagonists. Variation of the linker region identified diol 23 as a potent B1 antagonist, while modifications of the aryl moiety led to compound 26, both of which were efficacious in rabbit biochemical challenge and pain models.
Subject(s)
Bradykinin B1 Receptor Antagonists , Pain/drug therapy , Sulfones/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Bradykinin B2 Receptor Antagonists , Chronic Disease , Humans , Rabbits , Rats , Rats, Sprague-Dawley , Sulfones/administration & dosageABSTRACT
Inhibition of the PHD2 enzyme has been associated with increased red blood cell levels. From a screening hit, a series of novel hydroxyl-thiazoles were developed as potent PHD2 inhibitors.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/chemical synthesis , Thiazoles/chemistry , Chemistry, Pharmaceutical/methods , Diabetes Mellitus/drug therapy , Drug Design , Drug Evaluation, Preclinical/methods , Erythrocytes/enzymology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Inhibitory Concentration 50 , Models, Chemical , Models, Molecular , Myocardial Infarction/drug therapy , Peripheral Vascular Diseases/therapy , Stroke/drug therapy , Structure-Activity Relationship , Thiazoles/pharmacologyABSTRACT
UNLABELLED: Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but more sustained exposures to PTH. A PTH-Fc fusion protein with a longer half-life than PTH(1-34) increased cortical and cancellous BMD and bone strength with once- or twice-weekly injections. INTRODUCTION: The anabolic effects of PTH are currently achieved with, and thought to require, daily injections that result in brief exposure to the peptide. We hypothesized that less frequent but more sustained exposures to PTH could also be anabolic for bone, provided that serum levels of PTH were not constant. MATERIALS AND METHODS: PTH(1-34) was fused to the Fc fragment of human IgG1 to increase the half-life of PTH. Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH-Fc fusion protein. RESULTS: PTH-Fc and PTH(1-34) had similar effects on PTH/PTHrP receptor activation, internalization, and signaling in vitro. However, PTH-Fc had a 33-fold longer mean residence time in the circulation of rats compared with that of PTH(1-34). Subcutaneous injection of PTH-Fc once or twice per week resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats. These anabolic effects occurred in association with hypercalcemia and were significantly greater than those achievable with high concentrations of daily PTH(1-34). PTH-Fc also significantly improved cortical bone volume and density under conditions where daily PTH(1-34) did not. Antiresorptive co-therapy with estrogen further enhanced the ability of PTH-Fc to increase bone mass and strength in ovariectomized rats. CONCLUSIONS: These results challenge the notion that brief daily exposure to PTH is essential for its anabolic effects on cortical and cancellous bone. PTH-derived molecules with a sustained circulating half-life may represent a powerful and previously undefined anabolic regimen for cortical and cancellous bone.
Subject(s)
Anabolic Agents/administration & dosage , Anabolic Agents/pharmacology , Bone and Bones/drug effects , Immunoglobulin Fc Fragments/administration & dosage , Parathyroid Hormone/administration & dosage , Parathyroid Hormone/pharmacology , Recombinant Proteins/pharmacology , Aging/physiology , Anabolic Agents/pharmacokinetics , Animals , Arrestins/metabolism , Bone and Bones/metabolism , Cell Line , Cricetinae , Dose-Response Relationship, Drug , Estrogens/pharmacology , Half-Life , Humans , Male , Mice , Ovariectomy , Parathyroid Hormone/pharmacokinetics , Protein Transport , Rats , Receptor, Parathyroid Hormone, Type 1/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacokinetics , Time Factors , beta-ArrestinsABSTRACT
The bradykinin B1 receptor is induced following tissue injury and/or inflammation. Antagonists of this receptor have been studied as promising candidates for treatment of chronic pain. We have identified aryl sulfonamides containing a chiral chroman diamine moiety that are potent antagonists of the human B1 receptor. Our previously communicated lead, compound 2, served as a proof-of-concept molecule, but suffered from poor pharmacokinetic properties. With guidance from metabolic profiling, we performed structure-activity relationship studies and have identified potent analogs of 2. Variation of the sulfonamide moiety revealed a preference for 3- and 3,4-disubstituted aryl sulfonamides, while bulky secondary and tertiary amines were preferred at the benzylic amine position for potency at the B1 receptor. Modifying the beta-amino acid core of the molecule lead to the discovery of highly potent compounds with improved in vitro pharmacokinetic properties. The most potent analog at the human receptor, compound 38, was also active in a rabbit B1 receptor cellular assay. Furthermore, compound 38 displayed in vivo activity in two rabbit models, a pharmacodynamic model with a blood pressure readout and an efficacy model of inflammatory pain.
Subject(s)
Amides/chemical synthesis , Analgesics/chemical synthesis , Benzopyrans/chemical synthesis , Bradykinin B1 Receptor Antagonists , Chromans/chemical synthesis , Sulfonamides/chemical synthesis , Amides/pharmacokinetics , Amides/pharmacology , Analgesics/pharmacokinetics , Analgesics/pharmacology , Animals , Benzopyrans/pharmacokinetics , Benzopyrans/pharmacology , Blood Pressure/drug effects , CHO Cells , Calcium/metabolism , Chromans/pharmacokinetics , Chromans/pharmacology , Cricetinae , Cricetulus , Humans , In Vitro Techniques , Inflammation/drug therapy , Male , Microsomes/metabolism , Pain/drug therapy , Rabbits , Rats , Rats, Sprague-Dawley , Receptor, Bradykinin B1/agonists , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacologyABSTRACT
We report the discovery of chroman 28, a potent and selective antagonist of human, nonhuman primate, rat, and rabbit bradykinin B1 receptors (0.4-17 nM). At 90 mg/kg s.c., 28 decreased plasma extravasation in two rodent models of inflammation. A novel method to calculate entropy is introduced and ascribed approximately 30% of the gained affinity between "flexible" 4 (Ki = 132 nM) and "rigid" 28 (Ki = 0.77 nM) to decreased conformational entropy.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Bradykinin B1 Receptor Antagonists , Chromans/chemical synthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , CHO Cells , Capillary Permeability/drug effects , Chlorocebus aethiops , Chromans/pharmacokinetics , Chromans/pharmacology , Cricetinae , Cricetulus , Crystallography, X-Ray , Entropy , Humans , In Vitro Techniques , Models, Molecular , Molecular Conformation , Pleurisy/drug therapy , Rabbits , Rats , Species Specificity , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Conventional assay methods for discovering and profiling drug-target interactions are typically developed on a target-by-target basis and hence can be cumbersome to enable and orchestrate. Herein the authors report a solid-state ligand-binding assay that operates in a multiplexed mode to report compound activity against a micorarray-configured panel of G-protein-coupled receptor (GPCR) targets. The pharmacological fidelity of the system is high, and its miniaturized "plug-and-play" format provides improved efficiency both in terms of execution time and reagent consumption. Taken together, these features make the system ideally suited to explore the structure-activity relationship of compounds across a broad region of target class space.
Subject(s)
Drug Evaluation, Preclinical/methods , Protein Array Analysis/methods , Receptors, G-Protein-Coupled/drug effects , Fluorescent Dyes , In Vitro TechniquesABSTRACT
The bradykinin 1 (B1) receptor is upregulated during times of inflammation and is important for maintaining inflamed and chronic pain states. Blocking this receptor has been shown to reverse and/or ameliorate pain and inflammation in animal models. In this report, we describe a new class of B1 receptor antagonists that contain the piperidine acetic acid tetralin core. A structure-activity relationship for these analogs is described in this paper. The most potent compounds from this class have IC50s<20 nM in a B1 receptor functional assay. One of these compounds, 13g, shows modest oral bioavailability in rats.