ABSTRACT
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age. A frequent class of mutations in RDEB is premature termination codons (PTC), which appear in homozygosity or compound heterozygosity with other mutations. RDEB has no cure and current therapies are mostly palliative. Using patient-derived keratinocytes and a library of 8273 small molecules and 20,160 microbial extracts evaluated in a phenotypic screening interrogating C7 levels, we identified three active chemical series. Two of these series had PTC readthrough activity, and one upregulated C7 mRNA, showing synergistic activity when combined with the reference readthrough molecule gentamicin. These compounds represent novel potential small molecule-based systemic strategies that could complement topical-based treatments for RDEB.
Subject(s)
Collagen Type VII , Epidermolysis Bullosa Dystrophica , Epidermolysis Bullosa Dystrophica/genetics , Epidermolysis Bullosa Dystrophica/drug therapy , Collagen Type VII/genetics , Collagen Type VII/metabolism , Humans , Up-Regulation/drug effects , Keratinocytes/drug effects , Keratinocytes/metabolism , Small Molecule Libraries/pharmacology , Codon, Nonsense , Gentamicins/pharmacologyABSTRACT
Rational design of a novel template of naphthyridinones rapidly led to PDE4 inhibitors with subnanomolar enzymatic potencies. X-ray crystallography confirmed the binding mode of this novel template. We achieved compounds with double-digit picomolar enzymatic potencies through further structure-based design by targeting both the PDE4 enzyme metal-binding pocket and occupying the solvent-filled pocket. A strategy for lung retention and long duration of action based on low aqueous solubility was followed. In vivo efficacies were measured in a rat lung neutrophilia model by suspension microspray and dry powder administration. Suspension microspray of potent compounds showed in vivo efficacy with a clear dose-response. Despite sustained lung levels, dry powder administration performed much less well and without proper dose-response, highlighting clear differences between the two formulations. This indicates a deficiency in the low aqueous solubility strategy for long duration lung efficacy.
Subject(s)
Naphthyridines/chemical synthesis , Naphthyridines/pharmacology , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/pharmacology , Administration, Inhalation , Animals , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Design , Dry Powder Inhalers , Lipopolysaccharides/pharmacology , Lung/metabolism , Male , Naphthyridines/administration & dosage , Neutrophils/drug effects , Phosphodiesterase 4 Inhibitors/administration & dosage , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/bloodABSTRACT
Thymic stromal lymphopoietin (TSLP) is a cytokine released by human lung epithelium in response to external insult. Considered as a master switch in T helper 2 lymphocyte (Th2) mediated responses, TSLP is believed to play a key role in allergic diseases including asthma. The aim of this study was to use a phenotypic approach to identify new biological and chemical starting points for inhibition of TSLP production in human bronchial epithelial cells (NHBE), with the objective of reducing Th2-mediated airway inflammation. To this end, a phenotypic screen was performed using poly I:C / IL-4 stimulated NHBE cells interrogated with a 44,974 compound library. As a result, 85 hits which downregulated TSLP protein and mRNA levels were identified and a representative subset of 7 hits was selected for further characterization. These molecules inhibited the activity of several members of the MAPK, PI3K and tyrosine kinase families and some of them have been reported as modulators of cellular phenotypic endpoints like cell-cell contacts, microtubule polymerization and caspase activation. Characterization of the biological profile of the hits suggested that mTOR could be a key activity involved in the regulation of TSLP production in NHBE cells. Among other targeted kinases, inhibition of p38 MAPK and JAK kinases showed different degrees of correlation with TSLP downregulation, while Syk kinase did not seem to be related. Overall, inhibition of TSLP production by the selected hits, rather than resulting from inhibition of single isolated targets, appeared to be due to a combination of activities with different levels of relevance. Finally, a hit expansion exercise yielded additional active compounds that could be amenable to further optimization, providing an opportunity to dissociate TSLP inhibition from other non-desired activities. This study illustrates the potential of phenotypic drug discovery to complement target based approaches by providing new chemistry and biology leads.
Subject(s)
Cytokines/antagonists & inhibitors , Drug Discovery , Lung/drug effects , Cells, Cultured , Cytokines/biosynthesis , Epithelial Cells/drug effects , Humans , Lung/cytology , Lung/metabolism , Real-Time Polymerase Chain Reaction , Thymic Stromal LymphopoietinABSTRACT
Cyclic nucleotide cAMP is a ubiquitous secondary messenger involved in a plethora of cellular responses to biological agents involving activation of adenylyl cyclase. Its intracellular levels are tightly controlled by a family of cyclic nucleotide degrading enzymes, the PDEs. In recent years, cyclic nucleotide phosphodiesterase type 4 (PDE4) has aroused scientific attention as a suitable target for anti-inflammatory therapy in respiratory diseases, particularly in the management of asthma and COPD. Here we describe our efforts to discover novel, highly potent inhaled inhibitors of PDE4. Through structure based design, with the inclusion of a variety of functional groups and physicochemical profiles in order to occupy the solvent-filled pocket of the PDE4 enzyme, we modified the structure of our oral PDE4 inhibitors to reach compounds down to picomolar enzymatic potencies while at the same time tackling successfully an uncovered selectivity issue with the adenosine receptors. In vitro potencies were demonstrated in a rat lung neutrophilia model by administration of a suspension with a Penn-Century MicroSprayer Aerosolizer.
Subject(s)
Biphenyl Compounds/pharmacology , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Phosphodiesterase 4 Inhibitors/pharmacology , Pyridazines/pharmacology , Animals , Biphenyl Compounds/chemical synthesis , Biphenyl Compounds/chemistry , Dose-Response Relationship, Drug , Humans , Male , Models, Molecular , Molecular Structure , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/chemistry , Pyridazines/chemical synthesis , Pyridazines/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTh2) is a G protein-coupled receptor expressed on the leukocytes most closely associated with asthma and allergy like eosinophils, mast cells, Th2-lymphocytes and basophils. At present it is clear that CRTh2 mediates most prostaglandin D2 (PGD2) pro-inflammatory effects and as a result antagonists for this receptor have reached asthma clinical studies showing a trend of lung function improvement. The challenge remains to identify compounds with improved clinical efficacy when administered once a day. Herein we described the pharmacological profile of LAS191859, a novel, potent and selective CRTh2 antagonist. In vitro evidence in GTPγS binding studies indicate that LAS191859 is a CRTh2 antagonist with activity in the low nanomolar range. This potency is also maintained in cellular assays performed with human eosinophils and whole blood. The main differentiation of LAS191859 vs other CRTh2 antagonists is in its receptor binding kinetics. LAS191859 has a residence time half-life of 21h at CRTh2 that translates into a long-lasting in vivo efficacy that is independent of plasma levels. We believe that the strategy behind this compound will allow optimal efficacy and posology for chronic asthma treatment.
Subject(s)
Anti-Asthmatic Agents/pharmacology , Eosinophils/drug effects , Prostaglandin Antagonists/pharmacology , Pyridines/pharmacology , Pyrroles/pharmacology , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Animals , Anti-Asthmatic Agents/blood , Anti-Asthmatic Agents/chemistry , Anti-Asthmatic Agents/pharmacokinetics , CHO Cells , Cell Shape/drug effects , Chemotaxis, Leukocyte/drug effects , Cricetulus , Dogs , Dose-Response Relationship, Drug , Drug Design , Eosinophils/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guinea Pigs , Half-Life , Kinetics , Male , Mice , Prostaglandin Antagonists/blood , Prostaglandin Antagonists/chemistry , Prostaglandin Antagonists/pharmacokinetics , Protein Binding , Pyridines/blood , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyrroles/blood , Pyrroles/chemistry , Pyrroles/pharmacokinetics , Rats, Wistar , Receptors, Immunologic/blood , Receptors, Immunologic/genetics , Receptors, Prostaglandin/blood , Receptors, Prostaglandin/genetics , TransfectionABSTRACT
A knowledge-based design strategy led to the discovery of several new series of potent and orally bioavailable CRTh2 antagonists where a bicyclic heteroaromatic ring serves as the central core. Structure-kinetic relationships (SKR) opened up the possibility of long receptor residence times.
Subject(s)
Acetates/chemistry , Bridged Bicyclo Compounds/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Acetates/chemical synthesis , Acetates/pharmacokinetics , Animals , Half-Life , Humans , Hydrogen-Ion Concentration , Microsomes, Liver/metabolism , Rats , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Solubility , Structure-Activity RelationshipABSTRACT
Pyrrolopiperidinone acetic acids (PPAs) were identified as highly potent CRTh2 receptor antagonists. In addition, many of these compounds displayed slow-dissociation kinetics from the receptor. Structure-kinetic relationship (SKR) studies allowed optimisation of the kinetics to give potent analogues with long receptor residence half-lives of up to 23 h. Low permeability was a general feature of this series, however oral bioavailability could be achieved through the use of ester prodrugs.
Subject(s)
Acetates/chemistry , Acetates/pharmacology , Piperidines/chemistry , Pyrazoles/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Acetates/chemical synthesis , Acetates/pharmacokinetics , Administration, Oral , Animals , Caco-2 Cells , Cell Membrane Permeability/drug effects , Half-Life , Humans , Rats , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Structure-Activity RelationshipABSTRACT
Extensive structure-activity relationship (SAR) and structure-kinetic relationship (SKR) studies in the bicyclic heteroaromatic series of CRTh2 antagonists led to the identification of several molecules that possessed both excellent binding and cellular potencies along with long receptor residence times. A small substituent in the bicyclic core provided an order of magnitude jump in dissociation half-lives. Selected optimized compounds demonstrated suitable pharmacokinetic profiles.
Subject(s)
Acetates/chemistry , Bridged Bicyclo Compounds/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Acetates/chemical synthesis , Acetates/pharmacokinetics , Animals , Half-Life , Humans , Indoles/chemistry , Injections, Intravenous , Rats , Rats, Wistar , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Structure-Activity RelationshipABSTRACT
The correct positioning and orientation of an hydrogen bond acceptor (HBA) in the tail portion of the biaryl series of CRTh2 antagonists is a requirement for long receptor residence time. The HBA in combination with a small steric substituent in the core section (R(core) ≠ H) gives access to compounds with dissociation half-lives of ⩾ 24h.
Subject(s)
Acetates/chemistry , Bridged Bicyclo Compounds/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Acetates/chemical synthesis , Acetates/pharmacokinetics , Half-Life , Humans , Hydrogen Bonding , Models, Chemical , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Structure-Activity RelationshipABSTRACT
In this manuscript, the synthesis and biological activity of a series of pyrazole acetic acid derivatives as CRTh2 antagonists is presented. Biological evaluation in vitro revealed that the pyrazole core showed in several cases a different structure-activity relationship (SAR) to that of related indole acetic acid. A potent series of ortho-sulfonyl benzyl substituents was found, from which compounds 27 and 63 were advanced to in vivo profiling.
Subject(s)
Acetates/chemistry , Acetates/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Acetates/chemical synthesis , Humans , Pyrazoles/chemical synthesis , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Structure-Activity RelationshipABSTRACT
High throughput screening identified the pyrazole-4-acetic acid substructure as CRTh2 receptor antagonists. Optimisation of the compounds uncovered a tight SAR but also identified some low nanomolar inhibitors.
Subject(s)
Acetates/chemistry , Pyrazoles/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Acetates/chemical synthesis , Acetates/pharmacokinetics , Animals , Half-Life , High-Throughput Screening Assays , Protein Binding , Rats , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Structure-Activity RelationshipABSTRACT
Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.
Subject(s)
Corticotropin-Releasing Hormone/physiology , Protein Multimerization , Receptors, Corticotropin-Releasing Hormone/metabolism , Receptors, Vasopressin/metabolism , Vasopressins/physiology , Adrenal Glands/cytology , Animals , Antidiuretic Hormone Receptor Antagonists , Catecholamines/metabolism , Cattle , Cells, Cultured , Chromaffin Cells/metabolism , Corticotropin-Releasing Hormone/pharmacology , Cyclic AMP/metabolism , Enzyme Activation , Estrenes/pharmacology , HEK293 Cells , Humans , Hydrocarbons, Halogenated/pharmacology , Indoles/pharmacology , Inositol Polyphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/metabolism , Primary Cell Culture , Protein Binding , Pyrrolidines/pharmacology , Pyrrolidinones/pharmacology , Receptor Cross-Talk , Receptors, Corticotropin-Releasing Hormone/agonists , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Receptors, Vasopressin/agonists , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/metabolism , Second Messenger Systems , Thiazines/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism , Vasopressins/pharmacologyABSTRACT
The structure-activity relationships of a novel series of biaryl dihydroorotate dehydrogenase (DHODH) inhibitors related to teriflunomide are disclosed. These biaryl derivatives were the result of structure-based design and proved to be potent DHODH inhibitors which in addition showed good antiproliferative activities on peripheral blood mononuclear cells and good efficacies in vivo in the rat adjuvant-induced-arthritis model.
Subject(s)
Biphenyl Compounds/chemistry , Crotonates/chemistry , Enzyme Inhibitors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Toluidines/chemistry , Animals , Arthritis, Experimental/drug therapy , Binding Sites , Biphenyl Compounds/chemical synthesis , Biphenyl Compounds/therapeutic use , Computer Simulation , Dihydroorotate Dehydrogenase , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Humans , Hydroxybutyrates , Nitriles , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Protein Structure, Tertiary , Rats , Structure-Activity RelationshipABSTRACT
A series of pyrido[3',2':4,5]furo[3,2-d]pyrimidines (PFP) were synthesized and tested for phosphodiesterase type 4 (PDE4) inhibitory activity, with the potential to treat asthma and chronic obstructive pulmonary disease (COPD). Structure-activity relationships within this series, leading to an increase of potency on the enzyme, is presented. Both gem-dimethylcyclohexyl moieties fused to the pyridine ring and the substitution at the 5 position of the PFP scaffold, proved to be key elements in order to get a high affinity in the enzyme.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Phosphodiesterase 4 Inhibitors/chemistry , Phosphodiesterase 4 Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Animals , Asthma/drug therapy , Asthma/enzymology , Caco-2 Cells , Cell Membrane Permeability , Ferrets , Humans , Models, Molecular , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/pharmacokinetics , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/enzymology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Structure-Activity RelationshipABSTRACT
A series of pyrido[3',2':4,5]thieno[3,2-d]pyrimidines (PTP) has been synthesized and tested as phosphodiesterase IV inhibitors (PDE4), a target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Structure-activity relationships within this series, leading to an increase of potency on the enzyme, are presented. The gem-dimethylcycloalkyl moiety fused to the pyridine ring proved to be a key element of the scaffold in order to get a higher affinity in the enzyme.
Subject(s)
Phosphodiesterase 4 Inhibitors , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Thiophenes/chemical synthesis , Humans , Pichia , Pyridines/chemistry , Pyrimidines/chemistry , Recombinant Proteins/antagonists & inhibitors , Structure-Activity Relationship , Thiophenes/chemistryABSTRACT
For the first time we have investigated the natural ecosystem engineering capacity of stromatolitic microbial assemblages. Stromatolites are laminated sedimentary structures formed by microbial activity and are considered to have dominated the shallows of the Precambrian oceans. Their fossilised remains are the most ancient unambiguous record of early life on earth. Stromatolites can therefore be considered as the first recognisable ecosystems on the planet. However, while many discussions have taken place over their structure and form, we have very little information on their functional ecology and how such assemblages persisted despite strong eternal forcing from wind and waves. The capture and binding of sediment is clearly a critical feature for the formation and persistence of stromatolite assemblages. Here, we investigated the ecosystem engineering capacity of stromatolitic microbial assemblages with respect to their ability to stabilise sediment using material from one of the few remaining living stromatolite systems (Highborne Cay, Bahamas). It was shown that the most effective assemblages could produce a rapid (12-24 h) and significant increase in sediment stability that continued in a linear fashion over the period of the experimentation (228 h). Importantly, it was also found that light was required for the assemblages to produce this stabilisation effect and that removal of assemblage into darkness could lead to a partial reversal of the stabilisation. This was attributed to the breakdown of extracellular polymeric substances under anaerobic conditions. These data were supported by microelectrode profiling of oxygen and calcium. The structure of the assemblages as they formed was visualised by low-temperature scanning electron microscopy and confocal laser microscopy. These results have implications for the understanding of early stromatolite development and highlight the potential importance of the evolution of photosynthesis in the mat forming process. The evolution of photosynthesis may have provided an important advance for the niche construction activity of microbial systems and the formation and persistence of the stromatolites which came to dominate shallow coastal environments for 80% of the biotic history of the earth.
Subject(s)
Geologic Sediments/microbiology , Photosynthesis , Water Microbiology , Bahamas , Biological Evolution , Ecosystem , Evolution, Planetary , Fossils , Geologic Sediments/chemistry , Geology , Light , Microscopy, Confocal , Oxygen , PaleontologyABSTRACT
In this study, we characterized the bovine vasopressin V(1a), V(1b), V(2) receptor isoforms and compared their pharmacological properties to those of corresponding rat and human vasopressin receptor subtypes. Specific binding sites of high affinity for vasopressin were found in all bovine tissues tested (kidney, liver and pituitary). Using a large series of recent peptidic and non-peptidic selective vasopressin agonists or antagonists, we demonstrated the presence of vasopressin V(2), V(1a) or V(1b) receptors in the kidney, liver and pituitary bovine tissues, respectively. This extensive characterization of bovine vasopressin receptor isoforms validates the pharmacological vasopressin receptor classification earlier established for the rat and human species. As expected, the bovine vasopressin receptors look much more like human receptors than rat ones. Interestingly, among the three vasopressin receptor isoforms studied, the vasopressin V(1b) receptor subtype is the best conserved for the three species studied.
Subject(s)
Receptors, Vasopressin/agonists , Receptors, Vasopressin/metabolism , Animals , Cattle , Dose-Response Relationship, Drug , Humans , Protein Binding/drug effects , Protein Binding/physiology , Protein Isoforms/agonists , Protein Isoforms/metabolism , Rats , Species Specificity , Vasopressins/pharmacologyABSTRACT
1. The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V(1a) receptor subtype (K(i)=11 nM) and was selective for this receptor subtype. 2. Functional studies performed on CHO cells expressing human V(1a) receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K(act)) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V(2) and V(1b) receptor subtypes and an antagonist for the human OT receptor. 3. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V(1a) receptors, but weak affinity and non selective properties for rat V(1a) receptors. 4. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5. In conclusion, we demonstrate that F-180 is the first selective V(1a) agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V(1a) receptor subtypes.