ABSTRACT
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel are established as the primary causative factor in the devastating lung disease cystic fibrosis (CF). More recently, cigarette smoke exposure has been shown to be associated with dysfunctional airway epithelial ion transport, suggesting a role for CFTR in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, the identification and characterization of a high throughput screening hit 6 as a potentiator of mutant human F508del and wild-type CFTR channels is reported. The design, synthesis, and biological evaluation of compounds 7-33 to establish structure-activity relationships of the scaffold are described, leading to the identification of clinical development compound icenticaftor (QBW251) 33, which has subsequently progressed to deliver two positive clinical proofs of concept in patients with CF and COPD and is now being further developed as a novel therapeutic approach for COPD patients.
Subject(s)
Aminopyridines/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Administration, Oral , Aminopyridines/metabolism , Aminopyridines/therapeutic use , Animals , Cystic Fibrosis/drug therapy , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Disease Models, Animal , Drug Evaluation, Preclinical , Gene Deletion , Half-Life , Humans , Protein Binding , Pulmonary Disease, Chronic Obstructive/drug therapy , Rats , Rats, Sprague-Dawley , Solubility , Structure-Activity RelationshipABSTRACT
Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating the splicing of the related gene SMN2 to enhance the exon-7 inclusion and increase levels of the SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for the compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole, which provided additional opportunities for a conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than 50% increase in production of full-length SMN protein in a mouse model of SMA.
Subject(s)
Drug Design , RNA Splicing , Thiadiazoles/chemistry , Animals , Half-Life , Halogens/chemistry , Humans , Male , Mice , Molecular Conformation , Motor Neurons/metabolism , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/pathology , Oxygen/chemistry , Pyridazines/chemistry , RNA Splicing/drug effects , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Sulfur/chemistry , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 1 Protein/metabolism , Survival of Motor Neuron 2 Protein/genetics , Survival of Motor Neuron 2 Protein/metabolism , Thiadiazoles/metabolism , Thiadiazoles/pharmacologyABSTRACT
Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.
Subject(s)
Brain/drug effects , ERG1 Potassium Channel/metabolism , Muscular Atrophy, Spinal/drug therapy , Pyridazines/chemistry , Administration, Oral , Animals , Brain/metabolism , Cell Line , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , ERG1 Potassium Channel/antagonists & inhibitors , Humans , Mice, Inbred C57BL , Motor Neurons/drug effects , Muscular Atrophy, Spinal/genetics , Pyridazines/pharmacology , Quantitative Structure-Activity Relationship , RNA Splicing , Rats, Sprague-Dawley , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 1 Protein/metabolism , Survival of Motor Neuron 2 Protein/geneticsABSTRACT
Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.
Subject(s)
Alternative Splicing , Muscular Atrophy, Spinal/drug therapy , RNA, Double-Stranded/agonists , Ribonucleoprotein, U1 Small Nuclear/agonists , Small Molecule Libraries/pharmacology , Survival of Motor Neuron 2 Protein/metabolism , Animals , Binding Sites , Disease Models, Animal , Female , Gene Expression , Humans , Mice , Mice, Transgenic , Models, Molecular , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/mortality , Muscular Atrophy, Spinal/pathology , Protein Binding/drug effects , Protein Stability/drug effects , Proteolysis , RNA Precursors/agonists , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , Ribonucleoprotein, U1 Small Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/metabolism , Survival Analysis , Survival of Motor Neuron 2 Protein/chemistry , Survival of Motor Neuron 2 Protein/geneticsABSTRACT
Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.
Subject(s)
Interleukin-12/antagonists & inhibitors , Interleukin-23/antagonists & inhibitors , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Signal Transduction/drug effects , Toll-Like Receptors/metabolism , Triazines/pharmacology , Animals , Cell Line , Cytokines/metabolism , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Hydrazones , Mice , Morpholines/metabolism , Protein Binding , Pyrimidines , Substrate Specificity , Triazines/metabolismABSTRACT
Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.
Subject(s)
Acetamides/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrimidinones/pharmacology , Tankyrases/antagonists & inhibitors , Acetamides/administration & dosage , Acetamides/chemistry , Administration, Oral , Animals , Area Under Curve , Biological Availability , Enzyme Inhibitors/administration & dosage , Mice , Models, Molecular , Pyrimidinones/administration & dosage , Pyrimidinones/chemistry , Structure-Activity RelationshipABSTRACT
Identification of novel, validated targets remains a top priority in modern drug discovery. Chemical genetics represents a powerful approach to the discovery of new targets. Unlike the traditional target-based screen that relies on a predefined, sometimes poorly validated target, a chemical genetics-based phenotypic screen probes the entire molecular signaling pathway in an efficient and unbiased manner for the most drug-sensitive node. The most significant obstacle associated with this approach is identification of the efficacy targets of small-molecule probes. The huge potential of chemical genetics cannot be realized without the establishment of reliable mechanisms for target identification. In this article, we describe each essential element of the chemical genetics process, discuss common challenges that the field is facing, and critically review various biochemical and genetics approaches recently developed for target deconvolution. We also attempt to summarize lessons that we have collectively learned and provide a practical perspective to facilitate the advancement of chemical genetics.
Subject(s)
Drug Delivery Systems , Drug Discovery/methods , Gene Expression Profiling/methods , Pharmacogenetics/methods , Animals , Computational Biology/methods , Drug Design , Humans , Phenotype , Proteomics/methods , Signal Transduction , Transcription, GeneticABSTRACT
The application of chemical proteomics to new target discovery can lead to a rapid understanding of disease mechanism and new therapeutic methods. Successful application includes a thorough understanding of SAR and the validation of target relevance using multiple genetic and biochemical methods. This feature review highlights several successful applications of chemical proteomics and outlines the strategy and approaches that lead to the discovery of novel therapeutic targets.
Subject(s)
Drug Discovery , Molecular Targeted Therapy , Pharmacology/methods , Proteomics/methods , Animals , Drug Evaluation, Preclinical/methods , Drug Industry/methods , Humans , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
A new approach to (+)-cacospongionolide was developed to access conformationally restricted variants of the natural product. The flexible aliphatic region between the decalin and side chain portion of the natural product was replaced with alkenyl and alkynyl linkers to probe the influence of structural rigidity in the inhibition of secretary phospholipase A2 (sPLA2). It was found that when the aliphatic section is replaced with a Z-olefin or an alkyne, sPLA2 inhibitory activity suffered relative to the natural product; however, an E-olefin-containing analogue led to an enhanced activity. These results suggest that preferred sPLA2 binding conformation of the natural product is similar to the geometry of the E-olefin-containing analogue.
Subject(s)
4-Butyrolactone/analogs & derivatives , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Phospholipases A/antagonists & inhibitors , Porifera/chemistry , Pyrans/chemistry , Pyrans/chemical synthesis , 4-Butyrolactone/chemical synthesis , 4-Butyrolactone/chemistry , 4-Butyrolactone/pharmacology , Animals , Bee Venoms/enzymology , Enzyme Inhibitors/pharmacology , Furaldehyde/chemistry , Humans , Indicators and Reagents , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Phospholipases A2 , Pyrans/pharmacology , Recombinant Proteins/chemistryABSTRACT
The total syntheses of the antiinflammatory marine sponge metabolites (+)-cacospongionolide B and E are described. The pivotal steps in the synthetic route include a three-step sequence that couples the two main regions of the natural product, as well as generates the side chain dihydropyran ring. The activity of the synthetic analogues against bee venom phospholipase A2 suggests that the cacospongionolides have enantiospecific interactions with the enzyme that may be independent of the gamma-hydroxybutenolide moiety.
Subject(s)
4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Phospholipases A/antagonists & inhibitors , Pyrans/chemical synthesis , 4-Butyrolactone/pharmacology , Animals , Enzyme Inhibitors/pharmacology , Molecular Structure , Phospholipases A2 , Porifera/chemistry , Pyrans/pharmacology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The first total synthesis of the antiinflammatory marine sponge metabolite (+)-cacospongionolide B has been accomplished in 12 linear steps. The pivotal transformations include a three-step sequence coupling the two main regions of the natural product as well as generating the side chain dihydropyran ring. The activity of the synthetic analogues against bee venom phospholipase A(2) suggests that cacospongionolide B has an enantiospecific interaction with the enzyme that is independent of the gamma-hydroxybutenolide moiety.