ABSTRACT
The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.
Subject(s)
Drug Design , Drug Inverse Agonism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Pyridines/administration & dosage , Pyridines/pharmacology , Administration, Oral , Animals , Biological Availability , Drug Evaluation, Preclinical , Humans , Mice , Pyridines/pharmacokinetics , Th17 Cells/drug effects , Th17 Cells/metabolismABSTRACT
C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.
Subject(s)
Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/enzymology , Quinazolines/pharmacology , Animals , Cell Line , Disease Models, Animal , Enzyme Inhibitors/chemistry , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , Muscular Atrophy, Spinal/genetics , Promoter Regions, Genetic , Quinazolines/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival of Motor Neuron 2 Protein/deficiency , Survival of Motor Neuron 2 Protein/genetics , Survival of Motor Neuron 2 Protein/metabolismABSTRACT
The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.
Subject(s)
Drug Design , Endoribonucleases/antagonists & inhibitors , Muscular Atrophy, Spinal/drug therapy , Quinazolines/chemistry , Quinazolines/therapeutic use , RNA, Messenger/antagonists & inhibitors , Animals , Disease Models, Animal , Endoribonucleases/genetics , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , HEK293 Cells , Humans , Mice , Molecular Docking Simulation , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Quinazolines/pharmacokinetics , Quinazolines/pharmacology , RNA, Messenger/genetics , Survival of Motor Neuron 2 ProteinABSTRACT
Despite its diverse applications, such as identification of the protein binding partners of small molecules and investigation of intracellular drug-target engagement, photoaffinity labelling (PAL) is intrinsically challenging, primarily due to the difficulty in discovering functionally active photoaffinity probes. Here we describe the creation of a chemoproteomic library to discover a novel photoaffinity probe for DcpS, an mRNA decapping enzyme that is a putative target for Spinal Muscular Atrophy. This library approach expedites the discovery of photoaffinity probes and expands the chemical biology toolbox to include RNA cap-binding proteins.
Subject(s)
Endoribonucleases/metabolism , Molecular Probes/chemistry , Photoaffinity Labels/chemistry , Binding Sites , Endoribonucleases/chemistry , Gene Library , Humans , Models, Molecular , Molecular Probes/metabolism , Quinazolines/chemistryABSTRACT
This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.
Subject(s)
Endoribonucleases/metabolism , Enzyme Inhibitors/pharmacology , Molecular Probes/chemistry , Sulfinic Acids/chemistry , Tyrosine/metabolism , Catalytic Domain , Cells, Cultured , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Probes/chemical synthesis , Molecular Targeted Therapy/methods , Structure-Activity Relationship , Tyrosine/chemistryABSTRACT
The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Gram-Negative Bacteria/drug effects , Hydroxamic Acids/chemistry , Amidohydrolases/metabolism , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/chemistry , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Structure-Activity Relationship , Sulfones/chemistryABSTRACT
The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Hydroxamic Acids/chemical synthesis , Pyridones/chemical synthesis , Sulfonic Acids/chemical synthesis , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Crystallography, X-Ray , Humans , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/pharmacology , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Protein Conformation , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Pyridones/pharmacokinetics , Pyridones/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Sulfonic Acids/pharmacokinetics , Sulfonic Acids/pharmacologyABSTRACT
In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.
