ABSTRACT
Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.
Subject(s)
Estrogen Receptor alpha , Proteolysis , Von Hippel-Lindau Tumor Suppressor Protein , Humans , Estrogen Receptor alpha/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Female , Proteolysis/drug effects , Animals , Administration, Oral , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Mice , Antineoplastic Agents/pharmacology , Antineoplastic Agents/administration & dosageABSTRACT
Small molecules that induce protein degradation hold the potential to overcome several limitations of the currently available inhibitors. Monovalent or molecular glue degraders, in particular, enable the benefits of protein degradation without the disadvantages of high molecular weight and the resulting challenge in drug development that are associated with bivalent molecules like Proteolysis Targeting Chimeras. One key challenge in designing monovalent degraders is how to build in the degrader activityâhow can we convert an inhibitor into a degrader? If degradation activity requires very specific molecular features, it will be difficult to find new degraders and challenging to optimize those degraders toward drugs. Herein, we demonstrate that an unexpectedly wide range of modifications to the degradation-inducing group of the cyclin K degrader CR8 are tolerated, including both aromatic and nonaromatic groups. We used these findings to convert the pan-CDK inhibitors dinaciclib and AT-7519 to Cyclin K degraders, leading to a novel dinaciclib-based compound with improved degradation activity compared to CR8 and confirm the mechanism of degradation. These results suggest that general design principles can be generated for the development and optimization of monovalent degraders.
Subject(s)
Cyclins , Proteolysis , Cell Cycle Checkpoints , Cyclins/metabolismABSTRACT
BACKGROUND: SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn's disease (CD), suggesting a role in inflammation. RESULTS: We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. CONCLUSIONS: This study identifies SP140 as a druggable epigenetic therapeutic target for CD.
Subject(s)
Crohn Disease , Tumor Necrosis Factor Inhibitors , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , Crohn Disease/genetics , Crohn Disease/metabolism , Cytokines/genetics , Cytokines/metabolism , Epigenesis, Genetic , Humans , Macrophages , Transcription Factors/geneticsABSTRACT
Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.
Subject(s)
Aminoquinolines/chemistry , Drug Design , Proteins/metabolism , Administration, Oral , Aminoquinolines/metabolism , Aminoquinolines/pharmacokinetics , Aminoquinolines/therapeutic use , Animals , Benzoates/chemistry , Benzoates/metabolism , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Dogs , Half-Life , Humans , Male , Mice , Molecular Conformation , Molecular Dynamics Simulation , Neoplasms/drug therapy , Proteins/antagonists & inhibitors , Rats , Structure-Activity RelationshipABSTRACT
A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3) and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217).
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Protein Domains/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcription Factors/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Drug Design , Drug Discovery , Humans , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.
Subject(s)
Anti-Inflammatory Agents/chemistry , Ligands , Transcription Factors/antagonists & inhibitors , Administration, Oral , Amides/chemistry , Amides/metabolism , Amides/pharmacokinetics , Animals , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacokinetics , Binding Sites , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , Dogs , Half-Life , Humans , Hydrogen Bonding , Male , Molecular Dynamics Simulation , Protein Domains , Rats , Rats, Wistar , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
A palladium-catalysed Buchwald-Hartwig amination for lenalidomide-derived aryl bromides was optimised using high throughput experimentation (HTE). The substrate scope of the optimised conditions was evaluated for a range of alkyl- and aryl- amines and functionalised aryl bromides. The methodology allows access to new cereblon-based bifunctional proteolysis targeting chimeras with a reduced step count and improved yields.
Subject(s)
Amines/chemistry , Bromides/chemistry , Lenalidomide/chemistry , Proteolysis/drug effects , Amination , Ligands , Ubiquitin-Protein Ligases/metabolismABSTRACT
p300/CREB binding protein associated factor (PCAF/KAT2B) and general control nonderepressible 5 (GCN5/KAT2A) are multidomain proteins that have been implicated in retroviral infection, inflammation pathways, and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain. Herein, we report GSK4027 as a chemical probe for the PCAF/GCN5 bromodomain, together with GSK4028 as an enantiomeric negative control. The probe was optimized from a weakly potent, nonselective pyridazinone hit to deliver high potency for the PCAF/GCN5 bromodomain, high solubility, cellular target engagement, and ≥18000-fold selectivity over the BET family, together with ≥70-fold selectivity over the wider bromodomain families.
Subject(s)
Histone Acetyltransferases/chemistry , Molecular Probes/chemistry , Piperidines/chemistry , Pyridazines/chemistry , p300-CBP Transcription Factors/chemistry , Animals , Cell Membrane Permeability , Humans , Membranes, Artificial , Mice , Molecular Probes/chemical synthesis , Piperidines/chemical synthesis , Protein Domains , Pyridazines/chemical synthesis , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 µM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).
Subject(s)
Enzyme Inhibitors/chemistry , Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Pyridines/chemistry , Amination , Cell Line , Cell Membrane Permeability , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Histone Demethylases/chemistry , Histone Demethylases/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Models, Molecular , Pyridines/pharmacokinetics , Pyridines/pharmacologyABSTRACT
Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Cell Line , Cell Membrane Permeability , Crystallography, X-Ray , Enzyme Inhibitors/pharmacokinetics , Histone Demethylases/chemistry , Histone Demethylases/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Models, Molecular , Molecular Docking Simulation , Pyrimidinones/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.
Subject(s)
Aniline Compounds/chemistry , Aniline Compounds/therapeutic use , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Arthus Reaction/drug therapy , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Administration, Oral , Aniline Compounds/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Crystallography, X-Ray , Drug Discovery , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship , Syk KinaseABSTRACT
Herein we describe the medicinal chemistry programme to identify a potential back-up compound to the EP(1) receptor antagonist GW848687X. This work started with the lipophilic 1,2-biaryl benzene derivative 4 which displayed molecular weight of 414.9g/mol and poor in vivo metabolic stability in the rat and resulted in the identification of compound 7i (GSK345931A) which demonstrated good metabolic stability in the rat and lower molecular weight (381.9g/mol). In addition, 7i (GSK345931A) showed measurable CNS penetration in the mouse and rat and potent analgesic efficacy in acute and sub-chronic models of inflammatory pain.
Subject(s)
Inflammation/drug therapy , Pain/drug therapy , Pyridines/pharmacology , Receptors, Prostaglandin E/antagonists & inhibitors , Animals , Drug Evaluation, Preclinical , Pyridines/chemistry , Pyridines/therapeutic use , Rats , Receptors, Prostaglandin E, EP1 Subtype , Structure-Activity RelationshipABSTRACT
We describe the discovery of a series of pyrazole amide EP(1) receptor antagonists with good aqueous solubility and CNS penetration. In order to achieve solubility we investigated the incorporation of a basic group in the region of the molecule previously occupied by a carboxylic acid, which was known to be a key element of the pharmacophore. This study led to the identification of compounds such as 4h, 4j and 10b which demonstrated brain-to-blood ratios of 0.8:1-2.0:1 in addition to good solubility and metabolic stability.
Subject(s)
Brain/drug effects , Central Nervous System/drug effects , Pyrazoles/chemistry , Receptors, Prostaglandin E/antagonists & inhibitors , Amides/chemistry , Carboxylic Acids/chemistry , Chemistry, Pharmaceutical/methods , Drug Design , Humans , Inhibitory Concentration 50 , Isoquinolines/chemistry , Models, Chemical , Molecular Structure , Receptors, Prostaglandin E, EP1 Subtype , Solubility , Structure-Activity RelationshipABSTRACT
Replacement of the carboxylic acid group in a series of previously described methylene-linked pyrazole EP(1) receptor antagonists led to the discovery of amide, reversed amide and carbamate derivatives. Two compounds, 10a and 10b, were identified as brain penetrant compounds and both demonstrated efficacy in the CFA model of inflammatory pain.
