ABSTRACT
The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.
Subject(s)
Azabicyclo Compounds/pharmacology , Molecular Probes/pharmacology , Nuclear Proteins/antagonists & inhibitors , Pyridines/pharmacology , Transcription Factors/antagonists & inhibitors , Azabicyclo Compounds/chemical synthesis , Azabicyclo Compounds/chemistry , Crystallography, X-Ray , DNA-Binding Proteins , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Probes/chemical synthesis , Molecular Probes/chemistry , Molecular Structure , Nuclear Proteins/metabolism , Protein Processing, Post-Translational/drug effects , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship , Substrate Specificity , Transcription Factors/metabolismABSTRACT
Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/chemical synthesis , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Drug Design , Polypharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Blotting, Western , Calorimetry , Cell Line, Tumor , Crystallization , Drug Interactions , Drug Screening Assays, Antitumor , Epigenesis, Genetic , High-Throughput Screening Assays , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Proto-Oncogene Proteins c-myc/metabolism , Pteridines/pharmacology , Pyrrolidines/pharmacology , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Structure-Activity Relationship , Sulfonamides/pharmacologyABSTRACT
We tested the interaction of 72 kinase inhibitors with 442 kinases covering >80% of the human catalytic protein kinome. Our data show that, as a class, type II inhibitors are more selective than type I inhibitors, but that there are important exceptions to this trend. The data further illustrate that selective inhibitors have been developed against the majority of kinases targeted by the compounds tested. Analysis of the interaction patterns reveals a class of 'group-selective' inhibitors broadly active against a single subfamily of kinases, but selective outside that subfamily. The data set suggests compounds to use as tools to study kinases for which no dedicated inhibitors exist. It also provides a foundation for further exploring kinase inhibitor biology and toxicity, as well as for studying the structural basis of the observed interaction patterns. Our findings will help to realize the direct enabling potential of genomics for drug development and basic research about cellular signaling.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Catalysis , Drug Design , Enzyme Stability , High-Throughput Screening Assays , Humans , Protein Binding , Protein Kinase Inhibitors/classification , Protein Kinases/classification , Proteomics , Signal Transduction , Substrate SpecificityABSTRACT
Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor.
Subject(s)
Benzothiazoles/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Benzenesulfonates/pharmacology , Benzothiazoles/pharmacokinetics , Bone Marrow/drug effects , Bone Marrow/pathology , Carbazoles/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Furans , Humans , Mice , Mice, Nude , Mice, SCID , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacokinetics , Phosphorylation/drug effects , Piperazines/pharmacology , Prognosis , Protein Interaction Mapping , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyridines/pharmacology , Quinazolines/pharmacology , Sorafenib , Staurosporine/analogs & derivatives , Staurosporine/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.
