ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.
Subject(s)
Antineoplastic Agents/pharmacology , Intracellular Signaling Peptides and Proteins/chemistry , RNA-Binding Proteins/chemistry , Sulfonamides/pharmacology , Amino Acid Motifs , Calorimetry , Cloning, Molecular , Fluorometry , HCT116 Cells , HEK293 Cells , Humans , Image Processing, Computer-Assisted , Intracellular Signaling Peptides and Proteins/genetics , Kinetics , Nuclear Proteins/metabolism , Peptides/chemistry , Point Mutation , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Proteome , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Fusion Proteins, bcr-abl/chemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Animals , Antineoplastic Agents/metabolism , Antineoplastic Combined Chemotherapy Protocols , Benzamides , Binding Sites , Bone Marrow Transplantation , Cell Line, Tumor , Crystallization , Disease Models, Animal , Female , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Inhibitory Concentration 50 , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Male , Mass Spectrometry , Mice , Models, Molecular , Mutation/genetics , Piperazines/chemistry , Piperazines/pharmacology , Protein Structure, Tertiary , Pyrimidines/chemistry , Pyrimidines/metabolism , Pyrimidines/pharmacology , Transplantation, HeterologousABSTRACT
Kinase inhibitors that bind to the ATP cleft can be broadly classified into two groups: those that bind exclusively to the ATP site with the kinase assuming a conformation otherwise conducive to phosphotransfer (type I), and those that exploit a hydrophobic site immediately adjacent to the ATP pocket made accessible by a conformational rearrangement of the activation loop (type II). To date, all type II inhibitors were discovered by using structure-activity-guided optimization strategies. Here, we describe a general pharmacophore model of type II inhibition that enables a rational "hybrid-design" approach whereby a 3-trifluoromethylbenzamide functionality is appended to four distinct type I scaffolds in order to convert them into their corresponding type II counterparts. We demonstrate that the designed compounds function as type II inhibitors by using biochemical and cellular kinase assays and by cocrystallography with Abl.
Subject(s)
Enzyme Inhibitors/chemistry , Molecular Conformation , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Crystallography , Enzyme Inhibitors/metabolism , Hydrogen Bonding , Models, Molecular , Phosphorylation , Proto-Oncogene Proteins c-abl/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.
ABSTRACT
Inhibition of Bcr-Abl kinase activity by imatinib for the treatment of chronic myeloid leukemia (CML) currently serves as the paradigm for targeting dominant oncogenes with small molecules. We recently reported the discovery of GNF-2 (1) and GNF-5 (2) as selective non-ATP competitive inhibitors of cellular Bcr-Abl kinase activity that target the myristate binding site. Here, we used cell-based structure-activity relationships to guide the optimization and diversification of ligands that are capable of binding to the myristate binding site and rationalize the findings based upon an Abl-compound 1 cocrystal. We elucidate the structure-activity relationships required to obtain potent antiproliferative activity against Bcr-Abl transformed cells and report the discovery of new compounds (5g, 5h, 6a, 14d, and 21j-I) that display improved potency or pharmacological properties. This work demonstrates that a variety of structures can effectively target the Bcr-Abl myristate binding site and provides new leads for developing drugs that can target this binding site.