ABSTRACT
Indoleamine 2,3-dioxygenase-1 (IDO1) is a potential target for the next generation of cancer immunotherapies. We describe the development of two series of IDO1 inhibitors incorporating a N-hydroxy-thiophene-carboximidamide core generated by knowledge-based drug design. Structural modifications to improve the cellular activity and pharmacokinetic (PK) properties of the compounds synthesized, including extension of the side chain of the N-hydroxythiophene-2-carboximidamide core, resulted in compound 27a, a potent IDO1 inhibitor which demonstrated significant (51%) in vivo target inhibition on IDO1 in a human SK-OV-3 ovarian xenograft tumor mouse model. This strategy is expected to be applicable to the discovery of additional IDO1 inhibitors for the treatment of other diseases susceptible to modulation of IDO1.
Subject(s)
Amides/chemistry , Drug Design , Enzyme Inhibitors/chemistry , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Amides/metabolism , Animals , Binding Sites , Cell Line, Tumor , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Half-Life , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Mice , Mice, Inbred ICR , Molecular Docking Simulation , Neoplasms/drug therapy , Structure-Activity Relationship , Thiophenes/chemistry , Transplantation, HeterologousABSTRACT
Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.
Subject(s)
Aminohydrolases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Methylenetetrahydrofolate Dehydrogenase (NADP)/antagonists & inhibitors , Multifunctional Enzymes/antagonists & inhibitors , Xanthine/pharmacology , Allosteric Site/drug effects , Aminohydrolases/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Methylenetetrahydrofolate Dehydrogenase (NADP)/metabolism , Models, Molecular , Molecular Structure , Multifunctional Enzymes/metabolism , Structure-Activity Relationship , Xanthine/chemical synthesis , Xanthine/chemistryABSTRACT
Indoleamine 2,3-dioxygenase (IDO1) inhibitors are speculated to be useful in cancer immunotherapy, but a phase III clinical trial of the most advanced IDO1 inhibitor, epacadostat, did not meet its primary end point and was abandoned. In previous work, we identified the novel IDO1 inhibitor N-(4-chlorophenyl)-2-((5-phenylthiazolo[2,3-c][1,2,4]triazol-3-yl)thio)acetamide 1 through high-throughput screening (HTS). Herein, we report a structure-activity relationship (SAR) study of this compound, which resulted in the potent IDO1 inhibitor 1-(4-cyanophenyl)-3-(3-(cyclopropylethynyl)imidazo[2,1-b]thiazol-5-yl)thiourea 47 (hIDO IC50 = 16.4 nM). X-ray cocrystal structural analysis revealed that the basis for this high potency is a unique sulfur-aromatic interaction network formed by the thiourea moiety of 47 with F163 and F226. This finding is expected to inspire new approaches toward the discovery of potent IDO1 inhibitors in the future.
Subject(s)
Enzyme Inhibitors/chemistry , Imidazoles/chemistry , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Thiazoles/chemistry , Binding Sites , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Imidazoles/chemical synthesis , Imidazoles/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/chemistry , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Molecular Structure , Protein Binding , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/metabolismABSTRACT
Gastrointestinal stromal tumors (GISTs) are prototypes of stem cell factor receptor (c-KIT)-driven cancer. Two receptor tyrosine kinases, c-KIT and fms-tyrosine kinase (FLT3), are frequently mutated in acute myeloid leukemia (AML) patients, and these mutations are associated with poor prognosis. In this study, we discovered a multitargeted tyrosine kinase inhibitor, compound 15a, with potent inhibition against single or double mutations of c-KIT developed in GISTs. Moreover, crystal structure analysis revealed the unique binding mode of 15a with c-KIT and may elucidate its high potency in inhibiting c-KIT kinase activity. Compound 15a inhibited cell proliferation and induced apoptosis by targeting c-KIT in c-KIT-mutant GIST cell lines. The antitumor effects of 15a were also demonstrated in GIST430 and GIST patient-derived xenograft models. Further studies demonstrated that 15a inhibited the proliferation of c-KIT- and FLT3-driven AML cells in vitro and in vivo. The results of this study suggest that 15a may be a potential anticancer drug for the treatment of GISTs and AML.
Subject(s)
Antineoplastic Agents/pharmacology , Gastrointestinal Stromal Tumors/drug therapy , Leukemia, Myeloid, Acute/drug therapy , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Apoptosis , Cell Proliferation , Female , Gastrointestinal Neoplasms/drug therapy , Gastrointestinal Neoplasms/enzymology , Gastrointestinal Neoplasms/genetics , Gastrointestinal Neoplasms/pathology , Gastrointestinal Stromal Tumors/enzymology , Gastrointestinal Stromal Tumors/genetics , Gastrointestinal Stromal Tumors/pathology , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred ICR , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Phosphorylation , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-kit/genetics , Pyrimidines/chemistry , Rats, Sprague-Dawley , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Drug resistance due to acquired mutations that constitutively activate c-KIT is a significant challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identified 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the first structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state through a sequence of conformational changes. Moreover, 10a can effectively inhibit various c-KIT mutants and the proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its excellent efficacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Evaluation, Preclinical , Gastrointestinal Stromal Tumors/drug therapy , Gastrointestinal Stromal Tumors/pathology , Humans , Imatinib Mesylate/chemistry , Imatinib Mesylate/metabolism , Mice , Mice, Inbred ICR , Molecular Docking Simulation , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , Pyrimidines/chemistry , Structure-Activity Relationship , Urea/analogs & derivatives , Urea/metabolism , Urea/pharmacology , Urea/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Indoleamine 2,3-dioxygenase 1 (IDO1), promoting immune escape of tumors, is a therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have been identified, but only limited structural biology studies of IDO1 inhibitors are available to provide insights on the binding mechanism of IDO1. In this study, we present the structure of IDO1 in complex with 24, a NLG919 analogue with potent activity. The complex structure revealed the imidazole nitrogen atom of 24 to coordinate with the heme iron, and the imidazoleisoindole core situated in pocket A with the 1-cyclohexylethanol moiety extended to pocket B to interact with the surrounding residues. Most interestingly, 24 formed an extensive hydrogen bond network with IDO1, which is a distinct feature of IDO1/24 complex structure and is not observed in the other IDO1 complex structures. Further structure-activity relationship, UV spectra, and structural biology studies of several analogues of 24 demonstrated that extensive hydrophobic interactions and the unique hydrogen bonding network contribute to the great potency of imidazoleisoindole derivatives. These results are expected to facilitate the structure-based drug design of new IDO inhibitors.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Humans , Hydrogen Bonding , Models, Molecular , Structure-Activity RelationshipABSTRACT
The overexpression of Aurora kinases in multiple tumors makes these kinases appealing targets for the development of anticancer therapies. This study identified two small molecules with a furanopyrimidine core, IBPR001 and IBPR002, that target Aurora kinases and induce a DFG conformation change at the ATP site of Aurora A. Our results demonstrate the high potency of the IBPR compounds in reducing tumorigenesis in a colorectal cancer xenograft model in athymic nude mice. Human hepatoma up-regulated protein (HURP) is a substrate of Aurora kinase A, which plays a crucial role in the stabilization of kinetochore fibers. This study used the IBPR compounds as well as MLN8237, a proven Aurora A inhibitor, as chemical probes to investigate the molecular role of HURP in mitotic spindle formation. These compounds effectively eliminated HURP phosphorylation, thereby revealing the coexistence and continuous cycling of HURP between unphosphorylated and phosphorylated forms that are associated, respectively, with microtubules emanating from centrosomes and kinetochores. Furthermore, these compounds demonstrate a spatial hierarchical preference for HURP in the attachment of microtubules extending from the mother to the daughter centrosome. The finding of inequality in the centrosomal microtubules revealed by these small molecules provides a versatile tool for the discovery of new cell-division molecules for the development of antitumor drugs.
