ABSTRACT
The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
Subject(s)
Neoplasms/drug therapy , Nucleosides/pharmacology , Small Molecule Libraries/pharmacology , Sulfonamides/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Animals , Cell Line, Tumor , DNA Damage/drug effects , DNA Repair/drug effects , Humans , Imides/pharmacology , Mice , Neoplasms/genetics , Neoplasms/pathology , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/genetics , Protein Binding , Pyrazoles , Pyrimidines , Sulfides , Ubiquitin/antagonists & inhibitors , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/genetics , Xenograft Model Antitumor AssaysABSTRACT
Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.
Subject(s)
Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Sumoylation , Cell Proliferation/drug effects , Chromosome Segregation/drug effects , DNA Damage/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, myc , Humans , Mitosis/drug effects , Neoplasms/genetics , Neoplasms/pathology , Protein Processing, Post-Translational , Tumor Cells, CulturedABSTRACT
SUMOylation is a post-translational ubiquitin-like protein modification pathway that regulates important cellular processes including chromosome structure, kinetochore function, chromosome segregation, nuclear and sub-nuclear organization, transcription and DNA damage repair. There is increasing evidence that the SUMO pathway is dysregulated in cancer, raising the possibility that modulation of this pathway may have therapeutic potential. To investigate the importance of the SUMO pathway in the context of cancer cell proliferation and tumor growth, we applied lentivirus-based short hairpin RNAs (shRNA) to knockdown SUMO pathway genes in human cancer cells. shRNAs for SAE2 and UBC9 reduced SUMO conjugation activity and inhibited proliferation of human cancer cells. To expand upon these observations, we generated doxycycline inducible conditional shRNA cell lines for SAE2 to achieve acute and reversible SAE2 knockdown. Conditional SAE2 knockdown in U2OS and HCT116 cells slowed cell growth in vitro, and SAE2 knockdown induced multiple terminal outcomes including apoptosis, endoreduplication and senescence. Multinucleated cells became senescent and stained positive for the senescence marker, SA-ß Gal, and displayed elevated levels of p53 and p21. In an attempt to explain these phenotypes, we confirmed that loss of SUMO pathway activity leads to a loss of SUMOylated Topoisomerase IIα and the appearance of chromatin bridges which can impair proper cytokinesis and lead to multinucleation. Furthermore, knockdown of SAE2 induces disruption of PML nuclear bodies which may further promote apoptosis or senescence. In an in vivo HCT116 xenograft tumor model, conditional SAE2 knockdown strongly impaired tumor growth. These data demonstrate that the SUMO pathway is required for cancer cell proliferation in vitro and tumor growth in vivo, implicating the SUMO pathway as a potential cancer therapeutic target.
Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , Signal Transduction , Small Ubiquitin-Related Modifier Proteins/metabolism , Animals , Apoptosis/genetics , Cell Cycle/genetics , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation , Cellular Senescence/genetics , Disease Models, Animal , Gene Knockdown Techniques , Heterografts , Humans , Neoplasms/genetics , Phenotype , RNA Interference , Small Ubiquitin-Related Modifier Proteins/genetics , Sumoylation , Tumor Burden/geneticsABSTRACT
Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting ß-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Tankyrases/antagonists & inhibitors , Administration, Oral , Biological Availability , Dose-Response Relationship, Drug , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Tankyrases/metabolismABSTRACT
Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates ß-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of ß-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).
Subject(s)
Benzimidazoles/chemical synthesis , Oxazolidinones/chemical synthesis , Tankyrases/antagonists & inhibitors , Administration, Oral , Animals , Benzimidazoles/pharmacokinetics , Benzimidazoles/pharmacology , Binding Sites , Biological Availability , In Vitro Techniques , Mice , Microsomes, Liver/metabolism , Models, Molecular , Oxazolidinones/pharmacokinetics , Oxazolidinones/pharmacology , Rats , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benzimidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1 isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity, and acceptable pharmacokinetic properties.
Subject(s)
Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Casein Kinase I/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Benzimidazoles/pharmacokinetics , Binding Sites , Casein Kinase I/chemistry , Casein Kinase I/metabolism , Humans , Mice , Models, Molecular , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Rats , Structure-Activity Relationship , beta Catenin/metabolismABSTRACT
Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.
ABSTRACT
The insulin-like growth factor-1 receptor (IGF-1R) plays an important role in the regulation of cell growth and differentiation, and in protection from apoptosis. IGF-1R has been shown to be an appealing target for the treatment of human cancer. Herein, we report the synthesis, structure-activity relationships (SAR), X-ray cocrystal structure and in vivo tumor study results for a series of 2,4-bis-arylamino-1,3-pyrimidines.
Subject(s)
Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Quinolines/chemical synthesis , Receptor, IGF Type 1/antagonists & inhibitors , Animals , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Mice , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Quinolines/chemistry , Quinolines/pharmacokinetics , Receptor, IGF Type 1/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).
Subject(s)
Amides/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , T-Lymphocytes/drug effects , Administration, Oral , Amides/chemical synthesis , Amides/chemistry , Animals , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Design , Enzyme Activation/drug effects , Female , Humans , Interleukin-2/antagonists & inhibitors , Interleukin-2/metabolism , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , Stereoisomerism , Structure-Activity Relationship , T-Lymphocytes/metabolismABSTRACT
Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided design, synthesis, structure-activity relationships, and pharmacological characterization of 2-amino-6-phenylpyrimido[5',4':5,6]pyrimido[1,2- a]benzimidazol-5(6 H)-ones, a new class of compounds that are potent inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)amino)pyrimido[5',4':5,6]pyrimido-[1,2- a]benzimidazol-5(6 H)-one ( 25), exhibits potent inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in vivo models of T-cell activation and arthritis, respectively.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Arthritis/drug therapy , Benzimidazoles/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyrimidinones/chemical synthesis , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Crystallography, X-Ray , Disease Models, Animal , Drug Design , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Female , Injections, Intradermal , Interleukin-2/metabolism , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Reproducibility of Results , Stereoisomerism , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
N-3-(Phenylcarbamoyl)arylpyrimidine-5-carboxamides are a novel class of selective Lck inhibitors. This series of compounds derives its selectivity from a hydrogen bond with the gatekeeper Thr316 of the enzyme. X-ray co-crystal structural data, structure-activity relationships, and the synthesis of these inhibitors are reported herein.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Amides/chemistry , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Drug Design , Humans , Hydrogen Bonding , Molecular Conformation , Molecular Structure , Pyrimidines/chemistry , Structure-Activity Relationship , Threonine/chemistryABSTRACT
4-Amino-5,6-biaryl-furo[2,3-d]pyrimidines were identified as potent non-selective inhibitors of Lck. A novel, divergent, and practical synthetic route was developed to access derivatives from bifunctional intermediates. Lead optimization was guided by X-ray crystallographic data, and preliminary SAR led to the identification of compounds with improved cellular potency and selectivity.
Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Quantitative Structure-Activity Relationship , Animals , Anti-Inflammatory Agents/chemical synthesis , Humans , Inhibitory Concentration 50 , Interleukin-2/metabolism , Lymphocyte Culture Test, Mixed , Lymphocytes/drug effects , Pharmacokinetics , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
2,3-Diarylfuro[2,3-b]pyridine-4-amines are a novel class of potent and selective inhibitors of Lck. The discovery, synthesis, and structure activity relationships of this series of inhibitors are reported. The most promising compounds were also profiled to deduce their pharmacokinetic properties.
Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyridines/chemical synthesis , Pyridines/pharmacokinetics , Amines/chemical synthesis , Amines/pharmacokinetics , Animals , Humans , Inhibitory Concentration 50 , Pharmacokinetics , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Benzamides/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Quinazolines/chemical synthesis , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzamides/chemistry , Benzamides/pharmacology , Biological Availability , Cell Proliferation/drug effects , Cells, Cultured , Female , Humans , In Vitro Techniques , Interleukin-2/biosynthesis , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Quinazolines/chemistry , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.