ABSTRACT
O-GlcNAcase (OGA) has received increasing attention as an attractive therapeutic target for tau-mediated neurodegenerative disorders; however, its role in these pathologies remains unclear. Therefore, potent chemical tools with favorable pharmacokinetic profiles are desirable to characterize this enzyme. Herein, we report the discovery of a potent and novel OGA inhibitor, compound 5i, comprising an aminopyrimidine scaffold, identified by virtual screening based on multiple methodologies combining structure-based and ligand-based approaches, followed by sequential optimization with a focus on ligand lipophilicity efficiency. This compound was observed to increase the level of O-GlcNAcylated protein in cells and display suitable pharmacokinetic properties and brain permeability. Crystallographic analysis revealed that the chemical series bind to OGA via characteristic hydrophobic interactions, which resulted in a high affinity for OGA with moderate lipophilicity. Compound 5i could serve as a useful chemical probe to help establish a proof-of-concept of OGA inhibition as a therapeutic target for the treatment of tauopathies.
Subject(s)
Acetylglucosamine/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , Animals , Brain/metabolism , Cell Line , Computer Simulation , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacokinetics , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Neuroprotective Agents/pharmacokinetics , Structure-Activity Relationship , Tauopathies/drug therapyABSTRACT
An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells. ROS production after nifedipine treatment was inhibited by a NOX inhibitor (GKT136901) but not the mitochondrial NADH dehydrogenase inhibitor, rotenone, suggesting that nifedipine decreases NRK52E cell viability primarily through a NOX-mediated pathway. To understand the breadth of NOX-mediated ROS production, 12 commercially available compounds that are structurally and/or pharmacologically related to nifedipine as well as 172 internal Takeda candidate drugs, were also evaluated against these two cell types. Over 15 % of compounds not cytotoxic to HepG2 cells (below 50 µM) were cytotoxic to NRK52E cells. Our results suggest that a combination of cell viability data from both NRK52E and HepG2 cells was superior for the prediction of in vivo toxicity findings when compared to use of only one cell line. Further, the NRK52E cell viability assay is a good predictor of NOX-mediated ROS production and can be used as a follow up assay following a negative HepG2 response to aid in the selection of suitable compounds for in vivo toxicity studies.
Subject(s)
Epithelial Cells/drug effects , Kidney/drug effects , Reactive Oxygen Species/metabolism , Biological Assay , Cell Line , Cell Survival/drug effects , Drug Evaluation, Preclinical , Drugs, Investigational/toxicity , Epithelial Cells/metabolism , Epithelial Cells/pathology , Hep G2 Cells , Humans , Inhibitory Concentration 50 , Kidney/metabolism , Kidney/pathology , NADPH Oxidase 4/genetics , Nifedipine/toxicityABSTRACT
Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.
Subject(s)
Enzyme Inhibitors/chemistry , Indoles/chemistry , Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Thiophenes/chemistry , Allosteric Site , Crystallography, X-Ray , Drug Discovery , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Guanine/analogs & derivatives , Guanine/metabolism , High-Throughput Screening Assays , Humans , Indoles/chemical synthesis , Indoles/metabolism , Molecular Structure , NAD/metabolism , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Protein Binding , Protein Conformation/drug effects , Spermidine/metabolism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/metabolismABSTRACT
In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.
Subject(s)
Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazolones/therapeutic use , Pyrimidines/therapeutic use , Pyrimidinones/therapeutic use , Quinuclidines/therapeutic use , Thiophenes/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Drug Design , Drug Discovery , Formaldehyde/chemistry , Humans , Mice , Molecular Docking Simulation , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Pyrazolones/pharmacology , Pyrimidines/pharmacology , Pyrimidinones/chemical synthesis , Pyrimidinones/metabolism , Quinuclidines/chemical synthesis , Quinuclidines/metabolism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.
ABSTRACT
Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazolones/pharmacology , Pyrimidines/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation , Cell Separation , Cell Survival , Centrosome/drug effects , Chromosome Aberrations/drug effects , Computational Biology , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Female , HeLa Cells , Humans , Inhibitory Concentration 50 , Kaplan-Meier Estimate , Mice , Mice, Inbred BALB C , Mitosis/drug effects , Models, Animal , Mutation , Neoplasm Transplantation , Pancreatic Neoplasms/drug therapy , Protein Binding , Protein Kinase Inhibitors/pharmacology , Proteomics , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Retinoic acid receptor-related orphan receptor γt (RORγt) agonists are expected to provide a novel class of immune-activating anticancer drugs via activation of Th17 cells and Tc17 cells. Herein, we describe a novel structure-based functionality switching approach from in house well-optimized RORγt inverse agonists to potent RORγt agonists. We succeeded in the identification of potent RORγt agonist 5 without major chemical structure change. The biochemical response was validated by molecular dynamics simulation studies that showed a helix 12 stabilization effect of RORγt agonists. These results indicate that targeting helix 12 is an attractive and novel medicinal chemistry strategy for switching existing RORγt inverse agonists to agonists.
Subject(s)
Drug Design , Drug Inverse Agonism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Animals , High-Throughput Screening Assays , Molecular Dynamics Simulation , Structure-Activity Relationship , Th17 Cells/drug effectsABSTRACT
Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.
Subject(s)
Breast Neoplasms/drug therapy , Cell Survival , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/pharmacology , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Enzyme Inhibitors/chemistry , Female , Humans , Phosphorylation , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
It has been hypothesized that selective inhibition of phosphodiesterase (PDE) 2A could potentially be a novel approach to treat cognitive impairment in neuropsychiatric and neurodegenerative disorders through augmentation of cyclic nucleotide signaling pathways in brain regions associated with learning and memory. Following our earlier work, this article describes a drug design strategy for a new series of lead compounds structurally distinct from our clinical candidate 2 (TAK-915), and subsequent medicinal chemistry efforts to optimize potency, selectivity over other PDE families, and other preclinical properties including in vitro phototoxicity and in vivo rat plasma clearance. These efforts resulted in the discovery of N-((1S)-2-hydroxy-2-methyl-1-(4-(trifluoromethoxy)phenyl)propyl)-6-methyl-5-(3-methyl-1H-1,2,4-triazol-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (20), which robustly increased 3',5'-cyclic guanosine monophosphate (cGMP) levels in the rat brain following an oral dose, and moreover, attenuated MK-801-induced episodic memory deficits in a passive avoidance task in rats. These data provide further support to the potential therapeutic utility of PDE2A inhibitors in enhancing cognitive performance.
Subject(s)
Cognition Disorders/drug therapy , Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors , Drug Discovery , Phosphodiesterase Inhibitors/pharmacology , Pyrazines/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacology , 3T3 Cells , Administration, Oral , Animals , COS Cells , Cell Survival/drug effects , Chlorocebus aethiops , Cognition Disorders/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Dose-Response Relationship, Drug , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Molecular Structure , Phosphodiesterase Inhibitors/administration & dosage , Phosphodiesterase Inhibitors/chemistry , Powder Diffraction , Pyrazines/chemistry , Pyrazoles/chemistry , Pyridines/chemistry , Pyrimidines/chemistry , Rats , Rats, Long-Evans , Solubility , Structure-Activity Relationship , ThermodynamicsABSTRACT
Phosphodiesterase (PDE) 2A inhibitors have emerged as a novel mechanism with potential therapeutic option to ameliorate cognitive dysfunction in schizophrenia or Alzheimer's disease through upregulation of cyclic nucleotides in the brain and thereby achieve potentiation of cyclic nucleotide signaling pathways. This article details the expedited optimization of our recently disclosed pyrazolo[1,5-a]pyrimidine lead compound 4b, leading to the discovery of clinical candidate 36 (TAK-915), which demonstrates an appropriate combination of potency, PDE selectivity, and favorable pharmacokinetic (PK) properties, including brain penetration. Successful identification of 36 was realized through application of structure-based drug design (SBDD) to further improve potency and PDE selectivity, coupled with prospective design focused on physicochemical properties to deliver brain penetration. Oral administration of 36 demonstrated significant elevation of 3',5'-cyclic guanosine monophosphate (cGMP) levels in mouse brains and improved cognitive performance in a novel object recognition task in rats. Consequently, compound 36 was advanced into human clinical trials.
Subject(s)
Brain/drug effects , Cognition/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors , Phosphodiesterase Inhibitors/pharmacology , Phosphodiesterase Inhibitors/pharmacokinetics , Pyrazines/pharmacology , Pyrazines/pharmacokinetics , Animals , Brain/metabolism , Cognition Disorders/drug therapy , Cognition Disorders/metabolism , Crystallography, X-Ray , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 2/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Drug Design , Halogenation , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Phosphodiesterase Inhibitors/chemistry , Pyrazines/chemistry , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Most prostate cancers initially respond to androgen deprivation therapy, but then progress from androgen-dependent to androgen-independent prostate cancers. In the present study, a differential cytotoxicity screen of hormone-resistant prostate cancer LNCaP-hr cells and the parental LNCaP-FGC cells against normal MRC5 fibroblast cells, identified a small molecule compound, Aristeromycin (a derivative of 3-deazaneplanocin A (DZNeP)). The molecular target was shown to be S-adenosylhomocysteine hydrolase (AHCY), which catalyzes reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and L-homocysteine. DZNeP and Aristeromycin showed high inhibitory activity against AHCY. Treatment of the prostate cancer cells with DZNeP led to SAH accumulation and decreased levels of homocysteine and histone H3K27 methylation. SAH accumulation and cell growth inhibition were confirmed after siRNA-mediated AHCY knockdown. To further understand why AHCY inhibitors decreased prostate cancer cell growth, we performed microRNA expression profiling with LNCaP-hr cells. Mir-26a, which is involved in regulation of EZH2 expression, was upregulated in Aristeromycin-treated LNCaP-hr cells. A reporter assay established with the EZH2 3'-UTR confirmed that transfection of microRNA precursor molecules for miR-26a decreased the EZH2 3'-UTR luciferase activity. Meanwhile, an antisense microRNA inhibitor for miR-26a recovered the luciferase activity. The present findings suggest, at least in part, that miR-26a induced by an AHCY inhibitor can regulate oncogenic EZH2 expression, and could thus be an important mechanism of action for AHCY inhibitors in the treatment of prostate cancer.
Subject(s)
Adenosine/analogs & derivatives , MicroRNAs/genetics , Prostatic Neoplasms/pathology , Transcriptional Activation/drug effects , Adenosine/pharmacology , Adenosylhomocysteinase/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enhancer of Zeste Homolog 2 Protein/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Male , RNA, Small Interfering/geneticsABSTRACT
S-adenosylhomocysteine hydrolase (AHCY) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and l-homocysteine. This enzyme is frequently overexpressed in many tumor types and is considered to be a validated anti-tumor target. In order to enable the development of small molecule AHCY inhibitors as targeted cancer therapeutics we developed an assay based on a RapidFire high-throughput mass spectrometry detection system, which allows the direct measurement of AHCY enzymatic activity. This technique avoids many of the problems associate with the previously reported method of using a thiol-reactive fluorescence probes to measure AHCY activity. Screening of a â¼500,000 compound library using this technique identified multiple SAH competitive hits. Co-crystal structures of the hit compounds complexed with AHCY were obtained showing that the compounds indeed bind in the SAH site of the enzyme. In addition, some hit compounds increased the SAH levels in HCT116 cells and showed growth inhibition. These compounds could be promising starting points for the optimization of cancer treatments.
Subject(s)
Adenosylhomocysteinase/antagonists & inhibitors , Adenosylhomocysteinase/metabolism , Antineoplastic Agents/analysis , Enzyme Inhibitors/analysis , Mass Spectrometry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Cell Survival/drug effects , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HCT116 Cells , High-Throughput Screening Assays , Humans , Protein Binding , Protein Interaction MapsABSTRACT
In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50=0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/chemistry , Pyrimidinones/chemistry , Thiophenes/chemical synthesis , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/metabolism , Humans , Inhibitory Concentration 50 , Kinetics , Molecular Docking Simulation , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Pyrimidinones/chemical synthesis , Pyrimidinones/pharmacokinetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacokinetics , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.
Subject(s)
Alternative Splicing/drug effects , Imidazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Pyrimidines/pharmacology , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Exons , Gene Expression Profiling , Genome, Human , HCT116 Cells , Humans , Imidazoles/chemical synthesis , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Pyrimidines/chemical synthesis , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , Structure-Activity Relationship , Transcription, GeneticABSTRACT
Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors. Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor xenograft model.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Drug Design , Imidazoles/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pteridines/chemical synthesis , Animals , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , HT29 Cells , Heterografts , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Mice , Molecular Structure , Pteridines/chemistry , Pteridines/pharmacology , Structure-Activity Relationship , Polo-Like Kinase 1ABSTRACT
With the aim of discovering a selective kinase inhibitor targeting pan-RAF kinase inhibition, we designed novel 1,3-benzothiazole derivatives based on our thiazolo[5,4-b]pyridine class RAF/VEGFR2 inhibitor 1 and developed a regioselective cyclization methodology for the C-7-substituted 1,3-benzothiazole scaffold utilizing meta-substituted anilines. Eventually, we selected 7-cyano derivative 8B (TAK-632) as a development candidate and confirmed its binding mode by cocrystal structure with BRAF. Accommodation of the 7-cyano group into the BRAF-selectivity pocket and the 3-(trifluoromethyl)phenyl acetamide moiety into the hydrophobic back pocket of BRAF in the DFG-out conformation contributed to enhanced RAF potency and selectivity vs VEGFR2. Reflecting its potent pan-RAF inhibition and slow off-rate profile, 8B demonstrated significant cellular activity against mutated BRAF or mutated NRAS cancer cell lines. Furthermore, in both A375 (BRAF(V600E)) and HMVII (NRAS(Q61K)) xenograft models in rats, 8B demonstrated regressive antitumor efficacy by twice daily, 14-day repetitive administration without significant body weight loss.
Subject(s)
Benzothiazoles/chemical synthesis , Benzothiazoles/pharmacology , Drug Discovery , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Benzothiazoles/chemistry , Blood-Brain Barrier , Cell Line, Tumor , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Models, Molecular , Protein Kinase Inhibitors/chemistry , Surface Plasmon ResonanceABSTRACT
Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.
Subject(s)
Azepines/chemistry , Azepines/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , 4-Aminobenzoic Acid/chemistry , 4-Aminobenzoic Acid/pharmacokinetics , 4-Aminobenzoic Acid/pharmacology , Administration, Oral , Adult , Azepines/pharmacokinetics , Cell Line, Tumor , Drug Discovery , Humans , Protein Kinase Inhibitors/pharmacokinetics , Young Adult , Polo-Like Kinase 1ABSTRACT
Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase involved in key processes during mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and elevated levels of PLK1 have been associated with poor prognosis, making it an attractive target for anticancer therapy. TAK-960 [4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl) benzamide] is a novel, investigational, orally bioavailable, potent, and selective PLK1 inhibitor that has shown activity in several tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1). Consistent with PLK1 inhibition, TAK-960 treatment caused accumulation of G(2)-M cells, aberrant polo mitosis morphology, and increased phosphorylation of histone H3 (pHH3) in vitro and in vivo. TAK-960 inhibited proliferation of multiple cancer cell lines, with mean EC(50) values ranging from 8.4 to 46.9 nmol/L, but not in nondividing normal cells (EC(50) >1,000 nmol/L). The mutation status of TP53 or KRAS and MDR1 expression did not correlate with the potency of TAK-960 in the cell lines tested. In animal models, oral administration of TAK-960 increased pHH3 in a dose-dependent manner and significantly inhibited the growth of HT-29 colorectal cancer xenografts. Treatment with once daily TAK-960 exhibited significant efficacy against multiple tumor xenografts, including an adriamycin/paclitaxel-resistant xenograft model and a disseminated leukemia model. TAK-960 has entered clinical evaluation in patients with advanced cancers.
Subject(s)
Azepines/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , 4-Aminobenzoic Acid/chemistry , 4-Aminobenzoic Acid/pharmacology , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Azepines/chemistry , Biological Availability , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drugs, Investigational/chemistry , Drugs, Investigational/pharmacokinetics , Drugs, Investigational/pharmacology , Female , HT29 Cells , Histones/metabolism , Humans , K562 Cells , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Molecular Structure , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Xenograft Model Antitumor Assays , Polo-Like Kinase 1ABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder leading to a progressive loss of cognitive function and is pathologically characterized by senile plaques and neurofibrillary tangles. Glycogen synthase kinase-3 (GSK-3) is involved in AD pathogenesis. GSK-3 is reported not only to phosphorylate tau, a major component of neurofibrillary tangles, but also to regulate the production of amyloid ß, which is deposited in senile plaques. Therefore, pharmacological inhibition of GSK-3 is considered an attractive therapeutic approach. In this study, we report the pharmacological effects of a novel GSK-3 inhibitor, 2-methyl-5-(3-{4-[(S)-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole (MMBO), which displays high selectivity for GSK-3 and brain penetration following oral administration. MMBO inhibited tau phosphorylation in primary neural cell culture and also in normal mouse brain. When administered to a transgenic mouse model of AD, MMBO significantly decreased hippocampal tau phosphorylation at GSK-3 sites. Additionally, chronic MMBO administration suppressed tau pathology as assessed by AT8-immunoreactivity without affecting amyloid ß pathology. Finally, in behavioral assessments, MMBO significantly improved memory and cognitive deficits in the Y-maze and in novel object recognition tests in the transgenic AD mouse model. These results indicate that pharmacological GSK-3 inhibition ameliorates behavioral dysfunction with suppression of tau phosphorylation in an AD mouse model, and that MMBO might be beneficial for AD treatment.
Subject(s)
Cognition Disorders/drug therapy , Enzyme Inhibitors/therapeutic use , Glycogen Synthase Kinase 3/antagonists & inhibitors , tau Proteins/metabolism , Alzheimer Disease/complications , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Benzofurans/pharmacology , Benzofurans/therapeutic use , Brain/drug effects , Brain/metabolism , Cell Culture Techniques , Cerebral Cortex/cytology , Cognition Disorders/etiology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Exploratory Behavior/drug effects , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Maze Learning/drug effects , Mice , Mice, Transgenic , Mutation/genetics , Neurons/drug effects , Oxadiazoles/pharmacology , Oxadiazoles/therapeutic use , Peptide Fragments/metabolism , Phosphorylation/drug effects , Presenilin-1/genetics , Time Factors , tau Proteins/geneticsABSTRACT
Glycogen synthase kinase 3beta (GSK-3beta) inhibition is expected to be a promising therapeutic approach for treating Alzheimer's disease. Previously we reported a series of 1,3,4-oxadiazole derivatives as potent and highly selective GSK-3beta inhibitors, however, the representative compounds 1a,b showed poor pharmacokinetic profiles. Efforts were made to address this issue by reducing molecular weight and lipophilicity, leading to the identification of oxadiazole derivatives containing a sulfinyl group, (S)-9b and (S)-9c. These compounds exhibited not only highly selective and potent inhibitory activity against GSK-3beta but also showed good pharmacokinetic profiles including favorable BBB penetration. In addition, (S)-9b and (S)-9c given orally to mice significantly inhibited cold water stress-induced tau hyperphosphorylation in mouse brain.