Novel quinazolin-4(3H)-one based Cyclin K degraders regulate alternative polyadenylation activity.

Phenotypic screening is gaining attention as a powerful method for identifying compounds that regulate cellular phenotypes of interest through novel mechanisms of action. Recently, a new modality of compounds, called molecular glues, which can induce the degradation of target proteins by forming ternary complexes of E3 ligases, has emerged from phenotypic screening. In this study, using global proteomic analysis, we identified a novel Cyclin K degrader, T4, which was previously discovered through phenotypic screening for alternative polyadenylation regulation. Our detailed mechanistic analysis revealed that T4 induced Cyclin K degradation, leading to the regulation of alternative polyadenylation. Additionally, we generated a more potent Cyclin K degrader, TR-213, through a structure-activity relationship study of T4. T4 and TR-213 are structurally distinct from other Cyclin K degraders and can be used as novel chemical tools to further analyze the degradation of Cyclin K and the regulation of alternative polyadenylation.

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

Synthetic mRNA-based differentiation method enables early detection of Parkinson's phenotypes in neurons derived from Gaucher disease-induced pluripotent stem cells.

Gaucher disease, the most prevalent metabolic storage disorder, is caused by mutations in the glucocerebrosidase gene GBA1, which lead to the accumulation of glucosylceramide (GlcCer) in affected cells. Gaucher disease type 1 (GD1), although defined as a nonneuronopathic subtype, is accompanied by an increased risk of Parkinson's disease. To gain insights into the association of progressive accumulation of GlcCer and the Parkinson's disease phenotypes, we generated dopaminergic (DA) neurons from induced pluripotent stem cells (iPSCs) derived from a GD1 patient and a healthy donor control, and measured GlcCer accumulation by liquid chromatography-mass spectrometry. We tested two DA neuron differentiation methods: a well-established method that mimics a step-wise developmental process from iPSCs to neural progenitor cells, and to DA neurons; and a synthetic mRNA-based method that overexpresses a transcription factor in iPSCs. GD1-specific accumulation of GlcCer was detected after 60 days of differentiation by the former method, whereas it was detected after only 10 days by the latter method. With this synthetic mRNA-based rapid differentiation method, we found that the metabolic defect in GD1 patient cells can be rescued by the overexpression of wild-type GBA1 or the treatment with an inhibitor for GlcCer synthesis. Furthermore, we detected the increased phosphorylation of α-synuclein, a biomarker for Parkinson's disease, in DA neurons derived from a GD1 patient, which was significantly decreased by the overexpression of wild-type GBA1. These results suggest that synthetic mRNA-based method accelerates the analyses of the pathological mechanisms of Parkinson's disease in GD1 patients and possibly facilitates drug discovery processes.

Quantitative comparison of the mRNA content of human iPSC-derived motor neurons and their extracellular vesicles.

Extracellular vesicles (EVs) contain various cargo molecules, including RNAs and proteins. EVs, which include exosomes, are predicted to be suitable surrogates of their source cells for liquid biopsy to measure biomarkers. Several studies have performed qualitative comparisons of cargo molecule repertoires between source cells and their EVs. However, quantitative comparisons have not been reported so far. Furthermore, many studies analyzed microRNAs or proteins in EVs, but not mRNAs. In this study, we analyzed mRNAs in motor neurons and their EVs. Normal human-induced pluripotent stem cells were differentiated into motor neurons, and comprehensive analysis of mRNAs in the cells and their EVs was performed by RNA sequencing. Differential analysis between cellular and EV mRNAs was performed by edgeR after normalization of read count. The results suggest that signatures in the abundance of EV mRNAs were different from those of cellular mRNAs. Comparison of intracellular vesicle and EV mRNA abundance showed negatively and positively biased genes in the EVs. Gene Ontology analysis revealed that the genes showing negatively biased abundance in the EVs were enriched in many functions regarding neuronal development. In contrast, the positively biased genes were enriched in functions regarding cellular metabolism and protein synthesis. These results suggest that mRNAs in motor neurons are loaded into EVs to regulate certain mechanisms, which are yet to be elucidated.

A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling.

Early-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. We report two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca2+ response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. Our results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders.

A BET family protein degrader provokes senolysis by targeting NHEJ and autophagy in senescent cells.

Although cellular senescence acts primarily as a tumour suppression mechanism, the accumulation of senescent cells in vivo eventually exerts deleterious side effects through inflammatory/tumour-promoting factor secretion. Thus, the development of new drugs that cause the specific elimination of senescent cells, termed senolysis, is anticipated. Here, by an unbiased high-throughput screening of chemical compounds and a bio-functional analysis, we identify BET family protein degrader (BETd) as a promising senolytic drug. BETd provokes senolysis through two independent but integrated pathways; the attenuation of non-homologous end joining (NHEJ), and the up-regulation of autophagic gene expression. BETd treatment eliminates senescent hepatic stellate cells in obese mouse livers, accompanied by the reduction of liver cancer development. Furthermore, the elimination of chemotherapy-induced senescent cells by BETd increases the efficacy of chemotherapy against xenograft tumours in immunocompromised mice. These results reveal the vulnerability of senescent cells and open up possibilities for its control.

Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening.

The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.

Feedback activation of AMPK-mediated autophagy acceleration is a key resistance mechanism against SCD1 inhibitor-induced cell growth inhibition.

Elucidating the bioactive compound modes of action is crucial for increasing success rates in drug development. For anticancer drugs, defining effective drug combinations that overcome resistance improves therapeutic efficacy. Herein, by using a biologically annotated compound library, we performed a large-scale combination screening with Stearoyl-CoA desaturase-1 (SCD1) inhibitor, T-3764518, which partially inhibits colorectal cancer cell proliferation. T-3764518 induced phosphorylation and activation of AMPK in HCT-116 cells, which led to blockade of downstream fatty acid synthesis and acceleration of autophagy. Attenuation of fatty acid synthesis by small molecules suppressed the growth inhibitory effect of T-3764518. In contrast, combination of T-3764518 with autophagy flux inhibitors synergistically inhibited cellular proliferation. Experiments using SCD1 knock-out cells validated the results obtained with T-3764518. The results of our study indicated that activation of autophagy serves as a survival signal when SCD1 is inhibited in HCT-116 cells. Furthermore, these findings suggest that combining SCD1 inhibitor with autophagy inhibitors is a promising anticancer therapy.

Using a biologically annotated library to analyze the anticancer mechanism of serine palmitoyl transferase (SPT) inhibitors.

Mechanistic understanding is crucial to anticancer drug discovery. Here, we reveal that inhibition of serine palmitoyl transferase (SPT), the rate-limiting enzyme in sphingolipid synthesis, induced death in a lung cancer cell line via a necrosis-dependent pathway. To elucidate the mechanism of cell death induced by SPT inhibition, a biologically annotated library of diverse compounds was screened with an SPT inhibitor. This analysis identified suppressors of SPT inhibitor-mediated cell death. Further analysis using hit compounds from this screening revealed that SPT inhibitors induce COX-2 expression, leading to necrosis-dependent cell death. SPT inhibitors might therefore represent novel candidates for cancer therapy via necrosis pathway regulation. Our data illustrate that compound combination screening of biologically annotated libraries could be used for mechanistic elucidation.

MET Tyrosine Kinase Inhibition Enhances the Antitumor Efficacy of an HGF Antibody.

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. Mol Cancer Ther; 16(7); 1269-78. ©2017 AACR.

Vacuolin-1 inhibits autophagy by impairing lysosomal maturation via PIKfyve inhibition.

Lysosomal protein degradation via autophagy strictly regulates cellular protein homoeostasis. Herein we performed high-content screening to identify compounds that inhibit autophagy pathways. We obtained 11 hit compounds and performed cluster analysis using cellular morphological information. Vacuolin-1, which induces the formation of giant vacuoles and is a target unknown compound, clustered with the known PIKfyve inhibitor YM201636. We further confirmed that vacuolin-1 is a potent PIKfyve inhibitor, and we finally concluded that PIKfyve inhibitors are novel chemical tools for regulating autophagy.

Tubulin is a molecular target of the Wnt-activating chemical probe.

BACKGROUND: In drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential. RESULTS: 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization. CONCLUSION: These results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.

Structure-based design, synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors.

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC50=1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC50=5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C]=4%, po, 5mg/kg, once-daily) and COLO205 (T/C=13%, po, 15 mg/kg, once-daily) mouse xenograft models.

Design, synthesis, and evaluation of novel VEGFR2 kinase inhibitors: discovery of [1,2,4]triazolo[1,5-a]pyridine derivatives with slow dissociation kinetics.

For the purpose of discovering novel type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase, we designed and synthesized 5,6-fused heterocyclic compounds bearing a anilide group. A co-crystal structure analysis of imidazo[1,2-b]pyridazine derivative 2 with VEGFR2 revealed that the N1-nitrogen of imidazo[1,2-b]pyridazine core interacts with the backbone NH group of Cys919. To retain this essential interaction, we designed a series of imidazo[1,2-a]pyridine, [1,2,4]triazolo[1,5-a]pyridine, thiazolo[5,4-b]pyridine, and 1,3-benzothiazole derivatives maintaining a ring nitrogen as hydrogen bond acceptor (HBA) at the corresponding position. All compounds thus designed displayed strong inhibitory activity against VEGFR2 kinase, and the [1,2,4]triazolo[1,5-a]pyridine 13d displayed favorable physicochemical properties. Furthermore, 13d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited platelet-derived growth factor receptor (PDGFR) kinases. Oral administration of 13d showed potent anti-tumor efficacy in DU145 and A549 xenograft models in nude mice.

A novel inhibitor of c-Met and VEGF receptor tyrosine kinases with a broad spectrum of in vivo antitumor activities.

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are dysregulated in a wide variety of human cancers and are linked with tumorigenesis and metastatic progression. VEGF also plays a key role in tumor angiogenesis and progression by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor tyrosine kinases (VEGFR). Therefore, inhibiting both HGF/c-Met and VEGF/VEGFR signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of tumors. Toward this goal, we generated and characterized T-1840383, a small-molecule kinase inhibitor that targets both c-Met and VEGFRs. T-1840383 inhibited HGF-induced c-Met phosphorylation and VEGF-induced VEGFR-2 phosphorylation in cancer epithelial cells and vascular endothelial cells, respectively. It also inhibited constitutively activated c-Met phosphorylation in c-met-amplified cancer cells, leading to suppression of cell proliferation. In addition, T-1840383 potently blocked VEGF-dependent proliferation and capillary tube formation of endothelial cells. Following oral administration, T-1840383 showed potent antitumor efficacy in a wide variety of human tumor xenograft mouse models, along with reduction of c-Met phosphorylation levels and microvessel density within tumor xenografts. These results suggest that the efficacy of T-1840383 is produced by direct effects on tumor cell growth and by an antiangiogenic mechanism. Furthermore, T-1840383 showed profound antitumor activity in a gastric tumor peritoneal dissemination model. Collectively, our findings indicate the therapeutic potential of targeting both c-Met and VEGFRs simultaneously with a single small-molecule inhibitor for the treatment of human cancers.

Design and synthesis of novel pyrimido[4,5-b]azepine derivatives as HER2/EGFR dual inhibitors.

A novel 7,6 fused bicyclic scaffold, pyrimido[4,5-b]azepine was designed to fit into the ATP binding site of the HER2/EGFR proteins. The synthesis of this scaffold was accomplished by an intramolecular Claisen-type condensation. As the results of optimization lead us to 4-anilino and 6-functional groups, we discovered 6-substituted amide derivative 19b, which has a 1-benzothiophen-4-yloxy group attached to the 4-anilino group. An X-ray co-crystal structure of 19b with EGFR demonstrated that the N-1 and N-3 nitrogens of the pyrimido[4,5-b]azepine scaffold make hydrogen-bonding interactions with the main chain NH of Met793 and the side chain of Thr854 via a water-mediated hydrogen bond network, respectively. In addition, the NH proton at the 9-position makes an additional hydrogen bond with the carbonyl group of Met793, as we expected. Compound 19b revealed potent HER2/EGFR kinase (IC50: 24/36 nM) and BT474 cell growth (GI50: 18 nM) inhibitory activities based on its pseudo-irreversible (PI) profile.

Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor.

Vascular endothelial growth factor (VEGF) plays important roles in tumor angiogenesis, and the inhibition of its signaling pathway is considered an effective therapeutic option for the treatment of cancer. In this study, we describe the design, synthesis, and biological evaluation of 2-acylamino-6-phenoxy-imidazo[1,2-b]pyridazine derivatives. Hybridization of two distinct imidazo[1,2-b]pyridazines 1 and 2, followed by optimization led to the discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (23a, TAK-593) as a highly potent VEGF receptor 2 kinase inhibitor with an IC50 value of 0.95 nM. The compound 23a strongly suppressed proliferation of VEGF-stimulated human umbilical vein endothelial cells with an IC50 of 0.30 nM. Kinase selectivity profiling revealed that 23a inhibited platelet-derived growth factor receptor kinases as well as VEGF receptor kinases. Oral administration of 23a at 1 mg/kg bid potently inhibited tumor growth in a mouse xenograft model using human lung adenocarcinoma A549 cells (T/C=8%).

Structure-based discovery of cellular-active allosteric inhibitors of FAK.

In order to develop potent and selective focal adhesion kinase (FAK) inhibitors, synthetic studies on pyrazolo[4,3-c][2,1]benzothiazines targeted for the FAK allosteric site were carried out. Based on the X-ray structural analysis of the co-crystal of the lead compound, 8-(4-ethylphenyl)-5-methyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazine 4,4-dioxide 1 with FAK, we designed and prepared 1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin derivatives which selectively inhibited kinase activity of FAK without affecting seven other kinases. The optimized compound, N-(4-tert-butylbenzyl)-1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin-8-amine 4,4-dioxide 30 possessed significant FAK kinase inhibitory activities both in cell-free (IC50=0.64µM) and in cellular assays (IC50=7.1µM). These results clearly demonstrated a potential of FAK allosteric inhibitors as antitumor agents.

Anti-angiogenic and anti-tumor effects of TAK-593, a potent and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinase.

We recently reported that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK-593 exhibits a uniquely long-acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor ß (PDGFRß). In this study, we demonstrated that TAK-593 potently inhibits VEGF- and PDGF-stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK-593 also potently inhibits VEGF-induced tube formation of endothelial cells co-cultured with fibroblasts. Oral administration of TAK-593 exhibited strong anti-tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK-593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK-593. Immunohistochemical staining indicated that TAK-593 showed anti-proliferative and pro-apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast-enhanced magnetic resonance imaging revealed that TAK-593 reduced tumor vessel permeability prior to the onset of anti-tumor activity. In conclusion, TAK-593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti-angiogenic activity suggests therapeutic potential for the treatment of solid tumors.

Discovery and characterization of novel allosteric FAK inhibitors.

Focal adhesion kinase (FAK) regulates cell survival and proliferation pathways. Here we report the discovery of a highly selective series of 1,5-dihydropyrazolo[4,3-c][2,1]benzothiazines that demonstrate a novel mode of allosteric inhibition of FAK. These compounds showed slow dissociation from unphosphorylated FAK and were noncompetitive with ATP after long preincubation. Co-crystal structural analysis revealed that the compounds target a novel allosteric site within the C-lobe of the kinase domain, which induces disruption of ATP pocket formation leading to the inhibition of kinase activity. The potency of allosteric inhibition was reduced by phosphorylation of FAK. Coupled SAR analysis revealed that N-substitution of the fused pyrazole is critical to achieve allosteric binding and high selectivity among kinases.