Kinase Inhibitor Library Research Articles

Evaluation of STK17B as a cancer immunotherapy target utilizing highly potent and selective small molecule inhibitors.

The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy. To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo. In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model. In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

CDC7 Inhibition Potentiates Antitumor Efficacy of PARP Inhibitor in Advanced Ovarian Cancer.

Poly (ADP-ribose) Polymerase inhibitors (PARPi) have demonstrated remarkable clinical efficacy in treating ovarian cancer (OV) with BRCA1/2 mutations. However, drug resistance inevitably limits their clinical applications and there is an urgent need for improved therapeutic strategies to enhance the clinical utility of PARPi, such as Olaparib. Here, compelling evidence indicates that sensitivity of PARPi is associated with cell cycle dysfunction. Through high-throughput drug screening with a cell cycle kinase inhibitor library, XL413, a potent cell division cycle 7 (CDC7) inhibitor, is identified which can synergistically enhance the anti-tumor efficacy of Olaparib. Mechanistically, the combined administration of XL413 and Olaparib demonstrates considerable DNA damage and DNA replication stress, leading to increased sensitivity to Olaparib. Additionally, a robust type-I interferon response is triggered through the induction of thecGAS/STING signaling pathway. Using murine syngeneic tumor models, the combination treatment further demonstrates enhanced antitumor immunity, resulting in tumor regression. Collectively, this study presents an effective treatment strategy for patients with advanced OV by combining CDC7 inhibitors (CDC7i) and PARPi, offering a promising therapeutic approach for patients with limited response to PARPi.

The AKT inhibitor AZD5363 elicits synthetic lethality in ARID1A-deficient gastric cancer cells via induction of pyroptosis.

Gastric cancer (GC) is a deadly disease with poor overall survival and limited therapeutic options. Genetic alterations such as mutations and/or deletions in chromatin remodeling gene AT-rich interactive domain 1 A (ARID1A) occur frequently in GC. Although ARID1A mutations/deletions are not a druggable target for conventional treatments, novel therapeutic strategies based on a synthetic lethal approach may be effective for the treatment of ARID1A-deficient cancers. A kinase inhibitor library containing 551 compounds was screened in ARID1A isogenic GC cells for the ability to induce synthetic lethality effect. Selected hits' activity was validated, and the mechanism of the most potent candidate drug, AKT inhibitor AD5363 (capivasertib), on induction of the synthetic lethality with ARID1A deficiency was investigated. After robust vulnerability screening of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A-/- cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the Caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment.

Inactivated cGAS-STING Signaling Facilitates Endocrine Resistance by Forming a Positive Feedback Loop with AKT Kinase in ER+HER2- Breast Cancer.

Endocrine-resistant ER+HER2- breast cancer (BC) is particularly aggressive and leads to poor clinical outcomes. Effective therapeutic strategies against endocrine-resistant BC remain elusive. Here, analysis of the RNA-sequencing data from ER+HER2- BC patients receiving neoadjuvant endocrine therapy and spatial transcriptomics analysis both show the downregulation of innate immune signaling sensing cytosolic DNA, which primarily occurs in endocrine-resistant BC cells, not immune cells. Indeed, compared with endocrine-sensitive BC cells, the activity of sensing cytosolic DNA through the cGAS-STING pathway is attenuated in endocrine-resistant BC cells. Screening of kinase inhibitor library show that this effect is mainly mediated by hyperactivation of AKT1 kinase, which binds to kinase domain of TBK1, preventing the formation of a trimeric complex TBK1/STING/IRF3. Notably, inactivation of cGAS-STING signaling forms a positive feedback loop with hyperactivated AKT1 to promote endocrine resistance, which is physiologically important and clinically relevant in patients with ER+HER2- BC. Blocking the positive feedback loop using the combination of an AKT1 inhibitor with a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of endocrine-resistant tumors in humanized mice models, providing a potential strategy for treating patients with endocrine-resistant BC.

Screening of a kinase inhibitor library identified novel targetable kinase pathways in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly invasive breast cancer subtype that is challenging to treat due to inherent heterogeneity and absence of estrogen, progesterone, and human epidermal growth factor 2 receptors. Kinase signaling networks drive cancer growth and development, and kinase inhibitors are promising anti-cancer strategies in diverse cancer subtypes. Kinase inhibitor screens are an efficient, valuable means of identifying compounds that suppress cancer cell growth in vitro, facilitating the identification of kinase vulnerabilities to target therapeutically. The Kinase Chemogenomic Set is a well-annotated library of 187 kinase inhibitor compounds that indexes 215 kinases of the 518 in the known human kinome representing various kinase networks and signaling pathways, several of which are understudied. Our screen revealed 14 kinase inhibitor compounds effectively inhibited TNBC cell growth and proliferation. Upon further testing, three compounds, THZ531, THZ1, and PFE-PKIS 29, had the most significant and consistent effects across a range of TNBC cell lines. These cyclin-dependent kinase (CDK)12/CDK13, CDK7, and phosphoinositide 3-kinase inhibitors, respectively, decreased metabolic activity in TNBC cell lines and promote a gene expression profile consistent with the reversal of the epithelial-to-mesenchymal transition, indicating these kinase networks potentially mediate metastatic behavior. These data identified novel kinase targets and kinase signaling pathways that drive metastasis in TNBC.

TRPML1 triggers ferroptosis defense and is a potential therapeutic target in AKT-hyperactivated cancer.

Targeting ferroptosis for cancer therapy has slowed because of an incomplete understanding of ferroptosis mechanisms under specific pathological contexts such as tumorigenesis and cancer treatment. Here, we identify TRPML1-mediated lysosomal exocytosis as a potential anti-ferroptotic process through genome-wide CRISPR-Cas9 activation and kinase inhibitor library screening. AKT directly phosphorylated TRPML1 at Ser343 and inhibited K552 ubiquitination and proteasome degradation of TRPML1, thereby promoting TRPML1 binding to ARL8B to trigger lysosomal exocytosis. This boosted ferroptosis defense of AKT-hyperactivated cancer cells by reducing intracellular ferrous iron and enhancing membrane repair. Correlation analysis and functional analysis revealed that TRPML1-mediated ferroptosis resistance is a previously unrecognized feature of AKT-hyperactivated cancers and is necessary for AKT-driven tumorigenesis and cancer therapeutic resistance. TRPML1 inactivation or blockade of the interaction between TRPML1 and ARL8B inhibited AKT-driven tumorigenesis and cancer therapeutic resistance in vitro and in vivo by promoting ferroptosis. A synthetic peptide targeting TRPML1 inhibited AKT-driven tumorigenesis and enhanced the sensitivity of AKT-hyperactivated tumors to ferroptosis inducers, radiotherapy, and immunotherapy by boosting ferroptosis in vivo. Together, our findings identified TRPML1 as a therapeutic target in AKT-hyperactivated cancer.

4EBP1-mediated SLC7A11 protein synthesis restrains ferroptosis triggered by MEK inhibitors in advanced ovarian cancer.

Loss of ferroptosis contributes to the development of human cancer, and restoration of ferroptosis has been demonstrated as a potential therapeutic strategy in cancer treatment. However, the mechanisms of how ferroptosis escape contributes to ovarian cancer (OV) development are not well elucidated. Here, we show that ferroptosis negative regulation signatures correlated with the tumorigenesis of OV and were associated with poor prognosis, suggesting that restoration of ferroptosis represents a potential therapeutic strategy in OV. High-throughput drug screening with a kinase inhibitor library identified MEK inhibitors as ferroptosis inducers in OV cells. We further demonstrated that MEK inhibitor-resistant OV cells were less vulnerable to trametinib-induced ferroptosis. Mechanistically, mTOR/eIF4E binding protein 1 (4EBP1) signaling promoted solute carrier family 7 member 11 (SLC7A11) protein synthesis, leading to ferroptosis inhibition in MEK inhibitor-resistant cells. Dual inhibition of MEK and mTOR/4EBP1 signaling restrained the protein synthesis of SLC7A11 via suppression of the mTOR/4EBP1 axis to reactivate ferroptosis in resistant cells. Together, these findings provide a promising therapeutic option for OV treatment through ferroptosis restoration by the combined inhibition of MEK and mTOR/4EBP1 pathways.

Identification of a targeted ACSL4 inhibitor to treat ferroptosis-related diseases.

Ferroptosis is a form of iron-dependent, lipid peroxidation-driven regulatory cell death that has been implicated in the pathogenesis of multiple diseases, including organ injury, ischemia/reperfusion, and neurodegenerative diseases. However, inhibitors that directly and specifically target ferroptosis are not yet available. Here, we identify the compound AS-252424 (AS) as a potent ferroptosis inhibitor through kinase inhibitor library screening. Our results show that AS effectively inhibits lipid peroxidation and ferroptosis in both human and mouse cells. Mechanistically, AS directly binds to the glutamine 464 of ACSL4 to inhibit its enzymatic activity, resulting in the suppression of lipid peroxidation and ferroptosis. By using nanoparticle-based delivery systems, treatment with AS-loaded nanoparticles effectively alleviate ferroptosis-mediated organ injury in mouse models, including kidney ischemia/reperfusion injury and acute liver injury (ALI). Thus, our results identify that AS is a specific and targeted inhibitor of ACSL4 with remarkable antiferroptosis function, providing a potential therapeutic for ferroptosis-related diseases.

Discovery, Optimization, and Evaluation of Novel Pyridin-2(1H)-one Analogues as Potent TRK Inhibitors for Cancer Treatment.

Tropomyosin receptor kinase (TRK) fusion, an oncogenic form of kinase with pan-tumor occurrence, is a clinically validated important antitumor target. In this study, we screened our in-house kinase inhibitor library against TRK and identified a promising hit compound 4 with a novel pyridin-2(1H)-one scaffold. Through a combination of structure-based drug design and structure-activity relationship (SAR) study, compound 14q was identified as a potent TRK inhibitor with good kinase selectivity. It also blocked cellular TRK signaling, thereby inhibiting TRK-dependent cell viability. Additionally, 14q displayed acceptable pharmacokinetic properties with 37.8% oral bioavailability in mice. Strong in vivo tumor growth inhibition of 14q was observed in subcutaneous M091 and KM12 tumor xenograft models with TRK fusion, causing significant tumor inhibition or even complete tumor regression.

CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes.

Ferroptosis is iron-dependent oxidative cell death. Labile iron and polyunsaturated fatty acid (PUFA)-containing lipids are two critical factors for ferroptosis execution. Many processes regulating iron homeostasis and lipid synthesis are critically involved in ferroptosis. However, it remains unclear whether biological processes other than iron homeostasis and lipid synthesis are associated with ferroptosis. Using kinase inhibitor library screening, we discovered a small molecule named CGI1746 that potently blocks ferroptosis. Further studies demonstrate that CGI1746 acts through sigma-1 receptor (σ1R), a chaperone primarily located at mitochondria-associated membranes (MAMs), to inhibit ferroptosis. Suppression of σ1R protects mice from cisplatin-induced acute kidney injury hallmarked by ferroptosis. Mechanistically, CGI1746 treatment or genetic disruption of MAMs leads to defective Ca2+ transfer, mitochondrial reactive oxygen species (ROS) production and PUFA-containing triacylglycerol accumulation. Therefore, we propose a critical role for MAMs in ferroptosis execution.

Discovery of pyrazolo[3,4-d]pyrimidines as novel mitogen-activated protein kinase kinase 3 (MKK3) inhibitors

The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure–activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3′s role in various cancers.

Invasion-Block and S-MARVEL: A high-content screening and image analysis platform identifies ATM kinase as a modulator of melanoma invasion and metastasis.

Robust high-throughput assays are crucial for the effective functioning of a drug discovery pipeline. Herein, we report the development of Invasion-Block, an automated high-content screening platform for measuring invadopodia-mediated matrix degradation as a readout for the invasive capacity of cancer cells. Combined with Smoothen-Mask and Reveal, a custom-designed, automated image analysis pipeline, this platform allowed us to evaluate melanoma cell invasion capacity posttreatment with two libraries of compounds comprising 3840 U.S. Food and Drug Administration (FDA)-approved drugs with well-characterized safety and bioavailability profiles in humans as well as a kinase inhibitor library comprising 210 biologically active compounds. We found that Abl/Src, PKC, PI3K, and Ataxia-telangiectasia mutated (ATM) kinase inhibitors significantly reduced melanoma cell invadopodia formation and cell invasion. Abrogation of ATM expression in melanoma cells via CRISPR-mediated gene knockout reduced 3D invasion in vitro as well as spontaneous lymph node metastasis in vivo. Together, this study established a rapid screening assay coupled with a customized image-analysis pipeline for the identification of antimetastatic drugs. Our study implicates that ATM may serve as a potent therapeutic target for the treatment of melanoma cell spread in patients.

Abstract 17407: A Small-molecule Screening Strategy to Promote Cardiomyocyte Proliferation That Protects the Heart From Ischemic Injury

Restoring the ability of adult cardiac myocytes (ACMs) to proliferate and promote heart regeneration have been proposed as a promising therapeutic strategy. We have demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases ACM cell cycle activity, but proliferation was limited. To improve this, we performed a small-molecule screening in combination with KDM4D on neonatal rat cardiomyocytes (NRVMs), using a library of kinase inhibitors targeting different signaling pathways. Our study revealed that compounds targeting the PI3K and JAK signaling pathways significantly potentiated the effects of KDM4D and promote NRVM proliferation in vitro. Specifically, a JAK inhibitor (JAKi2) in combination with Ad-KDM4D treatment resulted in approximately 100%, 34%, and 62% increase in EdU+, phospho-H3+, and total cell number compared to control, respectively. To validate these findings in vivo, we tested JAKi2 in an inducible KDM4D transgenic mouse model (iKDM4D). The animals underwent ischemia/reperfusion (I/R) to induce infarction, and JAKi2 was administrated orally for up to 8 weeks. We found both early and late effects. 48 hours after I/R, the infarct size in iKDM4D+JAKi2 hearts was approximately 4-fold smaller than that in vehicle-treated or JAKi2 alone groups, and the combination increased EdU+, Ki67+, and phospho-H3+ CM number separately by 3.2-fold, 2.9-fold, and 6.5-fold. At 2 weeks post-I/R, the scar size was smaller in iKDM4D+JAKi2 hearts compared to vehicle-treated mice (9.0% vs 1.8%, P <0.05), and the combination resulted in a 3-fold, 5.5-fold, and 8.5-fold increases in EdU+, Ki67+, and phospho-H3+ CMs, respectively. Moreover, the iKDM4D+JAKi2 hearts demonstrated progressive improvement in left ventricular ejection fraction (26.8%, 27.3%, and 25.5%) and fraction shorten (37.9%, 38.1%, and 35.0%) at 2 weeks, 4 weeks, and 8 weeks post-I/R. The increased estimated ACM number and mononucleated CMs at 8 weeks post-I/R support that CM division was increased in the iKDM4D+JAKi2 hearts. These findings suggest that a combination therapy involving JAKi2, along with KDM4D, promotes robust CM proliferation and protects the heart after ischemic injury.

The potential of swine pseudorabies virus attenuated vaccine for oncolytic therapy against malignant tumors

BackgroundOncolytic viruses are now well recognized as potential immunotherapeutic agents against cancer. However, the first FDA-approved oncolytic herpes simplex virus 1 (HSV-1), T-VEC, showed limited benefits in some patients in clinical trials. Thus, the identification of novel oncolytic viruses that can strengthen oncolytic virus therapy is warranted. Here, we identified a live-attenuated swine pseudorabies virus (PRV-LAV) as a promising oncolytic agent with broad-spectrum antitumor activity in vitro and in vivo.MethodsPRV cytotoxicity against tumor cells and normal cells was tested in vitro using a CCK8 cell viability assay. A cell kinase inhibitor library was used to screen for key targets that affect the proliferation of PRV-LAV. The potential therapeutic efficacy of PRV-LAV was tested against syngeneic tumors in immunocompetent mice, and against subcutaneous xenografts of human cancer cell lines in nude mice. Cytometry by time of flight (CyTOF) and flow cytometry were used to uncover the immunological mechanism of PRV-LAV treatment in regulating the tumor immune microenvironment.ResultsThrough various tumor-specific analyses, we show that PRV-LAV infects cancer cells via the NRP1/EGFR signaling pathway, which is commonly overexpressed in cancer. Further, we show that PRV-LAV kills cancer cells by inducing endoplasmic reticulum (ER) stress. Moreover, PRV-LAV is responsible for reprogramming the tumor microenvironment from immunologically naïve (“cold”) to inflamed (“hot”), thereby increasing immune cell infiltration and restoring CD8+ T cell function against cancer. When delivered in combination with immune checkpoint inhibitors (ICIs), the anti-tumor response is augmented, suggestive of synergistic activity.ConclusionsPRV-LAV can infect cancer cells via NRP1/EGFR signaling and induce cancer cells apoptosis via ER stress. PRV-LAV treatment also restores CD8+ T cell function against cancer. The combination of PRV-LAV and immune checkpoint inhibitors has a significant synergistic effect. Overall, these findings point to PRV-LAV as a serious potential candidate for the treatment of NRP1/EGFR pathway-associated tumors.

Identification of tyrphostin AG879 and A9 inhibiting replication of chikungunya virus by screening of a kinase inhibitor library

Chikungunya virus (CHIKV) is a globally public health threat. There are currently no medications available to treat CHIKV infection. High-throughput screening of 419 kinase inhibitors was performed based on the cytopathic effect method, and six kinase inhibitors with reduced cytopathic effects, including tyrphostin AG879 (AG879), tyrphostin 9 (A9), sorafenib, sorafenib tosylate, regorafenib, and TAK-632, were identified. The anti-CHIKV activities of two receptor tyrosine kinase inhibitors, AG879 and A9, that have not been previously reported, were selected for further evaluation. The results indicated that 50% cytotoxic concentration (CC50) of AG879 and A9 in Vero cells were greater than 30 μM and 6.50 μM, respectively and 50% effective concentration (EC50) were 0.84 μM and 0.36 μM, respectively. The time-of-addition and time-of-removal assays illustrated that both AG879 and A9 function in the middle stage of CHIKV life cycle. Further, AG879 and A9 do not affect viral attachment; however, they inhibit viral RNA replication, and exhibit antiviral activity against CHIKV Eastern/Central/South African and Asian strains, Ross River virus and Sindbis virus in vitro.

Selective ROCK Inhibitor Enhances Blood Flow Recovery after Hindlimb Ischemia.

The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. Screening of 157 kinase inhibitors in wild-type (n=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (n=24 HCM and n=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (n=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

Phenotypic screening in Organ-on-a-Chip systems: a 1537 kinase inhibitor library screen on a 3D angiogenesis assay.

Modern drug development increasingly requires comprehensive models that can be utilized in the earliest stages of compound and target discovery. Here we report a phenotypic screening exercise in a high-throughput Organ-on-a-Chip setup. We assessed the inhibitory effect of 1537 protein kinase inhibitors in an angiogenesis assay. Over 4000 micro-vessels were grown under perfusion flow in microfluidic chips, exposed to a cocktail of pro-angiogenic factors and subsequently exposed to the respective kinase inhibitors. Efficacy of compounds was evaluated by reduced angiogenic sprouting, whereas reduced integrity of the main micro-vessel was taken as a measure for toxicity. The screen yielded 53 hits with high anti-angiogenicity and low toxicity, of which 44 were previously unassociated with angiogenic pathways. This study demonstrates that Organ-on-a-Chip models can be screened in high numbers to identify novel compounds and targets. This will ultimately reduce bias in early-stage drug development and increases probability to identify first in class compounds and targets for today's intractable diseases.

Identification of tau-tubulin kinase 1 inhibitors by microfluidics-based mobility shift assay from a kinase inhibitor library

Tau tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that phosphorylates multiple residues in tau protein. Hyperphosphorylated tau is the main cause of tauopathy, such as Alzheimer's disease (AD). Therefore, preventing tau phosphorylation by inhibiting TTBK1 has been proposed as a therapeutic strategy for AD. However, few substrates of TTBK1 are reported for a biochemical assay and few inhibitors targeting TTBK1 have been reported so far. In this study, we identified a fluorescein amidite (FAM)-labeled peptide 15 from a small peptide library as the optimal peptide substrate for human TTBK1 (hTTBK1). We then developed and validated a microfluidics-based mobility shift assay (MMSA) with peptide 15. We further confirmed that peptide 15 could also be used in the ADP-Glo kinase assay. The established MMSA was applied for screening of a 427-compound kinase inhibitor library, yielding five compounds with IC50s of several micro molars against hTTBK1. Among them, three compounds, AZD5363, A-674,563 and GSK690693 inhibited hTTBK1 in an ATP competitive manner and molecular docking simulations revealed that they enter the ATP pocket and form one or two hydrogen bonds to the hinge region with hTTBK1. Another hit compound, piceatannol, showed non-ATP competitive inhibitory effect on hTTBK1 and may serve as a starting point to develop highly selective hTTBK1 inhibitors. Altogether, this study provided a new in vitro platform for the development of novel hTTBK1 inhibitors that might have potential applications in AD prevention.

GSK3β-driven SOX2 overexpression is a targetable vulnerability in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest forms of human malignancy that currently lacks approved targeted therapeutics. Accumulating evidence suggests that SOX2 overexpression is a key driving factor for ESCC and various squamous cell carcinoma. Here, through screening a small-molecule kinase inhibitor library, we identified GSK3β as a kinase that is critically required for robust SOX2 expression in ESCC cells. GSK3β did not promote SOX2 transcriptionally but was required for SOX2 protein stability. We demonstrated that GSK3β interacts with and phosphorylates SOX2 at residue S251, which blocks SOX2 from ubiquitination and proteasome-dependent degradation instigated by ubiquitin E3 ligase CUL4ADET1-COP1. Pharmacological inhibition or knockdown of GSK3β by RNA interference selectively impaired SOX2-positive ESCC cell proliferation, cancer stemness, and tumor growth in mouse xenograft model, suggesting that GSK3β promotes ESCC tumorigenesis primarily by driving SOX2 overexpression. GSK3β was found to be frequently overexpressed in clinical esophageal tumors, and there was a positive correlation between GSK3β and SOX2 protein levels. Notably, we found that SOX2 enhanced GSK3β expression transcriptionally, suggesting the existence of a vicious cycle that drives a coordinated GSK3β and SOX2 overexpression in ESCC cells. Finally, we demonstrated in tumor xenograft model that GSK3β inhibitor AR-A014418 was effective in suppressing SOX2-positive ESCC tumor progression and inhibited tumor progression cooperatively with chemotherapeutic agent carboplatin. In conclusion, we uncovered a novel role for GSK3β in driving SOX2 overexpression and tumorigenesis and provided evidence that targeting GSK3β may hold promise for the treatment of recalcitrant ESCCs.