Activin receptor-like kinase 2 (ACVR1/ALK2) regulates bone morphogenetic protein signaling, and ALK2 modulation has been identified as a promising therapeutic strategy for conditions including fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG), and glioblastoma. Herein, we report on the development of first-in-class ALK2 degraders, including M4K3233 (13), a potent and selective compound that was utilized as a chemical tool to study the mechanism of ALK2 degradation. Subsequent optimization of this compound resulted in M4K3250 (20), a compound with improved ALK2 degradation potency. The compounds described have utility for studying the role of ALK2 in human disease and possess translational potential in drug discovery.

Chemically distinct PIKfyve inhibitors block proliferation and autophagy in PDAC cells

Combined inhibition of PIKfyve and RAS or MEK durably represses PDAC cell proliferation

A pivotal part of kinase chemical probe and drug development is assessment of the selectivity of a putative lead compound. While there is no consensus around the panel size or the type of assay(s) that are most appropriate, there is concurrence that gauging the number of on- and off-targets of a kinase inhibitor is essential. As pharmacology takes place in cells, we have compared profiling results for ten kinase inhibitors generated using the cell-free assays to those obtained when a panel of cellular target engagement NanoBRET assays is used to assess selectivity in intact cells. This is the first systematic comparison of these two approaches across a broad kinase panel. Comparison of the data sets demonstrates divergent results that can influence chemical probe prioritization. We identify unanticipated kinase interactions in cells for type II kinase inhibitors that are not observed in biochemical, cell-free systems. Furthermore, we characterize TPKI-39 as a DDR1, DDR2, and FLT1 chemical probe based on its in-cell selectivity profile.

Objectives/Goals: The Never-in-mitosis A-related (NEK) kinases are a family of 11 poorly explored kinases that play varying roles in cellular processes. Aberrant NEK activity is implicated in various human diseases, including cancer. We hypothesize that NEK9 drives triple-negative breast cancer (TNBC) motility. Methods/Study Population: Baseline NEK9 expression was determined across five breast cancer and three non-breast cancer cell lines by immunoblot. We expanded the analysis to six additional TNBC cell lines. To determine the impact of NEK9 on TNBC biology, we evaluated the effects of NEK9 pharmacological inhibition with novel inhibitor BA 03-53-11 (BA) at 0µM, 0.5µM, 1µM, and 2µM. We treated TNBC cell lines MDA-MB-231, BT-549, HS578T, and TNBC patient-derived cell line TUBcX-4IC and performed functional assays to assess impacts on cell motility (scratch assay), viability (crystal violet staining), and proliferation (Ki-67 staining). Results/Anticipated Results: NEK9 levels were elevated in the TNBC cell line compared to other breast cancer subtypes, osteosarcoma, and non-cancerous cells. Additionally, NEK9 protein expression was variable across TNBC cell lines. While pharmacological NEK9 inhibition did not significantly alter TNBC cell viability or proliferation, we observed that NEK9 inhibition suppressed cell motility in TNBC cells compared to vehicle control. Taken together, these preliminary results support a role for NEK9 in regulating TNBC progression. Our ongoing studies aim to test NEK9-mediated motility and proliferation in additional TNBC cell lines and evaluate NEK9 as a therapeutic target for this aggressive breast cancer subtype. Discussion/Significance of Impact: TNBC is an aggressive form of breast cancer that lacks targeted therapies. Because aberrant kinase activity underpins oncogenic transformation, kinase inhibitors have emerged as ideal therapeutic agents. Elucidation of NEK9 function in TNBC is essential for the development of novel therapeutics.

MEK inhibition synergizes with PIKfyvei to impair proliferation of PDAC cell lines

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. Inhibition of the KRAS-RAF-MEK-ERK pathway enhances autophagic flux and dependency, and concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK-MAPK pathway inhibitors can synergistically block PDAC growth. However, CQ is limited in terms of specificity and potency. To find alternative anti-autophagy strategies, in this study, we performed a CRISPR-Cas9 loss-of-function screen in PDAC cell lines that identified the lipid kinase phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) as a growth-promoting gene. PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition caused lysosomal dysfunction, reduced autophagic flux, and led to the accumulation of autophagy-related proteins. Furthermore, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and the RAS-MAPK pathway showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell-cycle arrest and induction of apoptosis following loss of inhibitor of apoptosis proteins. These findings indicate that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a therapeutic strategy for PDAC.Significance: PIKfyve inhibition effectively blocks autophagy in multiple models of KRAS-mutant pancreatic cancer and can synergize with inhibitors of members of the RAS-MAPK pathway, providing an effective combination strategy for pancreatic 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.

Objectives/Goals: The never in mitosis kinase (NEK) family regulates vital processes, namely cell cycle progression, but their potential as therapeutic targets in TNBC has not been fully explored. Our studies aim to develop a toolkit to investigate the functional roles of NEKs in pathologies including carcinogenesis. Methods/Study Population: To assess differential NEK expression in normal and tumor tissues and correlation of gene expression with patient survival, we used Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier Plotter (KMPlot) pan-cancer analysis, respectively. Basal NEK protein levels were determined by immunoblot across a panel of cell lines, including breast cancer, osteosarcoma, hepatocellular carcinoma, and non-cancerous cells, to identify appropriate systems for evaluation of NEK function. Doxycycline-inducible cell lines were generated by transduction with lentiviral stocks of NEK shRNA and overexpression constructs and antibiotic selection. Expression was analyzed by qPCR and immunoblot. Results/Anticipated Results: Expression of NEK2, 4, 5, 6, 8, and 11 was higher in breast tumors compared to normal tissue by GEPIA analysis. Further examination using KMPlot showed a correlation between elevated NEK6 expression and decreased overall survival in patients with aggressive cancers. As an initial proof-of-concept study, we analyzed NEK6 protein expression in breast cancer cells. Levels of NEK6 were elevated in TNBC cells (MDA-MB-231) compared to hormone receptor positive (HR+) breast cancer cells (MCF7). Using complementary approaches to investigate the functional role of NEK6 in breast cancer, we depleted NEK6 expression using shRNAs in TNBC cells and expressed NEK6 in HR+ cells Discussion/Significance of Impact: Because kinase dysregulation promotes oncogenesis and metastasis, targeting kinases is a key strategy in therapeutic development. A NEK-specific molecular toolkit allows researchers to elucidate NEK functions and contributions to carcinogenesis, promoting advancement of novel therapies.

The Never-in-Mitosis A-Related Kinase (NEK) family is an important, yet largely understudied, family of protein kinases involved in the regulation of a variety of critical cellular processes. Consequently, dysregulation of NEK function has been linked to the etiology and progression of several disorders, including cancer, ciliopathies, neurodegenerative disorders, inflammatory disorders, and other pervasive diseases. In this review, we have summarized recent findings to provide an overview of the NEK family and their diverse functions within various cellular contexts. In parallel, we have highlighted the emerging roles of NEK family members in human health, identifying potential therapeutic targets within the NEK family and exploring their potential for future clinical applications. Finally, we have addressed ongoing challenges and emerging research directions in this rapidly evolving field, aiming to pave the way for future discoveries and innovations.

Directional cell migration by pulmonary arterial cells (PACs) is one of the important features of diseases involving arterial remodeling, such as pulmonary arterial hypertension (PAH), a disease that is often characterized by reduced arterial compliance and increased extracellular matrix (ECM) stiffening. However, there are no therapeutics that can halt the directional cell migration of PACs in PAH. The inability to identify drug targets or drugs against the directional cell migration during PAH pathogenesis stems from an incomplete understanding of the process and a lack of effective translational models for screening of candidate small molecules. Here, for the first time, we introduce a bioengineered platform suitable for screening small molecule inhibitors targeting kinase pathways that are potentially linked to ECM-mediated directed cell migration in PAH. We used a photolithographic technique to develop mechanically patterned hydrogels with alternative stripes of soft and stiff bars representing the alternating stiffness regions of PAH ECM. Employing our bioengineered platform, we demonstrated the directional cell migration capacity of PACs and found that PAH-smooth muscle cells (SMCs) showed the highest ability to migrate from soft-stiff regions. Screening of different kinase inhibitors identified the role of JAK/STAT as a mechanosensor in the PAH-SMC-specific directional cell migration. Our study highlighted the use of a mechanically patterned bioengineering platform to identify new drug targets specific to the machinery involved in directional cell migration in PAH.

Candida albicans is a growing global health threat, causing 1.5 million invasive infections and 1 million deaths annually. Yeast casein kinase 2 (Yck2) in C. albicans has emerged as an antifungal target of the kinase inhibitor LY364947 (LY). Herein, we report Yck2 structure-activity relationships for 3,4- and 3,4,5-substituted pyrazole analogs of LY. X-ray crystallography and in vitro profiling revealed the importance of the hinge-binding heterocycle for Yck2 inhibition and fungal kinome selectivity. A hydrogen-bond network between the inhibitor, a bound water molecule, and catalytic residues within the ATP pocket was identified as a key determinant of selectivity over other fungal and human kinases. Phenol analog 11 showed remarkable selectivity for Yck2 and Yck22 over all other C. albicans protein kinases. Several of the LY analogs, including 11, demonstrated improved antifungal activity. These findings provide a framework for translating human kinase inhibitors into highly selective antifungal Yck2 inhibitors.

The novel (nua) kinases 1 and 2 are two of 12 AMP-activated protein-related kinases whose signaling pathways are involved in cancer progression, as well as neurologic, fibrotic, and inflammatory diseases. Currently, there are 80 Food and Drug Administration-approved kinase inhibitors which target roughly 24 of the 500+ known human kinases, leaving most kinases underexplored, including NUAK1 and NUAK2. Thus, there is a critical need for selective inhibition of NUAK1 and NUAK2 signaling. Here, we review the protein structure, known upstream regulators and downstream targets, and expression profiles of NUAK1 and NUAK2 in cancerous compared to noncancerous tissue. We also delineate the biological roles and signaling pathways of the NUAK kinases in a range of malignancies, focusing on cancer but also covering noncancerous physiology, and the therapeutic potential of NUAK kinase inhibition. We summarize the known small-molecule NUAK kinase inhibitors in preclinical models and one inhibitor in clinical trials. This review highlights the signaling mechanisms and therapeutic value of targeting NUAK kinase signaling pathways with specific, small-molecule NUAK inhibitors.

Candida albicans is a major cause of systemic candidiasis, a severe fungal infection with a ∼40% mortality rate. Yck2, a casein kinase 1 (CK1) in C. albicans, is targeted by antifungal inhibitors YK-I-02 (YK) and MN-I-157 (MN). Using multiplexed inhibitor beads and mass spectrometry (MIB/MS), the selectivity of these inhibitors was determined across the fungal kinome. The MIB matrix captured 89% of C. albicans protein kinases, revealing that YK and MN selectively engage three CK1 homologues (Yck2, Yck22, and Hrr25) and a human p38α homologue (Hog1). Chemoproteomics using a custom MN-kinobead confirmed the remarkable fungal kinome selectivity. To identify new Yck2 inhibitors with selectivity over Hog1, 13 human CK1 inhibitors were screened, leading to the discovery of a new chemotype with antifungal activity. These findings highlight the utility of MIB/MS in profiling nonhuman kinomes and developing selective fungal kinase inhibitors as antimicrobial agents.

The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against β-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-a]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.

Cilia are cellular signaling hubs. Given that human kinases are central regulators of signaling, it is not surprising that kinases are key players in cilia biology. In fact, many kinases modulate ciliogenesis, which is the generation of cilia, and distinct ciliary pathways. Several of these kinases are understudied with few publications dedicated to the interrogation of their function. Recent efforts to develop chemical probes for members of the cyclin-dependent kinase like (CDKL), never in mitosis gene A (NIMA) related kinase (NEK), and tau tubulin kinase (TTBK) families either have delivered or are working toward delivery of high-quality chemical tools to characterize the roles that specific kinases play in ciliary processes. A better understanding of ciliary kinases may shed light on whether modulation of these targets will slow or halt disease onset or progression. For example, both understudied human kinases and some that are more well-studied play important ciliary roles in neurons and have been implicated in neurodevelopmental, neurodegenerative, and other neurological diseases. Similarly, subsets of human ciliary kinases are associated with cancer and oncological pathways. Finally, a group of genetic disorders characterized by defects in cilia called ciliopathies have associated gene mutations that impact kinase activity and function. This review highlights both progress related to the understanding of ciliary kinases as well as in chemical inhibitor development for a subset of these kinases. We emphasize known roles of ciliary kinases in diseases of the brain and malignancies and focus on a subset of poorly characterized kinases that regulate ciliary biology.