Small-molecule Kinase Inhibitors Research Articles

The TNFSF12/TWEAK Modulates Colonic Inflammatory Fibroblast Differentiation and Promotes Fibroblast-Monocyte Interactions.

Fibroblasts acquire a proinflammatory phenotype in inflammatory bowel disease, but the factors driving this process and how fibroblasts contribute to mucosal immune responses are incompletely understood. TNF superfamily member 12 (TNFSF12, or TNF-like weak inducer of apoptosis [TWEAK]) has gained interest as a mediator of chronic inflammation. In this study, we explore its role as a driver of inflammatory responses in fibroblasts and its contribution to fibroblast-monocyte interaction using human primary colonic fibroblasts, THP-1 and primary monocytes. Recombinant human TWEAK induced the expression of cytokines, chemokines, and immune receptors in primary colonic fibroblasts. The TWEAK upregulated transcriptome shared 29% homology with a previously published transcriptional profile of inflammatory fibroblasts from ulcerative colitis. TWEAK elevated surface expression of activated fibroblast markers and adhesion molecules (podoplanin [PDPN], ICAM-1, and VCAM-1) and secretion of IL-6, CCL2, and CXCL10. In coculture, fibroblasts induced monocyte adhesion and secretion of CXCL1 and IL-8, and they promoted a CD14high/ICAM-1high phenotype in THP-1 cells, which was enhanced when fibroblasts were prestimulated with TWEAK. Primary monocytes in coculture with TWEAK-treated fibroblasts had altered surface expression of CD16 and triggering receptor expressed on myeloid cells-1 (TREM-1) as well as increased CXCL1 and CXCL10 secretion. Conversely, inhibition of the noncanonical NF-κB pathway on colonic fibroblasts with a NF-κB-inducing kinase small molecule inhibitor impaired their ability to induce a CD14high phenotype on monocytes. Our results indicate that TWEAK promotes an inflammatory fibroblast-monocyte crosstalk that may be amenable for therapeutic intervention.

Abstract PO3-27-02: A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells

Abstract Background. PTK6/Brk, a non-receptor tyrosine kinase, is an oncogenic driver for several tumor types, including breast, prostate, liver, and pancreatic cancers. PTK6 is expressed in all breast cancer subtypes and higher levels of expression are associated with worse patient outcomes. The optimal approach to targeting PTK6 for therapeutic benefit has not been established. Although several inhibitors of PTK6 kinase activity have been developed, none have thus far been successfully translated to clinical trials. This may be in part due to kinase activity-independent functions of PTK6, for which there is growing evidence, that are not effectively targeted by traditional small molecule kinase inhibitors. Downregulation of PTK6 using shRNA inhibits growth, invasion/migration, survival and metastasis of breast cancer cells, including those that are resistant to standard therapies. PTK6 kinase inhibitor treatment phenocopies effects of PTK6 downregulation with respect to some, but not all PTK6-driven oncogenic activities. Moreover, growth of PTK6 CRISPR-targeted cells can be rescued either with wild type or catalytically inactive PTK6, supporting kinase activity-independent activities of PTK6 in regulating growth and viability of breast cancer cells. Therefore, PROTAC degraders may be a better way to chemically target and downregulate PTK6 expression in cancer, thereby phenocopying the effects of PTK6 shRNA. Methods. We developed a first-in-class PTK6 degrader using PROTAC (proteolysis-targeting chimeric) technology to target PTK6 expression in breast cancer cells of diverse subtypes. Degrader controls were also synthesized to assess specificity of MS105’s effects. Results. PTK6 degrader, but not degrader controls, potently downregulates PTK6 expression in multiple breast cancer cell types. Proteomic and kinome profiling confirm the specificity of our PTK6 degrader. PTK6 PROTAC degrader inhibits viability and induces apoptosis of PTK6-expressing breast cancer cells, whereas treatment with parent kinase inhibitor or degrader controls that do not engage PTK6 or E3 ligase have minimal effect. Treatment with PTK6 degrader, but not kinase inhibitor or degrader controls, induces expression of pro-apoptotic Bim, phenocopying the effects observed with PTK6 shRNA. In contrast, PTK6 degrader and kinase inhibitor are comparably effective in inhibiting cell migration, supporting the differential kinase dependency of PTK6-dependent oncogenic functions. Therefore, our PTK6 PROTAC degrader effectively inhibits both kinase-dependent and independent oncogenic activities of PTK6. Our studies support the development of PTK6 PROTAC degraders as a preferred approach to clinically targeting PTK6 in cancer. Citation Format: Hanna Irie, Criseyda Martinez, Yan Xiong, Alison Bartkowski, Jessica Byerly, Jian Jin. A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-27-02.

Unravelling the Design and Mechanism: Potential of Substituted Quinolines as PASTA subunit 4 Inhibitors for Antimicrobial Activity

AbstractThe increasing incidences of antibiotic‐resistance in Microorganisms have become a critical global health challenge. Traditional treatments, often relying on multiple antibiotics, are plagued by issues like drug resistance, reduced effectiveness, and heightened toxicity. In response, there is a compelling demand for innovative anti‐microbial agents that offer novel mechanisms of action. The family of bacterial Penicillin‐binding protein And Serine/Threonine kinase‐Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to ‐lactam antibiotics. As such, small molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to ‐lactam therapy. Our study was primarily geared towards identifying novel PASTA Kinase inhibitors through a ligand‐based drug design approach. Subsequently, we curated a library of 12 molecules, incorporating chemical modifications guided by considerations like drug‐like properties, chemical accessibility, and synthetic feasibility. Molecular docking analyses conducted on this library pinpointed three molecules with noteworthy binding affinities. From this subset, KS_QD_04 and KS_QD_05 emerged as promising candidates, subsequently validated through MD simulation studies, bolstering their potential as lead compounds in the quest to develop PASTA Kinase inhibitors for combating raising microbial resistance towards antibiotics.

Pegylated-liposomes increase the efficacy of Idelalisib in lymphoma B-cells

New drugs and technologies are continuously developed to improve the efficacy and minimize the critical side effects of cancer treatments. The present investigation focuses on the development of a liposomal formulation for Idelalisib, a small-molecule kinase inhibitor approved for the treatment of lymphoid malignancies. Idelalisib is a potent and selective antitumor agent, but it is not indicated nor recommended for first-line treatment due to fatal and serious toxicities. Herein, liposomes are proposed as a delivery tool to improve the therapeutic profile of Idelalisib. Specifically, PEGylated liposomes were prepared, and their physicochemical and technological features were investigated. Light-scattering spectroscopy and cryo-transmission electron microscopy revealed nanosized unilamellar vesicles, which were proved to be stable in storage and in simulated biological fluids.The cytotoxicity of the liposome formulation was investigated in a human non-Hodgkin's lymphoma B cell line. Idelalisib was able to induce death of tumor cells if delivered by the nanocarrier system at increased efficacy. These findings suggest that combining Idelalisib and nanotechnologies may be a powerful strategy to increase the antitumor efficacy of the drug.

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

Meta-learning-based Inductive logistic matrix completion for prediction of kinase inhibitors

Protein kinases become an important source of potential drug targets. Developing new, efficient, and safe small-molecule kinase inhibitors has become an important topic in the field of drug research and development. In contrast with traditional wet experiments which are time-consuming and expensive, machine learning-based approaches for predicting small molecule inhibitors for protein kinases are time-saving and cost-effective, which are highly desired for us. However, the issue of sample scarcity (known active and inactive compounds are usually limited for most kinases) poses a challenge to the research and development of machine learning-based kinase inhibitors' active prediction methods. To alleviate the data scarcity problem in the prediction of kinase inhibitors, in this study, we present a novel Meta-learning-based inductive logistic matrix completion method for the Prediction of Kinase Inhibitors (MetaILMC). MetaILMC adopts a meta-learning framework to learn a well-generalized model from tasks with sufficient samples, which can fast adapt to new tasks with limited samples. As MetaILMC allows the effective transfer of the prior knowledge learned from kinases with sufficient samples to kinases with a small number of samples, the proposed model can produce accurate predictions for kinases with limited data. Experimental results show that MetaILMC has excellent performance for prediction tasks of kinases with few-shot samples and is significantly superior to the state-of-the-art multi-task learning in terms of AUC, AUPR, etc., various performance metrics. Case studies also provided for two drugs to predict Kinase Inhibitory scores, further validating the proposed method's effectiveness and feasibility.Scientific contributionConsidering the potential correlation between activity prediction tasks for different kinases, we propose a novel meta learning algorithm MetaILMC, which learns a prior of strong generalization capacity during meta-training from the tasks with sufficient training samples, such that it can be easily and quickly adapted to the new tasks of the kinase with scarce data during meta-testing. Thus, MetaILMC can effectively alleviate the data scarcity problem in the prediction of kinase inhibitors.

A highly selective KIT inhibitor MOD000001 suppresses IgE-mediated mast cell activation

BackgroundThe KIT receptor tyrosine kinase and its ligand, stem cell factor (SCF) control proliferation and survival of mast cells. Thus, targeting KIT signaling may show promise for the treatment of allergic diseases involving mast cells. Recently, we discovered a new compound MOD000001 as a potential small-molecule KIT kinase inhibitor by using an in silico approach. ObjectiveThis study determined whether MOD000001 is highly-selective to KIT, inhibits KIT signaling in mast cells, and affects IgE-mediated mast cell activation. MethodsThe interaction of MOD000001 with 468 human kinases and its inhibitory activity against KIT were profiled and evaluated by using KinomeScan and cell-free kinase assays, respectively. The effects of MOD000001 on SCF-dependent signaling were examined by using primary mouse and human mast cells. The effects of MOD000001 on SCF-induced degranulation and passive cutaneous anaphylactic (PCA) reaction were examined in mice. ResultsMOD000001 interacted with KIT and inhibited KIT kinase activity with high selectivity. MOD000001 suppressed SCF-induced KIT signaling in mouse and human mast cells and in mice. PCA reaction was suppressed in mice treated with MOD000001 both for a short-term (1 week) and a long-term (7 weeks). Mice treated with MOD000001 for a long-term, but not for a short-term, showed skin mast cell reduction. ConclusionsMOD000001 is a highly selective KIT inhibitor that can suppress IgE-mediated mast cell activation in vivo. MOD000001 may do so by reducing tissue mast cell numbers or by other unknown mechanisms. The findings suggest potential benefits of MOD000001 for allergic diseases involving IgE-mediated mast cell activation.

NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation invitro and invivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration invitro and invivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Abstract 608: NEOS-223 is a small molecule kinase inhibitor which induces apoptosis in selected cancer models

Abstract Due to their critical role in cancer signaling pathways, members of the kinase family have emerged as one of the most comprehensively pursued targets in pharmacological cancer research. Deregulation of kinase activities leads to a variety of upstream and downstream signaling pathway changes in cancer cell. The Ras/Raf/MEK/ERK and PI3K/Akt/mTOR cascades are activated by genetic alterations in selected upstream signaling molecules such as receptor tyrosine kinases. Recently developed therapies which target such kinases (EGFR, BRAF, MEK) with kinase inhibitors have notably improved the prognosis within various cancer patient groups. However, patients with different mutations of EGFR, RAS, and BRAF do not respond completely. In this study the potential of NEOS-223 as a new kinase-targeted therapy in selected cancer models was investigated. Multiple cancer cell lines were assessed for cell growth as well as the direct inhibitory effects of NEOS-223 alone and in combination with other clinically approved kinase inhibitors. NEOS-223 treatment caused cell growth inhibition, blocked colony growth, and induced apoptosis in lung, pancreatic, melanoma, and colon cancer cell lines in vitro and demonstrated potent antitumor activity in in vivo xenograft models of colon and lung cancer. We were particularly interested in examining different components of the ERK and PI3K signaling pathways in selected sensitive cell lines, it was identified that NEOS-223 treatment led to downregulation of the expression of pERK and pAKT signaling. Blockage of the PI3K/mTOR pathways showed inhibitory synergy with NEOS-223 in lung cancer in vitro. Most importantly, in vivo data showed that while administration of NEOS-223 alone decreased tumor growth, the addition of PI3K/Akt/mTOR inhibitor onatasertib significantly enhanced anticancer effect in a lung cancer model. In lung and pancreatic cancer models, inhibition of EGFR by the small molecule drug gefitinib strongly showed synergistic effect with NEOS-223 both in vitro and in vivo. This may be because EGFR potentially activates both the mitogen activated protein kinase (MAPK/ERK) and the PI3K/mTOR signaling cascades in different mutant cancer models. These studies reveal that NEOS-223 inhibits cell proliferation and induces apoptosis via suppressing the PI3K/Akt/mTOR pathway and further suggest that the combination of PI3K/Akt/mTOR and EGFR inhibitors and that NEOS-223 would be a strong potential chemotherapeutic strategy against lung and pancreatic cancers. Citation Format: Harish Potu, Sara A. Little, Stephanie Fedorchak, Sumathi Chittamuru, Timothy M Murphy, Roseanne Wexler, Sarah Di Croce, Melanie George, Laxman S. Desai. NEOS-223 is a small molecule kinase inhibitor which induces apoptosis in selected cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 608.

Abstract 603: BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models

Abstract Introduction: Immuno-suppressive micro-environment and abnormal signaling pathways are crucial for the proliferation of tumor cells. Hematopoietic progenitor kinase 1 (HPK1) inhibitor plays important roles in activation of exhausted T cells and enhancement of anti-tumor immune responses. VEGF/VEGFR2 pathway is the central therapeutic target in anti-angiogenic treatment in multiple cancers. Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family, and is reported to be essential for Wnt signaling and colorectal cancer (CRC) proliferation and progression. Blockade of critical kinases in multiple signaling pathways inhibits the proliferation, invasion and metastasis of cancer cells. Therefore, intergradation of these functions in a small molecule serves as the next generation of immune-oncology small molecules for cancer therapy. Methods: BB5523 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. CDX and syngeneic tumor models in mice were conducted to evaluate the in vivo efficacy of BB5523 as a single agent. Results: BB5523 shows high inhibition potency on HPK1 with good selectivity against other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 kinase activity (IC50 = 3.3 nM) by BB5523 strongly suppresses the phosphorylation of the downstream biomarker protein SLP-76 (IC50 = 30 nM); and boosts the IL-2 secretion in purified human T cells and human peripheral blood mononuclear cells. BB5523 also shows good activities on VEGFR2 (IC50 = 1.6 nM) and TNIK (IC50 = 0.7 nM) which are crucial for the proliferation of cancer cells. In in vitro experiments, BB5523 significantly inhibits the proliferation of various tumor cell lines at sub-micromolar concentrations. PK profile of BB5523 shows good oral bioavailability (F > 80%). In preliminary safety evaluation, no abnormal symptoms were observed in mice dosed up to 100 mpk for 14 days. BB5523 showed significant tumor-growth inhibition efficacy in TE-8, JIMT-1, B16-F10 and MC38 tumor models. Conclusion: As a potent and oral available small molecule kinase inhibitor, BB5523 boosts anti-tumor immunity through HPK1 inhibition, while also blocking crucial signaling pathways in tumor micro-environment. BB5523 shows promising in vitro and in vivo efficacy with a well-tolerated safety profile. The preclinical study of BB5523 is in progress. Citation Format: Gongping Duan, Min Li, Puyong Zhang, Wei Hua, Enlun Hao, Shipeng Wang, Xiaoyi Ma, Junheng Wang, Lijie Wei, Xingmin Zhang. BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 603.

Abstract 648: Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays

Abstract Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases. A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays. Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay. Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use. However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates. Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development. We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1. We compared the potency of a selection of WEE1 inhibitors when different substrates were used. While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly. Citation Format: Daniel G. Mueller, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kraemer, Thomas Weber, Michael H. Kubbutat. Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 648.

Abstract 4029: HPK1 inhibitor reinvigorates exhausted tumor-infiltrating CD8 T cells and synergizes with anti-PD-1 blockade in gynecologic malignancies

Abstract HPK1 (Hematopoietic Progenitor Kinase 1) is a negative intracellular immune checkpoint that interferes with the priming and activation of T cells. In recent years, several HPK1 inhibitors have been discovered as novel immune-oncology drugs with promising potential to improve anti-tumor immune responses. However, there has been no study showing the effects and potential application of HPK1 in gynecologic malignancies. Therefore, we investigated the expression and role of HPK1 in tumor-infiltrating CD8 T cells (CD8 TILs) and how the selective small molecule kinase inhibitor, FB849, affected the reinvigoration of exhausted CD8 TILs in gynecologic cancers. We isolated TILs from patients with newly diagnosed endometrial (n=53) and ovarian cancer (n=46). The immunological properties and HPK1 signaling pathway of CD8 TILs were explored using flow cytometry. TILs were in vitro stimulated with anti-CD3 in the presence of FB849 and/or anti-PD-1, and their proliferation was assessed. We observed that HPK1 marked severely exhausted PD-1+CD8 TILs, and high HPK1 expressers had more exhausted PD-1+CD8 TILs compared to low HPK1 expressers. In addition, FB849 could successfully restore the effector function of exhausted CD8 TILs. In particular, the reinvigorating capacity of FB849 was negatively correlated with phosphorylated SLP76+PD-1+CD8 TILs in endometrial cancer. In addition, FB849 further enhanced anti-PD-1-mediated reinvigoration of CD8 TILs in endometrial cancer. However, there was no synergistic effect of FB849 + anti-PD-1 in terms of proliferative capacity in ovarian cancer. Overall, the results of our present study provide a rationale for clinical trials investigating the anti-tumor efficacy of FB849 ± anti-PD-1 tailored to the respective properties of gynecologic malignancies. Citation Format: Junsik Park, Yong Jae Lee, Sunghoon Kim, Seungmook Lim, A Yeong Park, Jamie Jae Eun Kim, Jinhwa Lee, Sang Wun Kim, Su-Hyung Park, Jung-Yun Lee. HPK1 inhibitor reinvigorates exhausted tumor-infiltrating CD8 T cells and synergizes with anti-PD-1 blockade in gynecologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4029.

Abstract 616: Integrating organotypic tissue slices and polypharmacology-based screening to map the toxicity landscape of kinase inhibitors

Abstract Protein kinases play important role in the regulation of various biological processes, and their malfunction and dysregulation are intricately linked to the initiation and progression of cancer. The effectiveness of small-molecule kinase inhibitors (KIs) has been well-established in the therapeutic landscape, with the FDA approving over 70 KIs for the treatment of cancer and other diseases. However, the widespread utilization of KIs has been accompanied by reported adverse events, prominently featuring cardiac complications, alongside hepatic and renal function. While many studies have indicated on-target and off-target kinase specificity associated with organ damage, there is an urgent need for building predictive models that can pinpoint which specific kinases might be involved in different types of organ injuries. Such a model would not only facilitate mechanistic elucidation but also serve as a valuable tool for preemptively identifying potential adverse events associated with KI treatment. In this study, we performed polypharmacology-based screening of kinase inhibitors in organotypic tissue slices prepared from the normal heart, liver, and kidney of three distinct species—human, canine, and rodents. These tissue samples were treated individually using a curated selection of 32 KIs that are representative of a larger set of KIs and collectively target most of the 'kinome.' Subsequently, we developed Elastic Net Regularization models by utilizing the kinase inhibition profiles of ~300 kinases. These models were used to predict changes in tissue viability, measured as a percentage change, following the treatment with the respective kinase inhibitors. Using these models, we predicted the toxicity of 428 kinase inhibitors, both as single agents and in pairwise combinations, against three organs—heart, liver, and kidney—in humans, rodents, or canines. Our data show that the models accurately predicted the renal and cardiac toxicity of many FDA-approved KIs that were previously known. For example, the inhibition of RET kinase was identified to be linked with renal toxicity. Further, we compared the toxicity landscape of kinase KIs among different species and identified distinct sets of kinases that were recognized as essential indicators of toxicity within the same organ across species. These disparities underscore the potential distinctions in organ metabolism. In summary, our data provide a comprehensive landscape of kinase inhibitor toxicity. Our models could serve as a valuable resource for predicting kinase inhibitor toxicity across species by leveraging inhibitor profile data. Citation Format: Yuqi Kang, Marina Chan, Songli Zhu, Taranjit S. Gujral. Integrating organotypic tissue slices and polypharmacology-based screening to map the toxicity landscape of kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 616.

Potential protein kinase inhibitors that target G-quadruplex DNA structures in the human telomeric regions.

Telomeric regions contain Guanine-rich sequences arranged in a planar manner and connected by Hoogsteen hydrogen bonds that can fold into G-quadruplex (G4) DNA structures, and can be stabilized by monovalent metal cations. The presence of G4 DNA holds significance in cancer-related processes, especially due to their regulatory potential at transcriptional and translational levels of oncogene and tumor suppressor genes. The objective of this current research is to explore the evolving realm of FDA-approved protein kinase inhibitors, with a specific emphasis on their capacity to stabilize the G4 DNA structures formed at the human telomeric regions. This involves investigating the possibility of repurposing FDA-approved protein kinase inhibitors as a novel approach for targeting multiple cancer types. In this context, we have selected 16 telomeric G4 DNA structures as targets and 71 FDA-approved small-molecule protein kinase inhibitors as ligands. To investigate their binding affinities, molecular docking of human telomeric G4 DNA with nuclear protein kinase inhibitors and their corresponding co-crystalized ligands were performed. We found that Ponatinib and Lapatinib interact with all the selected G4 targets, the binding free energy calculations, and molecular dynamic simulations confirm their binding efficacy and stability. Thus, it is hypothesized that Ponatinib and Lapatinib may stabilize human telomeric G4 DNA in addition to their ability to inhibit BCR-ABL and the other members of the EGFR family. As a result, we also hypothesize that the stabilization of G4 DNA might represent an additional underlying mechanism contributing to their efficacy in exerting anti-cancer effects.

Light-Activatable Photocaged UNC2025 for Triggering TAM Kinase Inhibition in Bladder Cancer.

Photopharmacology is an emerging field that utilizes photo-responsive molecules to enable control over the activity of a drug using light. The aim is to limit the therapeutic action of a drug at the level of diseased tissues and organs. Considering the well-known implications of protein kinases in cancer and the therapeutic issues associated with protein kinase inhibitors, the photopharmacology is seen as an innovative and alternative solution with great potential in oncology. In this context, we developed the first photocaged TAM kinase inhibitors based on UNC2025, a first-in-class small molecule kinase inhibitor. These prodrugs showed good stability in biologically relevant buffer and rapid photorelease of the photoremovable protecting group upon UV-light irradiation (<10 min.). These light-activatable prodrugs led to a 16-fold decrease to a complete loss of kinase inhibition, depending on the protein and the position at which the coumarin-type phototrigger was introduced. The most promising candidate was the N,O-dicaged compound, showing the superiority of having two photolabile protecting groups on UNC2025 for being entirely inactive on TAM kinases. Under UV-light irradiation, the N,O-dicaged compound recovered its inhibitory potency in enzymatic assays and displayed excellent antiproliferative activity in RT112 cell lines.

Response to Dabrafenib Plus Trametinib in a Patient With an Uncommon Activating BRAF Mutation: A First in Non-Small Cell Lung Cancer.

Mutations in BRAF are present in 4% of non-small cell lung cancer (NSCLC), of which half are well-characterized activating variants affecting codon 600 (classified as class I). These mutations, most commonly BRAF V600E, have been associated with response to BRAF/MEK-directed small molecule kinase inhibitors. NSCLC with kinase-activating BRAF mutations occurring at other codons (class II variants) represent a substantial portion of BRAF-mutated NSCLC, but use of targeted therapy in these tumors is still under investigation. Class II mutations have been described in other tumor types and have been associated with response to BRAF/MEK-targeted agents, although optimal treatment strategies for these patients are lacking. This report presents a case of a woman with metastatic NSCLC harboring a class II BRAF p.N486_P490del variant who had a sustained clinical response to combination therapy with dabrafenib and trametinib. This first report of the use of BRAF/MEK-targeted therapy for this variant in NSCLC supports consideration of such treatment for tumors with class II BRAF variants.

Growth modulatory effects of fenretinide encompass keratinocyte terminal differentiation: A favorable outcome for oral squamous cell carcinoma chemoprevention.

Oral squamous cell carcinoma (OSCC) is world-wide health problem associated with high morbidity and mortality. From both the patient and socio-economic perspectives, prevention of progression of premalignant oral intraepithelial neoplasia (OIN) to OSCC is clearly the preferable outcome. Optimal OSCC chemopreventives possess a variety of attributes including high-tolerability, bioavailability, efficacy and preservation of an intact surface epithelium. Terminal differentiation, which directs oral keratinocytes leave the proliferative pool to form protective cornified envelopes, preserves the protective epithelial barrier while concurrently eliminating growth-aberrant keratinocytes. This study employed human premalignant oral keratinocytes and an OSCC cell line to evaluate the differentiation-inducing capacity of the synthetic retinoid, fenretinide (4HPR). Full thickness oral mucosal explants were evaluated for proof of concept differentiation studies. Results of this study characterize the ability of 4HPR to fulfill all requisite components for keratinocyte differentiation i.e. nuclear import via binding to CRABP-II (molecular modeling), binding to and subsequent activation of retinoic acid nuclear receptors (receptor activation assays), increased expression and translation of genes associated with keratinocyte differentiation (RT-PCR, immunoblotting) upregulation of a transglutaminase enzyme essential for cornified envelope formation (TGM3, functional assay) and augmentation of terminal differentiation in human oral epithelial explants (image-analyses quantified corneocyte desquamation). These data build upon the chemoprevention repertoire of 4HPR that includes function as a small molecule kinase inhibitor and inhibition of essential mechanisms necessary for basement membrane invasion. An upcoming clinical trial, which will assess whether a 4HPR-releasing mucoadhesive patch induces histologic, clinical and molecular regression in OIN lesions, will provide essential clinical insights.

Role and targeting of the AGC kinase family in pulmonary fibrosis (Review).

Pulmonary fibrosis (PF) is a progressive and irreversible pulmonary disease with a high mortality rate and limited treatment options. The cAMP-dependent protein kinase A, cGMP-dependent protein kinase G and phospholipid-dependent protein kinase C, collectively known as AGC kinases, are evolutionarily conserved protein kinases that are widely distributed among eukaryotes. AGC kinases serve a crucial role in a variety of cellular functions and pathological processes, including cancer, diabetes, inflammation and viral infections, where they have been implicated the pathogenesis of PF. The present review summarizes the evidence for the involvement of specific AGC kinases in the pathogenesis of PF, and provides a theoretical basis for the development of targeted AGC kinase small molecule inhibitors or targeted drugs, offering more effective treatment options and strategies for patients with PF.

Current status of immunological therapies for rheumatoid arthritis with a focus on antigen-specific therapeutic vaccines.

Rheumatoid arthritis (RA) is recognized as an autoimmune joint disease driven by T cell responses to self (or modified self or microbial mimic) antigens that trigger and aggravate the inflammatory condition. Newer treatments of RA employ monoclonal antibodies or recombinant receptors against cytokines or immune cell receptors as well as small-molecule Janus kinase (JAK) inhibitors to systemically ablate the cytokine or cellular responses that fuel inflammation. Unlike these treatments, a therapeutic vaccine, such as CEL-4000, helps balance adaptive immune homeostasis by promoting antigen-specific regulatory rather than inflammatory responses, and hence modulates the immunopathological course of RA. In this review, we discuss the current and proposed therapeutic products for RA, with an emphasis on antigen-specific therapeutic vaccine approaches to the treatment of the disease. As an example, we describe published results of the beneficial effects of CEL-4000 vaccine on animal models of RA. We also make a recommendation for the design of appropriate clinical studies for these newest therapeutic approaches, using the CEL-4000 vaccine as an example. Unlike vaccines that create or boost a new immune response, the clinical success of an immunomodulatory therapeutic vaccine for RA lies in its ability to redirect autoreactive pro-inflammatory memory T cells towards rebalancing the "runaway" immune/inflammatory responses that characterize the disease. Human trials of such a therapy will require alternative approaches in clinical trial design and implementation for determining safety, toxicity, and efficacy. These approaches include adaptive design (such as the Bayesian optimal design (BOIN), currently employed in oncological clinical studies), and the use of disease-related biomarkers as indicators of treatment success.

Kinome-Wide Virtual Screening by Multi-Task Deep Learning.

Deep learning is a machine learning technique to model high-level abstractions in data by utilizing a graph composed of multiple processing layers that experience various linear and non-linear transformations. This technique has been shown to perform well for applications in drug discovery, utilizing structural features of small molecules to predict activity. Here, we report a large-scale study to predict the activity of small molecules across the human kinome-a major family of drug targets, particularly in anti-cancer agents. While small-molecule kinase inhibitors exhibit impressive clinical efficacy in several different diseases, resistance often arises through adaptive kinome reprogramming or subpopulation diversity. Polypharmacology and combination therapies offer potential therapeutic strategies for patients with resistant diseases. Their development would benefit from a more comprehensive and dense knowledge of small-molecule inhibition across the human kinome. Leveraging over 650,000 bioactivity annotations for more than 300,000 small molecules, we evaluated multiple machine learning methods to predict the small-molecule inhibition of 342 kinases across the human kinome. Our results demonstrated that multi-task deep neural networks outperformed classical single-task methods, offering the potential for conducting large-scale virtual screening, predicting activity profiles, and bridging the gaps in the available data.