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A series of 5-benzylamine-substituted pyrimido[4,5-c]quinoline derivatives of the CSNK2A chemical probe SGC-CK2-2 were synthesized with the goal of improving kinase inhibitor cellular potency and antiviral phenotypic activity while maintaining aqueous solubility. Among the range of analogs, those bearing electron-withdrawing (4c and 4g) or donating (4f) substituents on the benzyl ring as well as introduction of non-aromatic groups such as the cyclohexylmethyl (4t) were shown to maintain CSNK2A activity. The CSNK2A activity was also retained with N-methylation of SGC-CK2-2, but α-methyl substitution of the benzyl substituent led to a 10-fold reduction in potency. CSNK2A inhibition potency was restored with indene-based compound 4af, with activity residing in the S-enantiomer (4ag). Analogs with the highest CSNK2A potency showed good activity for inhibition of Mouse Hepatitis Virus (MHV) replication. Conformational analysis indicated that analogs with the best CSNK2A inhibition (4t, 4ac, and 4af) exhibited smaller differences between their ground state conformation and their predicted binding pose. Analogs with reduced activity (4ad, 4ae, and 4ai) required more substantial conformational changes from their ground state within the CSNK2A protein pocket. [ABSTRACT FROM AUTHOR]

ABSTRACT Differentiated thyroid carcinoma (DTC) accounts for most cases of thyroid cancer, and the heterogeneity of DTC requires that management decisions be taken by a multidisciplinary team involving endocrinologists, head and neck surgeons, nuclear medicine physicians, pathologists, radiologists, radiation oncologists, and medical oncologists. It is important for nonspecialists to recognize and refer patients with DTC who will benefit from a specialized approach. Recent advances in knowledge and changes in management of DTC call for the need to raise awareness on the part of these nonspecialist physicians, including general endocrinologists and medical oncologists at large. We provide an overview of diagnostic and therapeutic principles in DTC, especially those that bear direct implication on day-to-day management of these patients by generalists. Patients with DTC may be broadly categorized as having localized, locally persistent/recurrent, or metastatic disease. Current recommendations for DTC include a three-tiered system that classifies patients with localized disease into low, intermediate, or high risk of persistent or recurrent disease. Risk stratification should be performed at baseline and repeated on an ongoing basis, depending on clinical evolution. One of the overarching goals in the management of DTC is the need to personalize treatment by tailoring its modality and intensity according to ongoing prognostic stratification, evolving knowledge about the disease, and patient characteristics and preference. In metastatic disease that is refractory to radioactive iodine, thyroid tumors are being reclassified into molecular subtypes that better reflect their biological properties and for which molecular alterations can be targeted with specific agents.

Aldehyde oxidase (AOX) is a molybdoenzyme that is primarily expressed in the liver and is involved in the metabolism of drugs and other xenobiotics. AOX-mediated metabolism can result in unexpected outcomes, such as the production of toxic metabolites and high metabolic clearance, which can lead to the clinical failure of novel therapeutic agents. Computational models can assist medicinal chemists in rapidly evaluating the AOX metabolic risk of compounds during the early phases of drug discovery and provide valuable clues for manipulating AOX-mediated metabolism liability. In this study, we developed a novel graph neural network called AOMP for predicting AOX-mediated metabolism. AOMP integrated the tasks of metabolic substrate/non-substrate classification and metabolic site prediction, while utilizing transfer learning from 13C nuclear magnetic resonance data to enhance its performance on both tasks. AOMP significantly outperformed the benchmark methods in both cross-validation and external testing. Using AOMP, we systematically assessed the AOX-mediated metabolism of common fragments in kinase inhibitors and successfully identified four new scaffolds with AOX metabolism liability, which were validated through in vitro experiments. Furthermore, for the convenience of the community, we established the first online service for AOX metabolism prediction based on AOMP, which is freely available at https://aomp.alphama.com.cn.

BackgroundSmall cell lung cancer (SCLC) transformation is one of the mechanisms of drug resistance to tyrosine kinase inhibitors (TKIs) in advanced epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) and represents an increasingly recognized clinical dilemma.MethodsWe performed a retrospective review of 964 cases at the University of California, San Diego of patients with EGFR sensitizing mutations. Nine patients had a biopsy-confirmed small cell transformation. The unique gene alterations and clinicopathologic features were collected and analyzed.ResultsNine cases (9/964, 0.9%) were identified, all with stage IV adenocarcinoma (ADC) at diagnosis, 7 were poorly differentiated, and 7 had an EGFR exon 19 deletion. All nine patients had tumor protein p53 (TP53) mutation. Among seven cases that had next-generation sequencing (NGS), 5 harbored retinoblastoma 1 (RB1) loss. WNK lysine deficient protein kinase 1 (WNK1) mutation was found in two patients that had longer survival. The median time from the initial diagnosis to transformation was 22.7 months (IQR: 15.1-25.1). After small cell transformation on EGFR inhibition, all patients were treated with etoposide/platinum, conferring a median progression-free survival (PFS) of 3.2 months (IQR, 2.2-6.5 months) and post-chemotherapy survival of 8.6 months (IQR, 4.0-19.0 months). Six patients, as they retained the initial EGFR mutations, resumed (did so after terminating chemotherapy)/continued (did so concomitantly with chemotherapy) TKIs with a median duration of 13.8 months (IQR, 3.8-27.7 months). Two patients received immunotherapy but had no benefit.ConclusionsIn our series, most patients with small cell transformation had poorly differentiated adenocarcinomas at baseline. RB1 loss was not universal in transformed patients in this series, though TP53 mutation was present in all tumor samples. WNK1 mutation may be a new resistance mechanism to TKIs that may be associated with improved survival.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

IntroductionApproximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy.MethodsTo compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition.ResultsRNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells.ConclusionsThe findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV− cases. [ABSTRACT FROM AUTHOR]

Aldehyde oxidase (AOX) is a molybdoenzyme that is primarily expressed in the liver and is involved in the metabolism of drugs and other xenobiotics. AOX-mediated metabolism can result in unexpected outcomes, such as the production of toxic metabolites and high metabolic clearance, which can lead to the clinical failure of novel therapeutic agents. Computational models can assist medicinal chemists in rapidly evaluating the AOX metabolic risk of compounds during the early phases of drug discovery and provide valuable clues for manipulating AOX-mediated metabolism liability. In this study, we developed a novel graph neural network called AOMP for predicting AOX-mediated metabolism. AOMP integrated the tasks of metabolic substrate/non-substrate classification and metabolic site prediction, while utilizing transfer learning from 13C nuclear magnetic resonance data to enhance its performance on both tasks. AOMP significantly outperformed the benchmark methods in both cross-validation and external testing. Using AOMP, we systematically assessed the AOX-mediated metabolism of common fragments in kinase inhibitors and successfully identified four new scaffolds with AOX metabolism liability, which were validated through in vitro experiments. Furthermore, for the convenience of the community, we established the first online service for AOX metabolism prediction based on AOMP, which is freely available at https://aomp.alphama.com.cn. A transfer learning enhanced graph neural network was established to predict the AOX-mediated metabolism. The proposed model achieved state-of-the-art performance and was applied to identify new scaffolds with AOX metabolism liability. [Display omitted] [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that poses a significant threat to the global livestock industry. However, specific antiviral treatments against FMDV are currently unavailable. This study aimed to evaluate the antiviral activity of anticancer drugs, including kinase and non-kinase inhibitors against FMDV replication in BHK-21 cells. Sorafenib, a multi-kinase inhibitor, demonstrated a significant dose-dependent reduction in FMDV replication. It exhibited a half maximal effective concentration (EC50) value of 2.46 µM at the pre-viral entry stage and 2.03 µM at the post-viral entry stage. Further intracellular assays revealed that sorafenib effectively decreased 3Dpol activity with a half maximal inhibitory concentration (IC50) of 155 nM, while not affecting 3Cpro function. The study indicates that sorafenib influences host protein pathways during FMDV infection, primarily by potentiating the c-RAF canonical pathway and AKT/PI3K pathway. Molecular docking analysis demonstrated specific binding of sorafenib to the active site of FMDV 3Dpol, interacting with crucial catalytic residues, including D245, D338, S298, and N307. Additionally, sorafenib exhibited significant binding affinity to the active site motifs of cellular kinases, namely c-RAF, AKT, and PI3K, which play critical roles in the viral life cycle. The findings suggest that sorafenib holds promise as a therapeutic agent against FMDV infection. Its mechanism of action may involve inhibiting FMDV replication by reducing 3Dpol activity and regulating cellular kinases. This study provides insights for the development of novel therapeutic strategies to combat FMDV infections. [ABSTRACT FROM AUTHOR]

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

Vinyl sulfones are a special sulfur-containing structural unit that have attracted considerable attention, owing to their important role in serving as key structural motifs of various biologically active compounds as well as serving as versatile building blocks for organic transformations. The synthetic strategy of vinyl sulfone derivatives has been substantially upgraded over the past 30 years, and the wide application of this functional group in drug design and discovery has been promoted. In this review, the authors review the application of vinyl sulfones in drug discovery and select optimized compounds which might have significant impact or potential inspiration for drug design. Vinyl sulfones have been reported to target various macromolecular targets via non-covalent or covalent interactions, including multiple kinases, tubulin, cysteine protease, transcription factor, and so on. Thus, it has been significantly applied as a privileged scaffold in the design of anticancer, anti-infective, anti-inflammatory, and neuroprotective agents. However, much work remains to be done to improve the drug-like properties, such as chemical and metabolic stability, ADME, and toxicity. Besides, the chemical space of vinyl sulfones needs to be expanded, including but not limited to the design of constrained endocyclic and exocyclic vinyl sulfones. [ABSTRACT FROM AUTHOR]

Kinase inhibitors (KIs) are important cancer drugs but often feature polypharmacology that is molecularly not understood. This disconnect is particularly apparent in cancer entities such as sarcomas for which the oncogenic drivers are often not clear. To investigate more systematically how the cellular proteotypes of sarcoma cells shape their response to molecularly targeted drugs, we profiled the proteomes and phosphoproteomes of 17 sarcoma cell lines and screened the same against 150 cancer drugs. The resulting 2550 phenotypic profiles revealed distinct drug responses and the cellular activity landscapes derived from deep (phospho) proteomes (9--10,000 proteins and 10--27,000 phosphorylation sites per cell line) enabled several lines of analysis. For instance, connecting the (phospho)proteomic data with drug responses revealed known and novel mechanisms of action (MoAs) of KIs and identified markers of drug sensitivity or resistance. All data is publicly accessible via an interactive web application that enables exploration of this rich molecular resource for a better understanding of active signalling pathways in sarcoma cells, identifying treatment response predictors and revealing novel MoA of clinical KIs. [ABSTRACT FROM AUTHOR]

Background and Objective: Kinome refers to the complete set of protein kinases encoded in the genome. The most common type of post-translational modification is phosphorylation, with more than two-thirds of all human encoded proteins being phosphorylated by protein kinases. Phosphorylation as an important regulatory factor in the activity of proteins greatly expands the flexibility of the epigenome. Thus, protein kinases often promote cell proliferation, survival, and migration by participating in intracellular pathways and are associated with carcinogenesis when overexpressed or activated. The main goal of this study is to investigate the role of kinase enzymes in causing cancer and to investigate their potential as drugs in the treatment of various types of cancer. Methods: In this review, by searching Scopus, PubMed, Web of Science and ScienceDirect databases, articles published between 2010 and 2022 were selected and analyzed using the keywords "cancer, human kinome, kinase and kinase inhibitor". Findings: 64 articles were selected according to the research topic and 11 articles were excluded due to the lack of relation to the main keywords. In this study, the role of kinases in cell proliferation and human cancers has been investigated, and kinase inhibitors in combination with other common treatments such as chemotherapy or radiation therapy can be mentioned as a new and promising approach in cancer treatment. Conclusion: Based on the results of this study, kinases are known as one of the most important components of cell growth and proliferation, so that they play an important role in the cancer process with excessive activity. This study shows that controlling and inhibiting the kinase family to overcome cancer cell growth has good benefits. [ABSTRACT FROM AUTHOR]

Among tyrosine kinase inhibitors, quinazoline-based compounds represent a large and well-known group of multi-target agents. Our previous studies have shown interesting kinases inhibition activity for a series of 4-aminostyrylquinazolines based on the CP-31398 scaffold. Here, we synthesised a new series of styrylquinazolines with a thioaryl moiety in the C4 position and evaluated in detail their biological activity. Our results showed high inhibition potential against non-receptor tyrosine kinases for several compounds. Molecular docking studies showed differential binding to the DFG conformational states of ABL kinase for two derivatives. The compounds showed sub-micromolar activity against leukaemia. Finally, in-depth cellular studies revealed the full landscape of the mechanism of action of the most active compounds. We conclude that S4-substituted styrylquinazolines can be considered as a promising scaffold for the development of multi-kinase inhibitors targeting a desired binding mode to kinases as effective anticancer drugs. [ABSTRACT FROM AUTHOR]

Inhibiting a specific target in cancer cells and reducing unwanted side effects has become a promising strategy in pancreatic cancer treatment. MAP4K4 is associated with pancreatic cancer development and correlates with poor clinical outcomes. By phosphorylating MKK4, proteins associated with cell apoptosis and survival are translated. Therefore, inhibiting MAP4K4 activity in pancreatic tumours is a new therapeutic strategy. Herein, we performed a structure-based virtual screening to identify MAP4K4 inhibitors and discovered the compound F389-0746 with a potent inhibition (IC50 120.7 nM). The results of kinase profiling revealed that F389-0746 was highly selective to MAP4K4 and less likely to cause side effects. Results of in vitro experiments showed that F389-0746 significantly suppressed cancer cell growth and viability. Results of in vivo experiments showed that F389-0746 displayed comparable tumour growth inhibition with the group treated with gemcitabine. These findings suggest that F389-0746 has promising potential to be further developed as a novel pancreatic cancer treatment. We identified a novel MAP4K4 inhibitor using a structure-based virtual screening strategy. Kinase profiling showed high selectivity towards MAP4K4. The identified inhibitor suppressed pancreatic cancer cell growth and viability in vitro. The inhibitor downregulated the MKK4-JNK signalling pathway. The inhibitor suppressed tumour growth in vivo. [ABSTRACT FROM AUTHOR]

Potassium 6-oxo-7,13,16,22-tetraazatetracyclo[12.6.2.18,12.017,21]tricosa-1(20),8(23),9,11,14,16,18,21-octaen-2-yne-15-carboxylate was synthesized through a multi-step pathway, starting from commercially available 3-iodo-1,2-phenylenediamine. Structure characterization of this new substituted macrocyclic quinoxaline compound was achieved using 1H NMR, 13C NMR, and HRMS spectral analysis. This new macrocyclic derivative demonstrated submicromolar potency on both Pim-1 and Pim-2 isoforms, with an interesting selectivity profile against a selected panel of human kinases. [ABSTRACT FROM AUTHOR]

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Protein kinases play vital roles in controlling cell behavior, and an array of kinase inhibitors are used successfully for treatment of disease. Typical drug development pipelines involve biological studies to validate a protein kinase target, followed by the identification of small molecules that effectively inhibit this target in cells, animal models, and patients. However, it is clear that protein kinases operate within complex signaling networks. These networks increase the resilience of signaling pathways, which can render cells relatively insensitive to inhibition of a single kinase, and provide the potential for pathway rewiring, which can result in resistance to therapy. It is therefore vital to understand the properties of kinase signaling networks in health and disease so that we can design effective multi-targeted drugs or combinations of drugs. Here, we outline how pharmacological and chemo-genetic approaches can contribute to such knowledge, despite the known low selectivity of many kinase inhibitors. We discuss how detailed profiling of target engagement by kinase inhibitors can underpin these studies; how chemical probes can be used to uncover kinase-substrate relationships, and how these tools can be used to gain insight into the configuration and function of kinase signaling networks.

BackgroundBone loss is the most common reason for broken bones among the elderly. An ideal agent for the treatment of bone loss should have both osteoclast inhibitory and osteoblast stimulatory functions. Leucine-rich repeat kinase 1 (LRRK1) is a novel target for alternative antiresorptive drugs to treat osteoporosis and osteoporotic fractures. Recently a chemical IN04, Methyl 3-[({([5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-thio}-acetyl)-amino]-benzoate, has been identified as a potential LRRK1 inhibitor.ObjectiveThe aim of this work is to investigate how the chemical IN04 interacts with LRRK1 and inhibits its activity.MethodsA structural model of the LRRK1 kinase domain was constructed with SWISS-MODEL. The human protein kinase ROCO4 (PDB ID: 4YZN) was chosen as the template based on sequence homology, structural and phylogenetic analysis. In addition, a homology model of the LRRK1 ROC domain was also prepared based on the LRRK2 ROC domain structure (PDB ID: 2ZEJ). The interactions of IN04 with the active sites in the LRRK1 kinase domain and ROC domain were investigated by SwissDock.ResultsIN04 was docked into the active site of the LRRK1 kinase domain with similar interactions as ATP comparable to the ligand bound to homologous kinases. Many rational binding modes of IN04 to LRRK1 kinase domain were investigated and the most likely binding pose containing multiple hydrogen bonds and a salt bridge was discovered. However, IN04 cannot fit into the GDP-binding site of the ROC domain.ConclusionChemical IN04 inhibits LRRK1 by binding to the active site of the kinase domain but not the ROC domain.

Kinases play a vital role in regulating essential cellular processes, including cell cycle progression, growth, apoptosis, and metabolism, by catalyzing the transfer of phosphate groups from adenosing triphosphate to substrates. Their dysregulation has been closely associated with numerous diseases, including cancer development, making them attractive targets for drug discovery. However, accurately predicting the binding affinity between chemical compounds and kinase targets remains challenging due to the highly conserved structural similarities across the kinome. To address this limitation, we present KinScan, a novel computational approach that leverages large-scale bioactivity data and integrates the Multi-Scale Context Aware Transformer framework to construct a virtual profiling model encompassing 391 protein kinases. The developed model demonstrates exceptional prediction capability, distinguishing between kinases by utilizing structurally aligned kinase binding site features derived from multiple sequence alignment for fast and accurate predictions. Through extensive validation and benchmarking, KinScan demonstrated its robust predictive power and generalizability for large-scale kinome-wide profiling and selectivity, uncovering associations with specific diseases and providing valuable insights into kinase activity profiles of compounds. Furthermore, we deployed a web platform for end-to-end profiling and selectivity analysis, accessible at https://kinscan.drugonix.com/softwares/kinscan. [ABSTRACT FROM AUTHOR]

Simple Summary: Genome-based cancer medicine is becoming the standard of care: the patient's tumor DNA is first analyzed to identify driver mutations, and this permits later selection of the most effective drugs. Treatment of lung cancer offers many examples. Activating mutations in the epidermal growth factor receptor (EGFR) gene, as well as in other receptor tyrosine kinases (e.g., ALK), are considered actionable candidates, and the respective drugs, called tyrosine kinase inhibitors, are relatively effective. Unfortunately, despite initial activity, the emergence of new, on-target mutations, along with adaptive processes, preempt the anti-cancer effects and necessitate switching to next-generation drugs. This review highlights recent progress in resolving the mechanisms that underlie acquisition of resistance. Specifically, we focus on the endogenous mutators that initiate emergence of new mutations and the potential clinical benefits that may be derived from this new understanding. Epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) have changed the landscape of lung cancer therapy. For patients who are treated with the new TKIs, the current median survival exceeds 3 years, substantially better than the average 20 month survival rate only a decade ago. Unfortunately, despite initial efficacy, nearly all treated patients evolve drug resistance due to the emergence of either new mutations or rewired signaling pathways that engage other receptor tyrosine kinases (RTKs), such as MET, HER3 and AXL. Apparently, the emergence of mutations is preceded by a phase of epigenetic alterations that finely regulate the cell cycle, bias a mesenchymal phenotype and activate antioxidants. Concomitantly, cells that evade TKI-induced apoptosis (i.e., drug-tolerant persister cells) activate an intrinsic mutagenic program reminiscent of the SOS system deployed when bacteria are exposed to antibiotics. This mammalian system imbalances the purine-to-pyrimidine ratio, inhibits DNA repair and boosts expression of mutation-prone DNA polymerases. Thus, the net outcome of the SOS response is a greater probability to evolve new mutations. Deeper understanding of the persister-to-resister transformation, along with the development of next-generation TKIs, EGFR-specific proteolysis targeting chimeras (PROTACs), as well as bispecific antibodies, will permit delaying the onset of relapses and prolonging survival of patients with EGFR+ lung cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: The FMS-like tyrosine kinase 3 (FLT3) gene mutated in 10–15% of pediatric acute myeloid leukemia (AML) is associated with an inferior outcome. We retrospectively analyzed the nationwide pediatric AML database from between 2005 and 2022. FLT3-ITD mutation was found in 10.7% of patients. An improvement in the outcome was found in the analyzed period of time. The treatment results in FLT3-ITD--positive patients treated under the AML-BFM 2012 and 2019 protocols were better in comparison to the AML-BFM 2004 protocol and better than previously reported by most authors. However, the outcome in patients with FLT3-ITD compared to children without this mutation was still significantly worse, with higher percentage of non-responders and relapses. It seems that SCT and FLT3 inhibitors have a beneficial impact on the prognosis; however, it should be confirmed in a larger group of patients. This gives hope for the improvement of the treatment results in pediatric AML with FLT3-ITD in the future. Background: The FMS-like tyrosine kinase 3 (FLT3) gene mutated in 10–15% of pediatric acute myeloid leukemia (AML) is associated with an inferior outcome. The aim of the study was to analyze the outcome and characteristics of FLT3-ITD-positive pediatric AML. Methods: We retrospectively analyzed the nationwide pediatric AML database from between 2005 and 2022. FLT3-ITD was found in 54/497 (10.7%) patients with available analysis. Three consecutive treatment protocols were used (AML-BFM 2004 Interim, AML-BFM 2012 Registry, AML-BFM 2019 recommendations). Results: Probabilities of 5-year overall (OS), event-free (EFS) and relapse-free survival were significantly lower in the FLT3-ITD-positive patients compared to FLT3-ITD-negative (0.54 vs. 0.71, p = 0.041; 0.36 vs. 0.59, p = 0.0004; 0.47 vs. 0.70, p = 0.0029, accordingly). An improvement in the outcome was found in the analyzed period of time, with a trend of better survival in patients treated under the AML-BFM 2012 and AML-BFM 2019 protocols compared to the AML-BFM 2004 protocol (5-year EFS 0.52 vs. 0.27, p = 0.069). There was a trend of improved outcomes in patients treated with FLT3 inhibitors (n = 9, 2-year EFS 0.67 vs. 0.33, p = 0.053) and those who received stem cell transplantation (SCT) (n = 26; 5-year EFS 0.70 vs. 0.27, p = 0.059). The co-occurrence of the WT1 mutation had a dismal impact on the prognosis (5-year EFS 0.23 vs. 0.69, p = 0.002), while the NPM1 mutation improved survival (5-year OS 1.0 vs. 0.44, p = 0.036). Conclusions: It seems that SCT and FLT3 inhibitors have a beneficial impact on the prognosis. Additional genetic alterations, like the WT1 and NPM1 mutations, significantly influence the outcome. [ABSTRACT FROM AUTHOR]

Abstract Background In the phase 2 double‐blind Study 211, a starting dose of lenvatinib 18 mg/day was compared with the approved starting dose of 24 mg/day in patients with radioiodine‐refractory differentiated thyroid cancer (RR‐DTC). Predefined criteria for noninferiority for efficacy in the 18 mg arm were not met; safety was similar in both arms. Impact of lenvatinib treatment on health‐related quality‐of‐life (HRQoL) was a secondary endpoint of Study 211. Methods Patients with RR‐DTC were randomly assigned to a blinded starting dose of lenvatinib 18 mg/day or 24 mg/day. HRQoL was assessed at baseline, every 8 weeks until Week 24, then every 16 weeks, and at the off‐treatment visit, using the EQ‐5D‐3L and FACT‐G instruments. Completion and compliance rates, mean change from baseline, and times to first and definitive deterioration were evaluated. Results Baseline EQ‐5D and FACT‐G scores, and overall changes from baseline, were comparable between patients in the lenvatinib 18 mg/day (n = 77) and 24 mg/day arms (n = 75). For the 18 mg versus 24 mg arms, least squares mean differences were −0.42 (95% CI −4.88, 4.03) for EQ‐5D‐VAS and 0.47 (95% CI −3.45, 4.39) for FACT‐G total. Time to first deterioration did not significantly favor either arm; EQ‐5D‐VAS HR [18 mg/24 mg] 0.93 (95% CI 0.61–1.40), EQ‐5D‐HUI HR [18 mg/24 mg] 0.68 (95% CI 0.44–1.05), FACT‐G total HR [18 mg/24 mg] 0.73 (95% CI 0.48–1.12). Time to definitive deterioration did not significantly favor either arm, though EQ‐5D‐VAS showed a trend in favor of the 24 mg arm (HR [18 mg/24 mg] 1.72; 95% CI 0.99–3.01); EQ‐5D‐HUI HR [18 mg/24 mg] was 0.96 (95% CI 0.57–1.63), FACT‐G total HR [18 mg/24 mg] was 0.72 (95% CI 0.43–1.21). Conclusions In Study 211, HRQoL for patients in the lenvatinib 18 mg/day arm was not statistically different from that of patients in the 24 mg/day arm. These data further support the use of the approved lenvatinib starting dose of 24 mg/day in patients with RR‐DTC. ClinicalTrials.gov Number NCT02702388.

Julita Chlebowicz,1 Radha Akella,1 John M Humphreys,1 Haixia He,1 Ashari R Kannangara,1 Shuguang Wei,2 Bruce Posner,2 Elizabeth J Goldsmith1 1Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX, USA; 2Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX, USACorrespondence: Elizabeth J Goldsmith, Department of Biophysics, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8816, USA, Tel +1 214 645 6376, Email Elizabeth.goldsmith@utsouthwestern.eduIntroduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors.Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors.Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity.Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.Graphical Abstract: Keywords: WNK, inhibition screen, kinase inhibitor, WNK3-specific, structure–activity relationship, crystallography

Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

Bioluminescence assays using luciferase enzymes are widely used in research to monitor gene expression and an array of other cell properties, and split luciferase enzymes can be used to measure protein interactions in biochemical assays and in living cells. When these methods are employed in chemical library screening efforts, it is vital that the activity of the luciferase enzyme itself is not strongly influenced by library components. Here, we developed a NanoBiT split luciferase assay to measure phosphorylation of Histone H3 peptides and used it to test the robustness of split luciferase to interference from two libraries of commonly used kinase inhibitors, including the Kinase Chemogenomic Set (KCGS). We found that NanoBiT luciferase is not significantly affected by the great majority of kinase inhibitors tested. However, the weak inhibition observed for a small minority of kinase inhibitors encourages the inclusion of suitable controls in NanoBiT (or NanoLuc) assays.

A series of N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles was synthesised and screened for their potential to inhibit kinases and exhibit anticancer activity against primary patient-derived glioblastoma 2D cells and 3D neurospheres. A collection of 10 compounds was evaluated against glioma cell lines, with compound 4j exhibiting promising glioma growth inhibitory properties. Compound 4j was screened against 139 purified kinases and exhibited low micromolar activity against kinase AKT2/PKBβ. AKT signalling is one of the main oncogenic pathways in glioma and is often targeted for novel therapeutics. Indeed, AKT2 levels correlated with glioma malignancy and poorer patient survival. Compound 4j inhibited the 3D neurosphere formation in primary patient-derived glioma stem cells and exhibited potent EC50 against glioblastoma cell lines. Although exhibiting potency against glioma cells, 4j exhibited significantly less cytotoxicity against non-cancerous cells even at fourfold–fivefold the concentration. Herein we establish a novel biochemical kinase inhibitory function for N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles and further report their anti-glioma activity in vitro for the first time.KEY MESSAGEAnti-glioma pyrano[2,3-c]pyrazole 4j inhibited the 3D neurosphere formation in primary patient-derived glioma stem cells. 4j also displayed PKBβ/AKT2 inhibitory activity. 4j is nontoxic towards non-cancerous cells.

11H-Indeno[1,2-b]quinoxalin-11-one oxime (IQ-1) and tryptanthrin-6-oxime are potent c-Jun N-terminal kinase 3 (JNK-3) inhibitors demonstrating neuroprotective, anti-inflammatory and anti-arthritic activity. However, the stereochemical configuration of the oxime carbon–nitrogen double bond (E- or Z-) in these compounds was so far unknown. In this contribution, we report the results of the determination of the double bond configuration in the solid state by single crystal X-ray diffraction and in solution by 1D and 2D NMR techniques and DFT calculations. It was found that both in the solid state and in solution, IQ-1 adopts the E-configuration stabilized by intermolecular hydrogen bonds, in contrast to previously assumed Z-configuration that could be stabilized only by an intramolecular hydrogen bond. [ABSTRACT FROM AUTHOR]

This study describes the synthesis and biological activity of new imadazopyrazines as first-in-class CDK9 inhibitors. The inhibition of CDK9 is a well-established therapeutic target in cancer therapy. The new compounds were assessed using an in vitro kinase assay against CDK9. In this assay, compound 1d exhibited the highest CDK9 inhibition with an IC50 of 0.18 µM. The cytotoxicity effect of the novel compounds was evaluated in three cancer cell lines: HCT116, K652, and MCF7. The results of this assay showed a correlation between the antiproliferative effect of the inhibitors and their CDK9 inhibitory effect in the biochemical assay. This suggests CDK9 inhibition as a mechanistic pathway for their anticancer effect. Several compounds demonstrated potent cytotoxic effects with single-digit micromolar IC50 values yielded through an MTT assay. The compounds with the most promising data were further assessed for their antiviral activity against human Coronavirus 229E. The results showed that compound 4a showed the highest antiviral potency with an IC50 of 63.28 µM and a selectivity index of 4.8. In silico target prediction data showed that 4a displayed a good affinity to proteases. The result of the docking studies of 4a with COVID-19 main protease revealed a high binding affinity, which confirmed the results obtained from in vitro study. The physiochemical and in silico pharmacokinetic parameters indicated reasonable drug-likeness properties of the new compounds, including solubility, lipophilicity, absorption, oral bioavailability, and metabolic stability. Further lead optimization of this novel scaffold could lead to a revolution of a new class of preclinical CDK9 agents. [ABSTRACT FROM AUTHOR]

Adaptor protein 2-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and plays a role in modulating receptor endocytosis. AAK1 was identified as a potential therapeutic target for the treatment of neuropathic pain when it was shown that AAK1 knock out (KO) mice had a normal response to the acute pain phase of the mouse formalin model, but a reduced response to the persistent pain phase. Herein we report our early work investigating a series of pyrrolo[2,1-f][1,2,4]triazines as part of our efforts to recapitulate this KO phenotype with a potent, small molecule inhibitor of AAK1. The synthesis, structure–activity relationships (SAR), and in vivo evaluation of these AAK1 inhibitors is described. [ABSTRACT FROM AUTHOR]

Kinases are key regulatory signalling proteins governing numerous essential biological processes and cellular functions. Dysregulation of many protein kinases is associated with cancer initiation and progression. Given their crucial roles, there has been increasing interest in harnessing kinases as prospective drug targets for cancer. In recent decades, numerous small‐molecule kinase inhibitors have been developed and revolutionized the cancer treatment landscape. Despite their great potential, challenges remain in developing highly selective and effective kinase inhibitors, with toxicity and resistance issues frequently arising. In this review, we first provide an overview of the role of kinases in carcinogenesis and describe the current progress with small‐molecule kinase inhibitors that have been approved for clinical use. We then discuss the application of mass spectrometry (MS)‐based proteomics strategies to help in the design of kinase inhibitors. Finally, we discuss the challenges and outlook concerning MS‐based proteomics techniques for kinase drug research. [ABSTRACT FROM AUTHOR]

The dihydrochloride salt of pexidartinib was synthesized and the crystal structure of its dihydrate was determined from single crystal data. The asymmetric unit consists of one formula unit. In the pexidartinib molecule, the N atom of the central pyridine ring and the pyridine-N atom of the pyrrolopyridine group are both protonated. The pexidartinib molecule forms N–H···O and N–H···Cl interactions with neighboring water molecules and chloride ions, respectively. Water/chloride (O–H···Cl) and water/water (O–H···O) interactions are also observed. Collectively, these intermolecular interactions result in a complex H-bonded framework structure. [ABSTRACT FROM AUTHOR]

Protein kinase C delta (PKCδ) is a multifunctional serine–threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, the molecular mechanism of PKCδ in colorectal cancer (CRC) remains unclear. In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild‐type (wt‐p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt‐p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53‐independent pathway and that PKCδ‐kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence‐like phenotypes, including increased senescence‐associated β‐galactosidase (SA‐β‐gal) staining, low LaminB1 expression, large nucleus size, and senescence‐associated secretory phenotype (SASP) detection. Finally, a kinase inhibitor of PKCδ suppressed senescence‐dependent tumorigenicity in a dose‐dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis. In addition, a novel therapeutic strategy for wt‐p53 CRC is proposed. [ABSTRACT FROM AUTHOR]

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

The proteolysis targeting chimera (PROTAC) technology is widely explored in cancer research and utilized in the treatment of various tumors and malignancies. In the design and optimization of PROTAC, linker is not only critical to the degradation activity of PROTACs, but also greatly affects the membrane permeability, metabolic stability and drug availability. In this study, fifteen new PROTACs based on Imatinib were designed, synthesized, and assessed for their anticancer activity against K562 cells. The CCK‐8 assay results showed that a new PROTAC with a 2‐oxoethyl linker (PROTAC 1) exhibited significant antiproliferative activity against K562 (IC50=0.62±0.02 μM) cells. Western blot analysis showed that PROTAC 1 regulated the protein levels of BCR‐ABL in a dose‐dependent manner and induced degradation of BCR‐ABL in a ubiquitin‐proteasome‐dependent manner in K562 cells. This study provides further evidence for the importance of linker selection in PROTAC design. [ABSTRACT FROM AUTHOR]

Introduction: Tyrosine kinase inhibitor drugs (TKIs) are highly effective cancer drugs, yet many TKIs are associated with various forms of cardiotoxicity. The mechanisms underlying these drug-induced adverse events remain poorly understood. We studied mechanisms of TKI-induced cardiotoxicity by integrating several complementary approaches, including comprehensive transcriptomics, mechanistic mathematical modeling, and physiological assays in cultured human cardiac myocytes. Methods: Induced pluripotent stem cells (iPSCs) from two healthy donors were differentiated into cardiac myocytes (iPSC-CMs), and cells were treated with a panel of 26 FDA-approved TKIs. Drug-induced changes in gene expression were quantified using mRNA-seq, changes in gene expression were integrated into a mechanistic mathematical model of electrophysiology and contraction, and simulation results were used to predict physiological outcomes. Results: Experimental recordings of action potentials, intracellular calcium, and contraction in iPSC-CMs demonstrated that modeling predictions were accurate, with 81% of modeling predictions across the two cell lines confirmed experimentally. Surprisingly, simulations of how TKI-treated iPSC-CMs would respond to an additional arrhythmogenic insult, namely, hypokalemia, predicted dramatic differences between cell lines in how drugs affected arrhythmia susceptibility, and these predictions were confirmed experimentally. Computational analysis revealed that differences between cell lines in the upregulation or downregulation of particular ion channels could explain how TKI-treated cells responded differently to hypokalemia. Discussion: Overall, the study identifies transcriptional mechanisms underlying cardiotoxicity caused by TKIs, and illustrates a novel approach for integrating transcriptomics with mechanistic mathematical models to generate experimentally testable, individual-specific predictions of adverse event risk. [ABSTRACT FROM AUTHOR]

The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion gene in non-small-cell lung cancer (NSCLC) was first identified in 2007. As the EML4-ALK fusion protein promotes carcinogenesis in lung cells, much attention has been paid to it, leading to the development of therapies for patients with NSCLC. These therapies include ALK tyrosine kinase inhibitors and heat shock protein 90 inhibitors. However, detailed information on the entire structure and function of the EML4-ALK protein remains deficient, and there are many obstacles to overcome in the development of novel anticancer agents. In this review, we describe the respective partial structures of EML4 and ALK that are known to date. In addition to their structures, noteworthy structural features and launched inhibitors of the EML4-ALK protein are summarized. Furthermore, based on the structural features and inhibitor-binding modes, we discuss strategies for the development of novel inhibitors targeting the EML4-ALK protein. [ABSTRACT FROM AUTHOR]

ALK-positive NSCLC coexisting with EGFR mutations is a frequently occurring clinical phenomenon. Targeting ALK and EGFR simultaneously may be an effective way to treat these cancer patients. In this study, we designed and synthesized ten new dual-target EGFR/ALK inhibitors. Among them, the optimal compound 9j exhibited good activity with IC50 values of 0.07829 ± 0.03 μM and 0.08183 ± 0.02 μM against H1975 (EGFR T790M/L858R) and H2228 (EML4-ALK) cells, respectively. Immunofluorescence assays indicated that the compound could simultaneously inhibit the expression of phosphorylated EGFR and ALK proteins. A kinase assay demonstrated that compound 9j could inhibit both EGFR and ALK kinases; thus, exerting an antitumor effect. Additionally, compound 9j induced apoptosis in a dose-dependent manner and inhibited the invasion and migration of tumor cells. All of these results indicate that 9j is worthy of further study. [ABSTRACT FROM AUTHOR]

Tyrosine kinase inhibitors (TKI) are known to be highly effective in the treatment of various cancers with kinase-domain mutations such as chronic myelogenous leukemia. However, they have important side effects such as increased vascular permeability and pulmonary hypertension. In patients undergoing pulmonary endarterectomy with deep hypothermic circulatory arrest, these side effects may exacerbate postoperative complications such as reperfusion edema and persistent pulmonary hypertension. We report on a simple modification of the perfusion strategy to increase intravascular oncotic pressure by retrograde autologous priming and the addition of packed cells and albumin in a patient treated with a TKI. [ABSTRACT FROM AUTHOR]

The traditional single-treatment strategy for cancer is frequently unsuccessful due to the complexity of cellular signaling. However, suppression of multiple targets is vital to defeat tumor cells. In this research, new compounds for the treatment of cancer were developed successfully as novel hybrid anticancer agents. Based on a molecular hybridization strategy, we designed hybrid agents that target multiple protein kinases to fight cancer cells. The proposed hybrid agents combined purine and isatin moieties in their structures with 4-aminobenzohydrazide and hydrazine as different linkers. Having those two moieties in one molecule enabled the capability to inhibit multiple kinases, such as human epidermal receptor (EGFR), human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 2 (CDK2). Anticancer activity was evaluated by performing cytotoxicity assays, kinase inhibition assays, cell cycle analysis, and BAX, Bcl-2, Caspase 3 and Caspase 9 protein level determination assays. The results showed that the designed hybrids tackled the cancer by inhibiting both cell proliferation and metastasis. A molecular docking study was performed to predict possible binding interactions in the active site of the investigated protein kinase enzymes. [ABSTRACT FROM AUTHOR]

Liver Fibrosis can be life-threatening if left untreated as it may lead to serious, incurable complications. The common therapeutic approach is to reverse the fibrosis while the intervention is still applicable. Celecoxib was shown to exhibit some antifibrotic properties in the induced fibrotic liver in rats. The present study aimed to investigate the possible antifibrotic properties in CCl4-induced liver fibrosis in male Sprague–Dawley rats compared to celecoxib of three novel methoxylated pyrazolo[3,4-d]pyrimidines. The three newly synthesized compounds were proved to be safe candidates. They showed a therapeutic effect against severe CCl4-induced fibrosis but at different degrees. The three compounds were able to partially reverse hepatic architectural distortion and reduce the fibrotic severity by showing antioxidant properties reducing MDA with increasing GSH and SOD levels, remodeling the extracellular matrix proteins and liver enzymes balance, and reducing the level of proinflammatory (TNF-α and IL-6) and profibrogenic (TGF-β) cytokines. The results revealed that the dimethoxy-analog exhibited the greatest activity in all the previously mentioned parameters compared to celecoxib and the other two analogs which could be attributed to the different methoxylation patterns of the derivatives. Collectively, the dimethoxy-derivative could be considered a safe promising antifibrotic candidate. [ABSTRACT FROM AUTHOR]

Calphostin C (Cal-C) is a protein kinase C (PKC) inhibitor that binds to its C1 domain. The aim of the present study was to elucidate the action of Cal-C in addition to PKC inhibition. First, we confirmed that Cal-C at low concentrations (<200 nM) inhibit phorbol ester-induced PKC translocation and G-protein-coupled receptor (GPCR)-mediated PKC activation. Cal-C at higher concentrations (>2 μM) increased intracellular calcium ion concentrations ([Ca2+] i) in a concentration-dependent manner. The origin of this increase is the mobilization of the endoplasmic reticulum (ER), which does not involve GPCR or ryanodine receptors. Cal-C at high concentrations also cause structural changes in the ER, such as the formation of vacuoles and aggregates, and calcium leakage from the ER. At 2 μM, Cal-C translocated a calcium-sensitive PKCα. Studies using a C-kinase activity reporter and a myristoylated alanine-rich protein kinase C substrate fused with green fluorescent protein (GFP) have also revealed that Cal-C at high concentrations activate PKC in living cells. Additionally, the PKC-activating effects of Cal-C were light-dependent. Finally, studies using Si-DMA, an indicator of singlet oxygen, showed that Cal-C at high concentrations generated singlet oxygen, causing structural changes in the ER and leakage of calcium into the cytosol, which triggered PKC activation. This study confirms the novel action of Cal-C, solely considered a PKC inhibitor. Cal-C acted as a PKC inhibitor at low concentrations and a PKC activator at high concentrations by generating singlet oxygen in a light-dependent manner, suggesting that Cal-C can be used in photodynamic therapy. • Cal-C, which has been considered a PKC inhibitor, exhibits PKC activating activity at high concentrations. • Cal-C at high concentrations activate PKC by inducing changes in endoplasmic reticulum morphology and an increase in intracellular calcium. • Cal-C at high cencetrations generate singlet oxygen in a light-dependent manner and are cytotoxic. • Normal cells and several cancer-derived cells showed similar effects. [ABSTRACT FROM AUTHOR]

• Combination TKIs with immunotherapy are safe and effective for unresectable ATC. • More than one-third of ATC patients could convert to operable status. • Most patients response well, including more than a quarter complete response. • Longer survival was observed in ATC patients with BRAF V600E mutations. Anaplastic thyroid carcinoma (ATC) is rare but has a very poor prognosis. New therapeutic options such as multikinase inhibitors and selective tyrosine kinase inhibitors have revolutionized the treatment of ATC, with immunotherapy also showing encouraging effects. This study evaluated the efficacy and safety of kinase inhibitors combined with an anti-PD-1 inhibitor as first-line treatment, as well as in the neoadjuvant setting for patients with unresectable ATC. This retrospective single-center study recruited consecutive patients with stage IVB and IVC ATC who received first-line kinase inhibitors plus immunotherapy between June 2021 and June 2023. The patients were treated with either selective or multi-kinase inhibitors (dabrafenib/trametinib, lenvatinib, or anlotinib) in combination with one immune checkpoint inhibitor (pembrolizumab, sintilimab, or camrelizumab). The endpoints included overall survival (OS), progression-free survival (PFS), response evaluation, and feasibility of R0/R1 resection. Eighteen patients were included in this analysis. The median OS (mOS) was 14.0 months and the 12-month survival rate was 55.6 %. The mOS in BRAF V600E mutated ATC was not reached, significantly longer than non-BRAF V600E mutated ATC (4.0 months [95 %CI, 1.1–6.9], p = 0.049). Among evaluable patients, 5 achieved a complete response (CR) and 6 patients achieved partial response (PR). The best ORR was 61.1 %. Surgical resection was feasible in 7/18 (38.9 %) patients. One grade 5 adverse event (AE) occurred. Most AEs were well tolerated. Combination kinase inhibitors with immunotherapy as first-line therapy are safe and effective for the treatment of unresectable ATC, especially with BRAF V600E mutation. [ABSTRACT FROM AUTHOR]

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies.

Background: Abemaciclib is a new oral targeted treatment option for patients with advanced breast cancer. The emerging field of oral antitumor therapeutics presents challenges for both patients and healthcare teams; non-adherence and high inter-individual pharmacokinetic variability can influence response rates. Methods: For monitoring abemaciclib in human sera, a rapid novel ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated. Sample preparation was based on a protein precipitation step followed by on-line solid phase extraction. Chromatographic separation was achieved using a biphenyl column and the isotope labeled standard abemaciclib-d8 was used for quantification. Results: The method showed linearity over a wide calibration range from 20.0 to 2500 ng/mL. With accuracies and precisions of ≤13.9% and ≤4.42%, respectively, the validation results were within the criteria of acceptance. The fitness of the method was tested by monitoring abemaciclib levels under compassionate use for a single individual. Conclusions: The novelty of the presented two dimensional isotope dilution UHPLC-MS/MS method is in the semi-automated sample preparation, which results in negligible matrix effects, thereby allowing the introduction of abemaciclib into robust routine therapeutic drug monitoring (TDM). This method provides an efficient tool to verify the usefulness of personalized anticancer therapy in clinical practice.

Cyclin-dependent kinase 9 (CDK9) activity is correlated with worse outcomes of triple-negative breast cancer (TNBC) patients. The heterodimer between CDK9 with cyclin T1 is essential for maintaining the active state of the kinase and targeting this protein–protein interaction (PPI) may offer promising avenues for selective CDK9 inhibition. Herein, we designed and generated a library of metal complexes bearing the 7-chloro-2-phenylquinoline CˆN ligand and tested their activity against the CDK9–cyclin T1 PPI. Complex 1 bound to CDK9 via an enthalpically-driven binding mode, leading to disruption of the CDK9–cyclin T1 interaction in vitro and in cellulo. Importantly, complex 1 showed promising anti-metastatic activity against TNBC allografts in mice and was comparably active compared to cisplatin. To our knowledge, 1 is the first CDK9–cyclin T1 PPI inhibitor with anti-metastatic activity against TNBC. Complex 1 could serve as a new platform for the future design of more efficacious kinase inhibitors against cancer, including TNBC.

Introduction: Tyrosine kinase inhibitor drugs (TKIs) are highly effective cancer drugs, yet many TKIs are associated with various forms of cardiotoxicity. The mechanisms underlying these drug-induced adverse events remain poorly understood. We studied mechanisms of TKI-induced cardiotoxicity by integrating several complementary approaches, including comprehensive transcriptomics, mechanistic mathematical modeling, and physiological assays in cultured human cardiac myocytes.Methods: Induced pluripotent stem cells (iPSCs) from two healthy donors were differentiated into cardiac myocytes (iPSC-CMs), and cells were treated with a panel of 26 FDA-approved TKIs. Drug-induced changes in gene expression were quantified using mRNA-seq, changes in gene expression were integrated into a mechanistic mathematical model of electrophysiology and contraction, and simulation results were used to predict physiological outcomes.Results: Experimental recordings of action potentials, intracellular calcium, and contraction in iPSC-CMs demonstrated that modeling predictions were accurate, with 81% of modeling predictions across the two cell lines confirmed experimentally. Surprisingly, simulations of how TKI-treated iPSC-CMs would respond to an additional arrhythmogenic insult, namely, hypokalemia, predicted dramatic differences between cell lines in how drugs affected arrhythmia susceptibility, and these predictions were confirmed experimentally. Computational analysis revealed that differences between cell lines in the upregulation or downregulation of particular ion channels could explain how TKI-treated cells responded differently to hypokalemia.Discussion: Overall, the study identifies transcriptional mechanisms underlying cardiotoxicity caused by TKIs, and illustrates a novel approach for integrating transcriptomics with mechanistic mathematical models to generate experimentally testable, individual-specific predictions of adverse event risk.