Discovery Of Inhibitors Research Articles

Discovery and Development of HDAC Inhibitors: Approaches for the Treatment of Cancer a Mini-review.

Histone deacetylase (HDAC) inhibitors have emerged as promising cancer therapeutics due to their ability to induce differentiation, cell cycle arrest, and apoptosis in cancer cells. In the present review, we have described the systemic discovery and development of HDAC inhibitors. Researchers across the globe have identified various small molecules like benzo[d][1,3]dioxol derivatives, belinostat analogs, pyrazine derivatives, quinazolin-4-one-based derivatives, 2,4-imidazolinedione derivatives, acridine hydroxamic acid derivatives, coumarin derivatives, tetrahydroisoquinoline derivatives, thiazole-5-carboxamide, salicylamide derivatives, β-peptoid-capped HDAC inhibitors, quinazoline derivatives, benzimidazole and benzothiazole derivatives, and β- elemene scaffold containing HDAC inhibitors. Most of the scaffolds have shown attractive IC50 (μM) in various cell lines like HDAC1, HDAC2, HDAC6, PI3K, HeLa, MDA-MB-231, MCF-10A, MCF-7, U937, K562 and Bcr-Abl cell lines.

Protein kinases as therapeutic targets for Alzheimer’s disease: a brief review

Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disorder, with an unknown etiology and a multifactorial pathophysiology characterized by protein misfolding, neuroinflammation, and neuronal loss. There are three well-discussed main hypotheses for the pathophysiology of AD, which are related to i) the accumulation of amyloid β (Aβ) protein aggregates in the extracellular space, ii) deposition of hyperphosphorylated tau fragments as neurofibrillary tangles, and iii) dysregulation of hemostasis of some neurotransmitters involved in the disease, such as acetylcholine (ACh) and glutamate. The association of all these factors is responsible for installing oxidative stress and neuroinflammation, which contribute to progressive neuronal death in specific brain regions. More recently, other remarkable pathological characteristics have been described, involving changes in all levels of cellular components, especially in the action and function of protein kinases. These enzymes are crucial for cellular regulation since they play a pivotal role in the phosphorylation of protein substrates by transferring a phosphate group from the ATP molecule to threonine, serine, or tyrosine residues. In more recent studies, some kinases have been especially reported by their role in inflammatory and oxidative processes associated to AD, such as cAMP-dependent protein kinase A (PKA), cyclin-dependent protein kinase 5 (CDK5), glycogen synthase kinase 3β (GSK-3β), and the microtubule affinity regulatory kinases (MARKs). Under homeostatic conditions, protein kinases act as cellular signals, directing physiological responses, but in AD pathogenesis, these enzymes have an exacerbated activity in the brain, justifying the need for a better comprehension of their function and role, and how new kinase inhibitors could lead to innovative drugs. In this context, this brief review aimed to compile the literature data related to the most recent efforts and strategies in Medicinal Chemistry in the discovery of new kinase inhibitors, opening new ways to AD therapeutics.

Discovery of novel NLRP3 inhibitors based on machine learning and physical methods

Discovery of novel NLRP3 inhibitors based on machine learning and physical methods

Target Fisher: a Consensus Structure-Based Target Prediction Tool, and its Application in the Discovery of Selective MAO-B Inhibitors.

In this work we introduce Target Fisher, a consensus structure-based target prediction tool that integrates molecular docking and machine learning with the aim to aid in the identification of potential biological targets and the optimization of the use of bioassays. Target Fisher uses per-residue energy decomposition profiles extracted from docking poses as fingerprints to train target-specific machine learning models. It provides predictions for a curated set of 37 protein targets, covering a diverse range of biological entities, and offers a user-friendly interface accessible via a web server (https://gqc.quimica.unlp.edu.ar/targetfisher/). In this sense, Target Fisher is a valuable tool to aid organic and medicinal chemistry groups in target identification, drug discovery and drug repurposing. As a case study, we demonstrate the efficacy of Target Fisher by screening a small library of assorted natural products for targets relevant to neurodegenerative diseases, which resulted in the identification and experimental validation of selective inhibitors of monoamine oxidase B (MAO-B).

Improving docking and virtual screening performance using AlphaFold2 multi-state modeling for kinases

Structure-based virtual screening (SBVS) is a crucial computational approach in drug discovery, but its performance is sensitive to structural variations. Kinases, which are major drug targets, exemplify this challenge due to active site conformational changes caused by different inhibitor types. Most experimentally determined kinase structures have the DFGin state, potentially biasing SBVS towards type I inhibitors and limiting the discovery of diverse scaffolds. We introduce a multi-state modeling (MSM) protocol for AlphaFold2 (AF2) kinase structures using state-specific templates to address these challenges. Our comprehensive benchmarks evaluate predicted model qualities, binding pose prediction accuracy, and hit compound identification through ensemble SBVS. Results demonstrate that MSM models exhibit comparable or improved structural accuracy compared to standard AF2 models, enhancing pose prediction accuracy and effectively capturing kinase-ligand interactions. In virtual screening experiments, our MSM approach consistently outperforms standard AF2 and AF3 modeling, particularly in identifying diverse hit compounds. This study highlights the potential of MSM in broadening kinase inhibitor discovery by facilitating the identification of chemically diverse inhibitors, offering a promising solution to the structural bias problem in kinase-targeted drug discovery.

Structure-Guided Discovery of Selective USP7 Inhibitors with In Vivo Activity.

Inhibition of ubiquitin-specific protease 7, USP7, has been proposed as a mechanism to affect many disease processes, primarily those implicated in oncology. The bound crystal structure of a published high-throughput screening hit with low-micromolar affinity for USP7 identified three regions of the compound for structure-guided optimization. Replacing one side of the compound with different aromatic moieties gave little improvement in affinity, and the central piperidine could not be improved. However, the binding site for the other side of the compound was poorly defined in the crystal structure, which suggested a wide variety of synthetically accessible options for optimization. These were assessed by screening reaction mixtures that introduced different substituents to this other side. Subsequent optimization led to a compound with low-nanomolar affinity for USP7, which showed target engagement in tumors, was tolerated in mice, and showed efficacy in xenograft models.

Medicinal chemistry insights in neuronal nitric oxide synthase inhibitors containing nitrogen heterocyclic compounds: a mini review.

Many scientific reports over the last two decades have focused on the discovery and development of novel nNOS inhibitors. The structural identity of isoforms, bioavailability, pharmacokinetic, and safety profile issues remain major obstacles in the discovery of more potent and selective nNOS inhibitors. This review aims to provide an in-depth overview of the molecular interaction patterns between nNOS active site and inhibitors containing structurally diverse nitrogen heterocyclic compounds and highlight the structural properties needed to develop selective nNOS inhibitors. Previously published data allowed the usage of the structure-driven approach in the designing of selective nNOS inhibitors, which relies on the specific structural features required to achieve isoform-selectivity towards nNOS. The incorporation of chiral pyrrolidine ring, two aminopyridine heads, or a specific amino tail group, along with the inhibitor's capacity to adopt the curled conformation in the nNOS environment significantly strengthens the molecular interaction between the inhibitor and nNOS residues by forming specific electrostatic interactions and non-bonded contacts that are vital for isoform selectivity. Additional structure-activity relationship investigations are necessary to elucidate more structural characteristics that will ultimately resolve the exact structural basis required for isoform-selective inhibition of nNOS.

High-Throughput Discovery of Synthetic Siderophores for Trojan Horse Antibiotics.

To cause infection, bacterial pathogens must overcome host immune factors and barriers to nutrient acquisition. Reproducing these aspects of host physiology in vitro has shown great promise for antibacterial drug discovery. When used as a bacterial growth medium, human serum replicates several aspects of the host environment, including innate immunity and iron limitation. We previously reported that a high-throughput chemical screen using serum as the growth medium enabled the discovery of novel growth inhibitors overlooked by conventional screens. Here, we report that a subset of compounds from this high-throughput serum screen display an unexpected growth enhancing phenotype and are enriched for synthetic siderophores. We selected 35 compounds of diverse chemical structure and quantified their ability to enhance bacterial growth in human serum. We show that many of these compounds chelate iron, suggesting they were acting as siderophores and providing iron to the bacteria. For two different pharmacophores represented among these synthetic siderophores, conjugation to the β-lactam antibiotic ampicillin imparted iron-dependent enhancement in antibacterial activity. Conjugation of the most potent growth-enhancing synthetic siderophore with the monobactam aztreonam produced MLEB-22043, a broad-spectrum antibiotic with significantly improved activity against Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. This synthetic siderophore-monobactam conjugate uses multiple TonB-dependent transporters for uptake into P. aeruginosa. Like aztreonam, MLEB-22043 demonstrated activity against metallo-β-lactamase expressing bacteria, and, when combined with the β-lactamase inhibitor avibactam, was active against clinical strains coexpressing the NDM-1 metallo-β-lactamase and serine β-lactamases. Our work shows that human serum is an effective bacterial growth medium for the high-throughput discovery of synthetic siderophores, enabling the development of novel Trojan Horse antibiotics.

MetaCGRP is a high-precision meta-model for large-scale identification of CGRP inhibitors using multi-view information

Migraine is considered one of the debilitating primary headache conditions with an estimated worldwide occurrence of approximately 14–15%, contributing highly to factors responsible for global disability. Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a crucial role in the pathophysiology of migraines and thus, its inhibition can help relieve migraine symptoms. However, conventional process of CGRP drug development has been laborious and time-consuming with incurred costs exceeding one billion dollars. On the other hand, machine learning (ML)-based approaches that are capable of accurately identifying CGRP inhibitors could greatly facilitate in expediting the discovery of novel CGRP drugs. Therefore, this study proposes a novel and high-accuracy meta-model, namely MetaCGRP, that can precisely identify CGRP inhibitors. To the best of our knowledge, MetaCGRP is the first SMILES-based approach that has been developed to identify CGRP inhibitors without the use of 3D structural information. In brief, we initially employed different molecular representation methods coupled with popular ML algorithms to construct a pool of baseline models. Then, all baseline models were optimized and used to generate multi-view features. Finally, we employed the feature selection method to optimize the multi-view features and determine the best feature subset to enable the construction of the meta-model. Both cross-validation and independent tests indicated that MetaCGRP clearly outperforms several conventional ML classifiers, with accuracies of 0.898 and 0.799 on the training and independent test datasets, respectively. In addition, MetaCGRP in conjunction with molecular docking was utilized to identify five potential natural product candidates from Thai herbal pharmacopoeia and analyze their binding affinity and interactions to CGRP. To facilitate community-wide efforts in expediting the discovery of novel CGRP inhibitors, a user-friendly web server for MetaCGRP is freely available at https://pmlabqsar.pythonanywhere.com/MetaCGRP.

In Silico Discovery of a Novel PI3Kδ Inhibitor Incorporating 3,5,7-Trihydroxychroman-4-one Targeting Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, and it is highly aggressive and heterogeneous. Targeted therapy is still the main treatment method used in clinic due to its lower risk of side effects and personalized medication. Excessive activation of PI3Kδ in DLBCL leads to abnormal activation of the PI3K/Akt pathway, promoting the occurrence and development of DLBCL. The side effects of existing PI3Kδ inhibitors limit their clinical application. The discovery of PI3Kδ inhibitors with novel structures and minimal side effects is urgently needed. This study constructed a PI3Kδ inhibitor screening model to screen natural product libraries. Revealing the mechanism of natural product therapy for DLBCL through network pharmacology, kinase assays, and molecular dynamics. The results of molecular docking indicated that Silibinin had a high docking score and a good binding mode with PI3Kδ. The results of network pharmacology indicated that Silibinin could exert therapeutic effects on DLBCL by inhibiting PI3Kδ activity and affecting the PI3K/Akt pathway. The kinase assays indicated that Silibinin concentration dependently inhibited the activity of PI3Kδ. The results of molecular dynamics indicated that Silibinin could stably bind to PI3Kδ. Silibinin was a structurally novel 3,5,7-trihydroxychroman-4-one PI3Kδ inhibitor, providing valuable information for the subsequent discovery of PI3Kδ inhibitors.

Structure-activity relationship of amino acid analogs to probe the binding pocket of sodium-coupled neutral amino acid transporter SNAT2.

The sodium-coupled neutral amino acid transporter SNAT2 (SLC38A2) has been shown to have important physiological functions and is implicated in various diseases like cancer. However, few compounds targeting this transporter have been identified and little is known about the structural requirements for SNAT2 binding. In this study, the aim was to establish the basic structure-activity relationship for SNAT2 using amino acid analogs. These analogs were first studied for their ability to inhibit SNAT2-mediated 3H-glycine uptake in hyperosmotically treated PC-3 cells. Then to identify substrates a FLIPR membrane potential assay and o-phthalaldehyde derivatization of intracellular amino with subsequent quantification using HPLC-Fl was used. The results showed that ester derivatives of the C-terminus maintained SNAT2 affinity, suggesting that the negative charge was less important. On the other hand, the positive charge at the N-terminus of the substrate and the ability to donate at least two hydrogen bonds to the binding site appeared important for SNAT2 recognition of the amine. Side chain charged amino acids generally had no affinity for SNAT2, but their non-charged derivatives were able to inhibit SNAT2-mediated 3H-glycine uptake, while also showing that amino acids of a notable length still had affinity for SNAT2. Several amino acid analogs appeared to be novel substrates of SNAT2, while γ-benzyl L-glutamate seemed to be inefficiently translocated by SNAT2. Elaborating on this structure could lead to the discovery of non-translocated inhibitors of SNAT2. Thus, the present study provides valuable insights into the basic structural binding requirements for SNAT2 and can aid the future discovery of compounds that target SNAT2.

Discovery of Trypanosoma brucei inhibitors enabled by a unified synthesis of diverse sulfonyl fluorides

Sets of electrophilic probes are generally prepared using a narrow toolkit of robust reactions, which tends to limit both their structural and functional diversity. A unified synthesis of skeletally-diverse sulfonyl fluorides was developed that relied upon photoredox-catalysed dehydrogenative couplings between hetaryl sulfonyl fluorides and hydrogen donor building blocks. A set of 32 diverse probes was prepared, and then screened against Trypanosoma brucei. Four of the probes were found to have sub-micromolar anti-trypanosomal activity. A chemical proteomic approach, harnessing an alkynylated analogue and broad-spectrum fluorophosphonate tools, provided insights into the observed anti-trypanosomal activity, which likely stems from covalent modification of multiple protein targets. It is envisaged that the unified diversity-oriented approach may enable the discovery of electrophilic probes that have value in the elucidation of biological and biomedical mechanisms.

Pharmacophore‐Based Modeling, Synthesis, and Biological Evaluation of Novel Quinazoline/Quinoline Derivatives: Discovery of EGFR Inhibitors with Low Nanomolar Activity

AbstractThe main aim of this study is to obtain novel molecules that are more selective on cancer cells compared to healthy cells. For this purpose, four hit molecules are identified using 11 new pharmacophore hypotheses followed by scanning the in‐house database. Then, based on those hit molecules, the synthesis and analysis of four different series (three quinazolines and one quinoline series) are carried out, and their anticancer activity is investigated. Finally, by using molecular docking and dynamics simulation methods, binding mode and structure–activity relationship are examined. Among the quinazolin‐4(3H)‐one derivatives, those containing halogen atom are found to be potentially effective, while the best epidermal growth factor receptor (EGFR) inhibition and apoptosis induction are displayed by compounds containing 4‐amino‐1,2,4‐triazole moiety. Notably, four compounds (4h, 8d, 8l, and 8m) show EGFR inhibition activity at 5.298 ± 0.164, 5.46 ± 0.221, 2.670 ± 0.124, and 2.191 ± 0.908 × 10−9 m, their inhibitory activity is similar to or stronger than gefitinib (IC50: 4.169 ± 0.156 × 10−9 m). In addition, EGFR inhibitor concentration of 4g, 8e, and 8o is determined as 27588 ± 6.945, 52.41 ± 2.312, and 33657 ± 8.512 × 10−9 m. These findings indicate that generated pharmacophore hypotheses successfully determine new EGFR inhibitors. In conclusion, four novel compounds, more active than gefitinib with fewer side effects, are reached, and the structure–activity relationships are clarified.

Structure-based discovery of first inhibitors targeting the helicase activity of human PIF1.

PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.

Thermal proteome profiling reveals fructose-1,6-bisphosphate as a phosphate donor to activate phosphoglycerate mutase 1

Deep understanding of sugar metabolite-protein interactions should provide implications on sugar metabolic reprogramming in human physiopathology. Although tremendous efforts have been made for determining individual event, global profiling of such interactome remains challenging. Here we describe thermal proteome profiling of glycolytic metabolite fructose-1,6-bisphosphate (FBP)-interacting proteins. Our results reveal a chemical signaling role of FBP which acts as a phosphate donor to activate phosphoglycerate mutase 1 (PGAM1) and contribute an intrapathway feedback for glycolysis and cell proliferation. At molecular level, FBP donates either C1-O-phosphate or C6-O-phosphate to the catalytic histidine of PGAM1 to form 3-phosphate histidine (3-pHis) modification. Importantly, structure-activity relationship studies facilitate the discovery of PGAM1 orthostatic inhibitors which can potentially restrain cancer cell proliferation. Collectively we have profiled a spectrum of FBP interactome, and discovered a unique covalent signaling function of FBP that supports Warburg effect via histidine phosphorylation which inspires the development of pharmacological tools targeting sugar metabolism.

Discovery of Janus Kinase and Histone Deacetylase Dual Inhibitors as a New Strategy to Treat Psoriasis.

Psoriasis is a common, chronic, recurrent, and inflammatory skin disease, which causes physical and psychological problems in patients and lacks effective and economic therapeutics. Herein, we designed Janus kinase (JAK) and histone deacetylase (HDAC) dual inhibitors as a new strategy for the treatment of psoriasis. In particular, compound 11i was identified with excellent inhibitory activity toward JAKs (JAK2 IC50 = 0.49 nM) and HDACs (HDAC6 IC50 = 12 nM). Moreover, it exhibited potent activities in inhibiting the proliferation of TNF-α-induced HaCAT cells and the production of nitric oxide. Importantly, compound 11i significantly ameliorated psoriasis-like skin lesions in an imiquimod-induced murine model with low toxicity, which was superior to JAK inhibitor momelotinib, HDAC inhibitor vorinostat, and their combination. This work provided a proof-of-concept for JAK/HDAC dual inhibitors as a promising strategy for the treatment of psoriasis.

Discovery of Novel Thiazole-Based SIRT2 Inhibitors as Anticancer Agents: Molecular Modeling, Chemical Synthesis and Biological Assays.

The search and development of effective sirtuin small molecule inhibitors (SIRTIs) continues to draw great attention due to their wide range of pharmacological applications. Based on SIRTs' involvement in different biological pathways, their ligands were investigated for many diseases, such as cancer, neurodegenerative disorders, diabetes, cardiovascular diseases and autoimmune diseases. The elucidation of a substantial number of SIRT2-ligand complexes is steering the identification of novel and more selective modulators. Among them, SIRT2 in the presence of the SirReal2 analog series was the most studied. On this basis, we recently reported structure-based analyses leading to the discovery of thiazole-based compounds acting as SIRT2 inhibitors (T1, SIRT2 IC50 = 17.3 µM). Herein, ligand-based approaches followed by molecular docking simulations allowed us to evaluate in silico a novel small series of thiazoles (3a-3d and 5a, 5d) as putative SIRT2 inhibitors. Results from the computational studies revealed comparable molecular interaction fields (MIFs) and docking positionings of most of these compounds with respect to reference SIRT2Is. Biochemical and biological assays validated this study and pointed to compound 5a (SIRT2 IC50 = 9.0 µM) as the most interesting SIRT2I that was worthy of further development as an anticancer agent.

Emerging Role of Natural Topoisomerase Inhibitors as Anticancer agents

Abstract: Topoisomerases I and II are the functionally two forms of DNA topoisomerase. In anticancer research, novel anticancer chemotherapeutical capable of blocking topoisomerase enzymes have been discovered. Most commonly, topoisomerase causes replication fork arrest and doublestrand breaks, and this is how a clinically successful topoisomerase-targeting anticancer medicines work. Unfortunately, this novel mechanism of action has been linked to the development of secondary malignancies as well as cardiotoxicity. The specific binding locations and mechanisms of topoisomerase poisons have been identified by studying the structures of topoisomerase-drug-DNA ternary complexes. Recent breakthroughs in science have revealed that isoform-specific human topoisomerase II poison could be created as safer anticancer drug molecules. It may also be able to develop catalytic inhibitors of topoisomerases by focusing on their inactive conformations. In addition to this, the discovery of new bacterial topoisomerase inhibitor molecules and regulatory proteins could lead to the discovery of new human topoisomerase inhibitors. As a result, biologists, organic chemists, and medicinal chemists worldwide have been identifying, designing, synthesizing, and testing a variety of novel topoisomerase-targeting bioactive compounds. This review focused on topoisomerase inhibitors, their mechanisms of action, and different types of topoisomerase inhibitors that have been developed during the last ten years.

Discovery of Novel PTP1B Inhibitors by High-throughput Virtual Screening.

To Discover novel PTP1B inhibitors by high-throughput virtual screening Background: Type 2 Diabetes is a significant global health concern. According to projections, the estimated number of individuals affected by the condition will reach 578 million by the year 2030 and is expected to further increase to 700 million deaths by 2045. Protein Tyrosine Phosphatase 1B is an enzymatic protein that has a negative regulatory effect on the pathways involved in insulin signaling. This regulatory action ultimately results in the development of insulin resistance and the subsequent elevation of glucose levels in the bloodstream. The proper functioning of insulin signaling is essential for maintaining glucose homeostasis, whereas the disruption of insulin signaling can result in the development of type 2 diabetes. Consequently, we sought to utilize PTP1B as a drug target in this investigation. The purpose of our study was to identify novel PTP1B inhibitors as a potential treatment for managing type 2 diabetes. To discover potent PTP1B inhibitors, we have screened the Maybridge HitDiscover database by SBVS. Top hits have been passed based on various drug-likeness rules, toxicity predictions, ADME assessment, Consensus Molecular docking, DFT, and 300 ns MD Simulations. Two compounds have been identified with strong binding affinity at the active site of PTP1B along with drug-like properties, efficient ADME, low toxicity, and high stability. The identified molecules could potentially manage T2DM effectively by inhibiting PTP1B, providing a promising avenue for therapeutic strategies.

Discovery of CLKs inhibitors for the treatment of non-small cell lung cancer

Targeted inhibition of the Wnt pathway is a promising strategy for treating NSCLC. CDC2-like kinase 2 (CLK2), a dual-specificity kinase responsible for phosphorylating serine/arginine-rich (SR) proteins, can modulate Wnt signaling through the alternative splicing of Wnt target genes, making CLK2 an attractive therapeutic target for NSCLC. In this study, we report the synthesis, optimization, and evaluation of CLK2 inhibitors that effectively suppress the proliferation of NSCLC cells, with the identification of the lead compound LBM22. Notably, compound LBM22 demonstrated potent inhibition of CLK2 (IC50 = 3.9 nM), leading to broad suppression of NSCLC cells proliferation and induction of apoptosis. Furthermore, LBM22 dose-dependently suppressed SR protein phosphorylation (pSRSF4, pSRSF5, and pSRSF6) in NSCLC cells, while downregulating the expression of Wnt pathway-related proteins (p-β-catenin, Axin 2, and c-Myc) as well as anti-apoptotic proteins (Bcl-2 and Mcl-1). Additionally, significant antiproliferative activity was observed for LBM22 in 3D cultured H1975OR cells. In conclusion, LBM22 emerges as a promising CLK2 inhibitor for the treatment of NSCLC.