Cytotoxic activity and cell specificity of a novel LHRH peptide drug conjugate, D-Cys6-LHRH vedotin, against ovarian cancer cell lines.

Ovarian cancer is the most deadly female gynaecological malignancy in developed countries and new treatments are urgently needed. The luteinising hormone releasing hormone (LHRH) peptide drug conjugate Zoptarelin doxorubicin is one such potential new drug modality that entered clinical trials for treating LHRH receptor-positive gynaecological cancers. However, development stopped after disappointing Phase 3 results in 2017. We believe the lack of efficacy was due to linker instability and payload potency. In this work, we replaced its linker-toxin with vedotin (MC-VC-PABC-MMAE), yielding the novel peptide drug conjugate D-Cys6-LHRH vedotin. A GI50 and cell specificity comparison against cancerous and non-cancerous ovarian cell lines showed significantly superior bioactivity and selectivity over Zoptarelin doxorubicin (GI50 4 vs. 453 nM) and other chemotherapeutic drugs used for treating ovarian cancers. Our results suggest D-Cys6-LHRH vedotin can potentially be used as a treatment for ovarian cancer.

Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines.

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction.

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

MicroRNA Expression Profiles in Human Samples and Cell Lines Revealed Nine miRNAs Associated with Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

Metastasis and drug resistance are major contributors to cancer-related fatalities worldwide. In ovarian cancer (OC), a staggering 70% develop resistance to the front-line therapy, cisplatin. Despite proposed mechanisms, the molecular events driving cisplatin resistance remain unclear. Dysregulated microRNAs (miRNAs) play a role in OC initiation, progression, and chemoresistance, yet few studies have compared miRNA expression in OC samples and cell lines. This study aimed to identify key miRNAs involved in the cisplatin resistance of high-grade-serous-ovarian-cancer (HGSOC), the most common gynecological malignancy. MiRNA expression profiles were conducted on RNA isolated from formalin-fixed-paraffin-embedded human ovarian tumor samples and HGSOC cell lines. Nine miRNAs were identified in both sample types. Targeting these with oligonucleotide miRNA inhibitors (OMIs) reduced proliferation by more than 50% for miR-203a, miR-96-5p, miR-10a-5p, miR-141-3p, miR-200c-3p, miR-182-5p, miR-183-5p, and miR-1206. OMIs significantly reduced migration for miR-183-5p, miR-203a, miR-296-5p, and miR-1206. Molecular pathway analysis revealed that the nine miRNAs regulate pathways associated with proliferation, invasion, and chemoresistance through PTEN, ZEB1, FOXO1, and SNAI2. High expression of miR-1206, miR-10a-5p, miR-141-3p, and miR-96-5p correlated with poor prognosis in OC patients according to the KM plotter database. These nine miRNAs could be used as targets for therapy and as markers of cisplatin response.

Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

Wide-Targeted Semi-Quantitative Analysis of Acidic Glycosphingolipids in Cell Lines and Urine to Develop Potential Screening Biomarkers for Renal Cell Carcinoma.

Glycosphingolipids (GSLs), mainly located in the cell membrane, play various roles in cancer cell function. GSLs have potential as renal cell carcinoma (RCC) biomarkers; however, their analysis in body fluids is challenging because of the complexity of numerous glycans and ceramides. Therefore, we applied wide-targeted lipidomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM) based on theoretical mass to perform a comprehensive measurement of GSLs and evaluate their potency as urinary biomarkers. In semi-quantitative lipidomics, 240 SRM transitions were set based on the reported/speculated structures. We verified the feasibility of measuring GSLs in cells and medium and found that disialosyl globopentaosylceramide (DSGb5 (d18:1/16:0)) increased GSL in the ACHN medium. LC-MS/MS analysis of urine samples from clear cell RCC (ccRCC) patients and healthy controls showed a significant increase in the peak intensity of urinary DSGb5 (d18:1/16:0) in the ccRCC group compared with that in the control group. Receiver operating characteristic analysis indicated that urinary DSGb5 could serve as a sensitive and specific marker for RCC screening, with an AUC of 0.89. This study demonstrated the possibility of urinary screening using DSGb5 (d18:1/16:0). In conclusion, urinary DSGb5 (d18:1/16:0) was a potential biomarker for cancer screening, which could contribute to the treatment of RCC patients.

Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Proximity-Based Labeling Identifies MHC Class II and CD37 as B Cell Receptor-Proximal Proteins with Immunological Functions.

The BCR allows for Ag-driven B cell activation and subsequent Ag endocytosis, processing, and presentation to recruit T cell help. Core drivers of BCR signaling and endocytosis are motifs within the receptor's cytoplasmic tail (primarily CD79). However, BCR function can be tuned by other proximal cellular elements, such as CD20 and membrane lipid microdomains. To identify additional proteins that could modulate BCR function, we used a proximity-based biotinylation technique paired with mass spectrometry to identify molecular neighbors of the murine IgM BCR. Those neighbors include MHC class II molecules, integrins, various transporters, and membrane microdomain proteins. Class II molecules, some of which are invariant chain-associated nascent class II, are a readily detected BCR neighbor. This finding is consistent with reports of BCR-class II association within intracellular compartments. The BCR is also in close proximity to multiple proteins involved in the formation of membrane microdomains, including CD37, raftlin, and Ig superfamily member 8. Known defects in T cell-dependent humoral immunity in CD37 knockout mice suggest a role for CD37 in BCR function. In line with this notion, CRISPR-based knockout of CD37 expression in a B cell line heightens BCR signaling, slows BCR endocytosis, and tempers formation of peptide-class II complexes. These results indicate that BCR molecular neighbors can impact membrane-mediated BCR functions. Overall, a proximity-based labeling technique allowed for identification of multiple previously unknown BCR molecular neighbors, including the tetraspanin protein CD37, which can modulate BCR function.

A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

A Custom Panel for Profiling Microglia Gene Expression.

Despite being immune cells of the central nervous system (CNS), microglia contribute to CNS development, maturation, and homeostasis, and microglia dysfunction has been implicated in several neurological disorders. Recent advancements in single-cell studies have uncovered unique microglia-specific gene expression. However, there is a need for a simple yet elegant multiplexed approach to quantifying microglia gene expression. To address this, we have designed a NanoString nCounter technology-based murine microglia-specific custom codeset comprising 178 genes. We analyzed RNA extracted from ex vivo adult mouse microglia, primary mouse microglia, the BV2 microglia cell line, and mouse bone marrow monocytes using our custom panel. Our findings reveal a pattern where homeostatic genes exhibit heightened expression in adult microglia, followed by primary cells, and are absent in BV2 cells, while reactive markers are elevated in primary microglia and BV2 cells. Analysis of publicly available data sets for the genes present in the panel revealed that the panel could reliably reflect the changes in microglia gene expression in response to various factors. These findings highlight that the microglia panel used offers a swift and cost-effective means to assess microglial cells and can be used to study them in varying contexts, ranging from normal homeostasis to disease models.

Proceedings of the 2023 Viral Clearance Symposium, Session 1: Regulatory Updates.

At the time of the 2023 Viral Clearance Symposium in Vienna, the ongoing revision of ICH Guideline Q5A(R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin clearly was the dominant regulatory topic. At the symposium, the changes expected for Q5A(R2) to mirror advances of scientific knowledge, for example, the inclusion of new products, including viral-vector-derived ones, that can be subject to virus clearance, deliberations around continuous manufacturing processes, the use of prior knowledge to supplement or in part replace virus validation studies, and new molecular methods for detection of adventitious viruses, were discussed by a European and a US regulator as well as representatives from industry associations that had been involved with the drafting process.

Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.

Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.

Tyrosine hydroxylase inhibits HCC progression by downregulating TGFß/Smad signaling.

The alteration of metabolic processes has been found to have significant impacts on the development of hepatocellular carcinoma (HCC). Nevertheless, the effects of dysfunction of tyrosine metabolism on the development of HCC remains to be discovered. This research demonstrated that tyrosine hydroxylase (TH), which responsible for the initial and limiting step in the bio-generation of the neuro-transmitters dopamine and adrenaline, et al. was shown to be reduced in HCC. Increased expression of TH was found facilitates the survival of HCC patients. In addition, decreased TH indicated larger tumor size, much more numbers of tumor, higher level of AFP, and the presence of cirrhosis. TH effectively impairs the growth and metastasis of HCC cells, a process dependent on the phosphorylation of serine residues (S19/S40). TH directly binds to Smad2 and hinders the cascade activation of TGFß/Smad signaling with the treatment of TGFß1. In summary, our study uncovered the non-metabolic functions of TH in the development of HCC and proposes that TH might be a promising biomarker for diagnosis as well as an innovative target for metastatic HCC.

Unraveling the significance of PPP1R1A gene in pancreatic ß-cell function: A study in INS-1 cells and human pancreatic islets.

BACKGROUND AND AIMS: The protein phosphatase 1 regulatory inhibitor subunit 1A (PPP1R1A) has been linked with insulin secretion and diabetes mellitus. Yet, its full significance in pancreatic ß-cell function remains unclear. This study aims to elucidate the role of the PPP1R1A gene in ß-cell biology using human pancreatic islets and rat INS-1 (832/13) cells. RESULTS: Disruption of Ppp1r1a in INS-1 cells was associated with reduced insulin secretion and impaired glucose uptake; however, cell viability, ROS, apoptosis or proliferation were intact. A significant downregulation of crucial ß-cell function genes such as Ins1, Ins2, Pcsk1, Cpe, Pdx1, Mafa, Isl1, Glut2, Snap25, Vamp2, Syt5, Cacna1a, Cacna1d and Cacnb3, was observed upon Ppp1r1a disruption. Furthermore, silencing Pdx1 in INS-1 cells altered PPP1R1A expression, indicating that PPP1R1A is a target gene for PDX1. Treatment with rosiglitazone increased Ppp1r1a expression, while metformin and insulin showed no effect. RNA-seq analysis of human islets revealed high PPP1R1A expression, with α-cells showing the highest levels compared to other endocrine cells. Muscle tissues exhibited greater PPP1R1A expression than pancreatic islets, liver, or adipose tissues. Co-expression analysis revealed significant correlations between PPP1R1A and genes associated with insulin biosynthesis, exocytosis machinery, and intracellular calcium transport. Overexpression of PPP1R1A in human islets augmented insulin secretion and upregulated protein expression of Insulin, MAFA, PDX1, and GLUT1, while silencing of PPP1R1A reduced Insulin, MAFA, and GLUT1 protein levels. CONCLUSION: This study provides valuable insights into the role of PPP1R1A in regulating ß-cell function and glucose homeostasis. PPP1R1A presents a promising opportunity for future therapeutic interventions.

Dysfunction in IGF2R Pathway and Associated Perturbations in Autophagy and WNT Processes in Beckwith-Wiedemann Syndrome Cell Lines.

Beckwith-Wiedemann Syndrome (BWS) is an imprinting disorder characterized by overgrowth, stemming from various genetic and epigenetic changes. This study delves into the role of IGF2 upregulation in BWS, focusing on insulin-like growth factor pathways, which are poorly known in this syndrome. We examined the IGF2R, the primary receptor of IGF2, WNT, and autophagy/lysosomal pathways in BWS patient-derived lymphoblastoid cell lines, showing different genetic and epigenetic defects. The findings reveal a decreased expression and mislocalization of IGF2R protein, suggesting receptor dysfunction. Additionally, our results point to a dysregulation in the AKT/GSK-3/mTOR pathway, along with imbalances in autophagy and the WNT pathway. In conclusion, BWS cells, regardless of the genetic/epigenetic profiles, are characterized by alteration of the IGF2R pathway that is associated with the perturbation of the autophagy and lysosome processes. These alterations seem to be a key point of the molecular pathogenesis of BWS and potentially contribute to BWS's characteristic overgrowth and cancer susceptibility. Our study also uncovers alterations in the WNT pathway across all BWS cell lines, consistent with its role in growth regulation and cancer development.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment. METHODS: By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG's catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition. RESULTS: Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors. CONCLUSIONS: We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

Neuroprotective mechanisms of luteolin in glutamate-induced oxidative stress and autophagy-mediated neuronal cell death.

Neurodegenerative diseases, characterized by progressive neuronal dysfunction and loss, pose significant health challenges. Glutamate accumulation contributes to neuronal cell death in diseases such as Alzheimer's disease. This study investigates the neuroprotective potential of Albizia lebbeck leaf extract and its major constituent, luteolin, against glutamate-induced hippocampal neuronal cell death. Glutamate-treated HT-22 cells exhibited reduced viability, altered morphology, increased ROS, and apoptosis, which were attenuated by pre-treatment with A. lebbeck extract and luteolin. Luteolin also restored mitochondrial function, decreased mitochondrial superoxide, and preserved mitochondrial morphology. Notably, we first found that luteolin inhibited the excessive process of mitophagy via the inactivation of BNIP3L/NIX and inhibited lysosomal activity. Our study suggests that glutamate-induced autophagy-mediated cell death is attenuated by luteolin via activation of mTORC1. These findings highlight the potential of A. lebbeck as a neuroprotective agent, with luteolin inhibiting glutamate-induced neurotoxicity by regulating autophagy and mitochondrial dynamics.

Development, synthesis and validation of improved c-Myc/Max inhibitors.

The pathophysiological foundations of various diseases are often subject to alteration through the utilization of small compounds, rendering them invaluable tools for the exploration and advancement of novel therapeutic strategies. Within the scope of this study, we meticulously curated a diverse library of novel small compounds meticulously designed to specifically target the c-Myc/Max complex. We conducted in vitro examinations of novel c-Myc inhibitors across a spectrum of cancer cell lines, including PANC1 (pancreatic adenocarcinoma), MCF7 (breast carcinoma), DU-145 (prostate carcinoma), and A549 (lung cancer). The initial analysis involved a 25 µM dose, which enabled the identification of potent anticancer compounds effective against a variety of tumour types. We identified c-Myc inhibitors with remarkable potency, featuring IC50 values as low as 1.6 µM and up to 40 times more effective than the reference molecule in diminishing cancer cell viability. Notably, c-Myc-i7 exhibited exceptional selectivity, displaying 37-fold and 59-fold preference for targeting prostate and breast cancers, respectively, over healthy cells. Additionally, we constructed drug-likeness models. This study underscores the potential for in vitro investigations of various tumour types using novel c-Myc inhibitors to yield ground-breaking and efficacious anticancer compounds.

High-Throughput Cell-Based Screening of Small Molecule KRAS Signaling Inhibitors Using a Homogeneous Time-Resolved Fluorescence (HTRF) Assay.

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.