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Results for “Drug Evaluation, Preclinical”

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At least 19 recordsLinked to original sources

PubMed2025

The limitations of small molecule and genetic screening in phenotypic drug discovery.

Phenotypic screens carried out with functional genomics or small molecules have led to novel biological insights, revealed previously unknown targets for drug discovery programs, and provided starting points for the development of first-in-class therapies. Despite being valuable research tools, genetic and compound screening also have significant limitations. This perspective aims to shed a light on those limitations and provide mitigation strategies when available, with a goal of helping phenotypic screening practitioners gain an understanding of how and when to best utilize either approach.

Drug Discovery↗

PubMed2025

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗

PubMed2025

An adjuvant database for preclinical evaluation of vaccines and immunotherapeutics.

Adjuvants are immunostimulators used to enhance vaccine efficacy against infectious diseases. However, current methods for evaluating their efficacy and safety are limited, hindering large-scale screening. To address this, we developed a prototype Adjuvant Database (ADB) containing transcriptome data, generated using the same protocols as the widely used Open TG-GATEs (OTG) toxicogenomics database, covering 25 adjuvants across multiple species, organs, time points, and doses. This enabled cross-database integration of ADB and OTG. Transcriptomic patterns successfully distinguished each adjuvant regardless of organs or species. Using both databases, we built machine learning models to predict adjuvanticity and hepatotoxicity. Notably, we identified colchicine's adjuvant activity and FK565's liver toxicity through data-driven analysis. Overall, ADB combined with OTG offers a framework for transcriptomics-based, data-driven screening of adjuvant candidates.

Animals↗

PubMed2025

Construction and In Vitro and In Vivo Analysis of Coxsackievirus B4 Reporter Viruses: Attenuated Virulence but Highly Efficient for Antiviral Drug Screening and Evaluation.

Coxsackievirus B4 (CVB4) is an enterovirus with one of the highest mortality rates following infection, yet research on it remains limited. To enhance the efficiency of CVB4 research, we developed the rCVB4-EGFP and rCVB4-NanoLuc reporter viruses. The replication kinetics of these reporter viruses in SH-SY5Y and HeLa cells were essentially consistent with those of the wild-type CVB4. A strong correlation was observed between the fluorescence and bioluminescence signals of rCVB4-EGFP and rCVB4-NanoLuc and viral titers at specific times postinfection. When evaluating the anti-CVB4 drug fluoxetine using these reporter viruses, the half-maximal effective concentrations derived from fluorescence signals, bioluminescence signal intensities, and viral genome copies were consistent. In In Vivo drug evaluations, because CVB4 can infect various tissues and organs, the bioluminescence signal of rCVB4-NanoLuc effectively demonstrated the antiviral effects of drugs, offering significant advantages over traditional tissue viral titer analysis. The reporter viruses exhibited reduced virulence compared with wild-type CVB4 both In Vitro, in SH-SY5Y and HeLa cells, and In Vivo, in ICR suckling mice. Although this reduced virulence may limit their application for studying pathogenic mechanisms, these reporter viruses can serve as highly efficient tools for high-throughput screening and evaluation of anti-CVB4 drugs, vaccines, and neutralizing antibodies.

Humans↗

PubMed2027

Screening of Antiviral Agents Against CHIKV Using Reporter Virus.

Chikungunya virus (CHIKV) causes a disease characterized by chronic musculoskeletal inflammation for which specific antivirals are not yet available. Currently, a supportive therapy to alleviate fever and pain is used, but it does not limit viral replication or the persistence of chronic arthritis symptoms. Thus, the identification and development of new active molecules against CHIKV is urgently needed. Here, we present a cell-based methodology that enables the implementation of a rapid and cost-effective strategy for high- and medium-throughput screening (HTS) of compounds, including repurposed drugs or novel molecules. This methodology allows for the identification of novel antiviral hits with a good activity and selectivity profile against CHIKV.

Antiviral Agents↗

PubMed2026

Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus.

In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.

Animals↗

PubMed2026

In silico screening of anti-atherosclerotic compounds from Morus alba leaves by machine learning and network pharmacology.

OBJECTIVE: This study integrates machine learning with network pharmacology, molecular docking, and molecular dynamics simulations to screen bioactive compounds from Mulberry leaves and elucidate their potential mechanisms against atherosclerosis (AS). METHODS: A training dataset of anti-AS active compounds was compiled and encoded as Morgan fingerprints. Three machine learning classifiers, specifically Random Forest (RF), Support Vector Machine (SVM), and Extreme Gradient Boosting (XG-Boost), were constructed and evaluated using multiple performance metrics. Potential active components from Mulberry leaves and AS-related targets were retrieved, followed by protein-protein interaction network construction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was then performed to evaluate binding affinities between core targets and candidate compounds, and the most stable complex was subjected to molecular dynamics simulations using GROMACS (2025). RESULTS: The RF model achieved superior performance (accuracy= 0.8354, F1 = 0.8408, AUC = 0.9119) with 100% external validation accuracy. Thirteen anti-AS candidates were prioritized from mulberry leaves, four of which have been previously documented. Network pharmacology revealed AKT1 and IL6 as core targets, enriched in pathways such as endocrine resistance. Molecular docking and dynamics simulations confirmed strong binding between oxysanguinarine and AKT1, with the complex exhibiting high stability. CONCLUSION: The RF model provides a reliable computational tool for prioritizing anti-AS compounds from Mulberry leaves. The integrated analysis reveals that Mulberry leaves exert anti-atherosclerotic effects through multi-target (e.g., AKT1, IL6) and multi-pathway (e.g., PI3K-Akt) mechanisms, offering a framework for further experimental validation.

Morus↗

PubMed2026

Identification and characterization of anti-chikungunya virus compounds using a biosafe toolkit.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus for which no specific antiviral therapy is currently available. During the large outbreak in Foshan, Guangdong Province, China, in July 2025, CHIKV rapidly spread to neighboring regions and caused more than 16,000 confirmed cases. In this study, the predominant outbreak strain of CHIKV was selected as the reference sequence to establish a panel of complementary biosafe tools for antiviral compound screening and mechanistic investigation. A virus replicon particle (VRP) system for CHIKV was first constructed and applied to compound library screening, resulting in the identification of three candidate antiviral compounds: MDL-12330A, bazedoxifene acetate, and anidulafungin. To further validate their antiviral activities and investigate their potential mechanisms, CHIKV functional evaluation systems were subsequently established, including vesicular stomatitis virus (VSV)- and murine leukemia virus (MLV)-based pseudovirus systems for viral entry, a replicon RNA system for post-entry replication-associated processes, a replication-defective nsP4 mutant replicon RNA system for primary translation, and a virus-like particle (VLP) system for viral particle assembly and budding assessment. Using these complementary systems, we systematically evaluated the antiviral profiles of the three candidate compounds across multiple stages of the CHIKV life cycle. This analysis revealed distinct stage-specific inhibitory patterns and provided insights into their potential antiviral mechanisms, which warrant validation using authentic CHIKV infection to assess their translational potential.

Chikungunya virus↗

PubMed2026

Dual HBV cccDNA-linked HiBiT reporter hepatocyte models for screening of candidate cccDNA modulators.

Chronic hepatitis B remains difficult to cure because the viral covalently closed circular DNA (cccDNA) minichromosome can persist and sustain viral transcription, creating a need for scalable, reporter readouts that facilitate early discovery of cccDNA-modulating agents. Here, we developed two complementary hepatocyte HiBiT reporter models: a replication-competent HBV reporter in HepaRG cells (HepaRG-Hibit16), in which a secreted split-NanoLuc HiBiT signal is linked to cccDNA-associated expression, and a Cre/Lox-based recombinant cccDNA (rcccDNA) reporter in HepG2 cells (HepG2-Rccc1a) that rapidly generates rcccDNA with a matched HiBiT readout. Screening of 1,403 FDA-approved compounds across both models identified 13 concordant, non-cytotoxic hits. Palovarotene, a retinoic acid receptor-γ agonist, was selected as an exemplar concordant hit and reduced HBV antigens, HBV DNA, and cccDNA and inhibited HBV infection in multiple hepatocyte-based in vitro systems without overt cytotoxicity at the tested concentrations. Together, this dual-reporter strategy supports efficient cross-model triage of candidate cccDNA modulators for subsequent orthogonal validation.

Humans↗

PubMed2026

Quantitative Fluorescence Imaging of Alphavirus Infection for Antiviral Screenings.

Fluorescence microscopy offers a highly sensitive and versatile approach for investigating alphavirus infection at the cellular level. By combining fluorescently labeled viruses with quantitative image analysis, this method enables detailed spatial and temporal characterization of infection dynamics, including the detection of subtle differences in replication kinetics and cell-to-cell spread. A central aim of this protocol is its application in antiviral screening assays. Image-based quantification of fluorescence intensity provides a robust and reproducible means to assess the efficacy of antiviral compounds, allowing early and sensitive detection of inhibitory effects in infected cells. This facilitates the identification of promising antiviral hits and supports the evaluation of dose-dependent responses. The approach is also well-suited for comparative studies of different alphavirus strains or mutants, as variations in replication behavior and dissemination patterns become readily apparent. Its flexibility, compatibility with multiple cell lines, and straightforward integration into automated imaging platforms makes the method scalable and suitable for high-throughput screening campaigns. Overall, this protocol advances the discovery and evaluation of antiviral strategies. Given that several alphaviruses cause significant human and veterinary diseases, lack approved antiviral therapies, and continue to expand geographically with emerging outbreaks, the identification of novel antivirals remains an urgent priority. Therefore, this fluorescence-based workflow represents a valuable and timely contribution to modern alphavirus research.

Antiviral Agents↗

PubMed2026

A Fibroblast-Based Adenoviral Reporter System Driven by the Mouse Collagen Type I Alpha 1 Promoter for Antifibrotic Drug Screening.

Cardiac fibrosis, characterized by aberrant fibroblast activation and excessive extracellular matrix deposition, lacks target-specific therapies, largely due to the absence of longitudinal, scalable, and non-destructive in vitro screening platforms. Traditional end-point assays and resource-intensive stem cell models inherently preclude real-time monitoring of fibrotic progression. To overcome these limitations, this protocol describes the generation, optimization, and validation of a mouse collagen type I alpha 1 (Col1a1) promoter-driven adenoviral mCherry fluorescent reporter system (Ad-mCol1a1p-mCherry) in NIH/3T3 fibroblasts. The critical steps for recombinant adenovirus packaging, transduction optimization (multiplicity of infection) to minimize cytotoxicity, and the establishment of a robust transforming growth factor beta (TGF-β)-induced fibrosis model are detailed. By circumventing the need for cell fixation, this system enables direct and longitudinal monitoring of collagen transcription in live cells. The model's specificity and reliability are pharmacologically validated using the TGF‑β type I receptor (ALK5) inhibitor SB431542, with fluorescent readouts correlating with endogenous fibrotic markers quantified via reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Ultimately, this cost-effective platform provides an accessible tool for the high-throughput screening of novel antifibrotic agents, thereby accelerating translational cardiovascular research.

Animals↗

PubMed2026

In vitro evaluation of sacituzumab govitecan in non-small cell lung cancer with actionable genomic alterations.

PURPOSE: The TROP2-directed antibody-drug conjugate sacituzumab govitecan (SG) has shown substantial therapeutic benefit in several malignancies; however, preclinical evidence supporting its activity in non-small cell lung cancer (NSCLC) is rare. MATERIALS AND METHODS: We evaluated 16 NSCLC cell lines harboring actionable genomic alterations for TROP2 expression and treated them with SG or its unconjugated payload, SN-38, for 3 days to determine cytotoxic effects. Apoptosis and DNA damage signaling were assessed using flow cytometry and western blot. SG internalization and lysosomal trafficking were visualized by confocal microscopy. RESULTS: SG had greater cytotoxic potency than SN-38, across all NSCLC cell lines, independent of genomic subtype or TROP2 expression level. Cell lines that were sensitive to SN-38 showed enhanced vulnerability to SG (P < 0.0001). Higher SLFN11 expression, a recognized determinant of SN-38 responsiveness, correlated with lower SG IC50 values. Both SG and SN-38 triggered apoptotic and DNA damage responses within 6-48 h, with SG inducing stronger activation of these pathways than SN-38. SG was efficiently taken up in CUTO17 and SNU-3173 adenocarcinoma cells, with more than 60% of the conjugate internalized within 3 h and subsequently localized to lysosomes. CONCLUSION: Our study provides in vitro evidence supporting the potential activity of SG in NSCLC with actionable genomic alterations. The efficacy of SG closely paralleled intrinsic sensitivity to the SN-38 payload, suggesting that DNA-damage responses, rather than oncogenic drivers, predominantly contribute to SG activity.

Actionable genomic alterations↗

PubMed2026

Systematic Identification of Therapeutic Targets and Repurposed Drugs for Stroke: From Genome Causal Analysis to Multilevel Validation.

BACKGROUND: Stroke is a severe cerebrovascular disease characterized by narrow time windows and complications. This study aimed to identify novel drug targets and repurposed drugs for stroke. METHODS: This study used expression quantitative trait loci data from druggable genes in brain and blood as instrumental variables. Mendelian randomization, colocalization, and phenome-wide Mendelian randomization were applied to evaluate causal relationships and potential side effects, with stroke and ischemic stroke as primary outcomes. Preclinical validation used oxygen-glucose deprivation/reperfusion and middle cerebral artery occlusion/reperfusion models. Pharmacological and behavioral assessments evaluated the therapeutic potential of candidate targets and drugs. Additionally, proteomic sequencing was performed following GGCX (γ-glutamyl carboxylase) overexpression to explore its biological functions. RESULTS: Elevated GGCX expression in brain and blood was potentially causally associated with reduced risk of stroke and ischemic stroke, supported by colocalization evidence, although potential cardiovascular risks could not be excluded. Drug repositioning identified ifenprodil as a candidate agent that reduced infarction volume, improved motor and cognitive functions, and reversed GGCX downregulation in mice. Ifenprodil treatment and GGCX overexpression alleviated oxygen-glucose deprivation/reperfusion-induced injury and upregulated GGCX expression. Mechanistically, GGCX conferred neuroprotection by regulating protein homeostasis, suppressing inflammation, promoting metabolic recovery, and modulating nuclear transcriptional regulation. CONCLUSIONS: This study established a potential causal link between GGCX and stroke risk, particularly ischemic stroke. GGCX represents a promising therapeutic target for ischemic stroke. Targeted GGCX expression upregulation and drug repurposing, particularly ifenprodil, may offer novel therapeutic avenues. Further validation is warranted to assess clinical efficacy and safety.

Animals↗

PubMed2026

Efficacy of pharmacological and microbiota-based therapies in preclinical models of autism spectrum disorder: a systematic review.

BACKGROUND: Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental condition in which pharmacological and microbiota-targeted interventions are emerging as promising therapeutic avenues. Animal models are the main tool to investigate etiology, molecular mechanisms and screening for pharmacological therapies. Methodological differences, outcome measure variability, incomplete reporting, biological confounders, and overgeneralization of the results made evaluating innovative pharmacological agents challenging. These limitations in the field highlight a need for systematic and standardized research to reliably assess and translate pharmacological interventions from ASD animal models to human clinical relevance. SUBJECTS: This systematic review synthesized efficacy evidence for pharmacological and microbiota-based therapies across established ASD animal models. RESULTS: We identified 52 recent (2010-2025) studies that reported key ASD behavioral outcomes after pharmacological or microbiota-focused treatments. Interventions were grouped into therapeutic classes - including oxytocinergic agents, E/I balance therapeutic targets, metabolic drugs, cannabinoids, purine-based interventions and emerging targets - alongside microbiota-directed strategies such as probiotics, prebiotics, and fecal microbiota transplantation. By integrating effect directions and robustness across models, we identified most potential drug candidates, evaluated the efficacy of novel strategies, and recognized critical translational gaps. The reviewed studies demonstrate that ASD-like behavioral deficits in preclinical models can be modulated through interventions targeting diverse biological systems, including neurotransmission, neuroinflammation, metabolism, and the gut-brain axis. CONCLUSIONS: These findings support the multifactorial nature of ASD pathophysiology which arises from a network of interacting systemic processes rather than a single molecular defect. It could explain the limited success of traditionally narrowly targeted interventions and suggest a paradigm shift into a more systemic approach.

Animals↗

PubMed2026

Kinesins in Cancer Drug Resistance: Mechanisms, Therapeutic Targeting, and Translational Potential.

Drug resistance in cancer remains a major barrier to durable therapeutic benefits and limits the effectiveness of chemotherapy, targeted therapy, and combination treatment in multiple malignancies. Increasing evidence indicates that specific kinesin superfamily proteins contribute to tumor adaptation and therapeutic response in a context-dependent manner through their roles in mitotic regulation, intracellular transport, and stress-response pathways. Aberrant expression of multiple kinesin family members has been documented across diverse cancers and is frequently associated with aggressive clinicopathological features, poor prognosis, and resistance to treatment. However, expression alterations alone do not establish functional dependency, and mechanistic validation is required to distinguish true resistance drivers from adaptive tumor states. In this review, we summarize the classification, biological functions, and abnormal expression patterns of kinesins in cancer; discuss the major mechanisms through which they contribute to drug resistance; and examine strategies for targeting kinesins, including natural-product-derived direct inhibitors, small-molecule inhibitor development, rational combination approaches, and structure-guided and computational optimization strategies. We also evaluate the biomarker potential of kinesin dysregulation and the value of advanced preclinical models for mechanistic and translational investigations. Finally, we highlight the major challenges that hinder clinical translation, including target specificity, compensatory resistance, insufficient biomarker validation, and tumor heterogeneity. Future progress will require integration of functional genomics, multiomics profiling, and mechanism-guided therapeutic strategies to determine when kinesin inhibition represents a clinically actionable approach for resistant malignancies.

biomarker potential↗

PubMed2026

A Patient-Derived Xenograft Repository Capturing Clinical and Molecular Heterogeneity of Large B-cell Lymphoma.

UNLABELLED: Large B-cell lymphomas (LBCL) are a clinically and molecularly diverse group of malignancies with a rapidly evolving therapeutic landscape that has introduced new areas of clinical need, such as post-CD19 chimeric antigen receptor T (CART19) progression. Patient-derived xenograft (PDX) models are an important tool for mechanistic studies and preclinical evaluation of new therapies and can be generated from a variety of clinical contexts that capture tumor-intrinsic resistance mechanisms. We therefore undertook a comprehensive effort to generate PDX models that encompass the molecular landscape of LBCLs and include important clinical scenarios for new drug development. Here, we describe the first 48 models within this publicly available repository, capturing the transcriptional and genetic subsets of LBCL. These models also include 23 generated from post-CART19 progression patient biopsies, which reproduce patterns of progression driven by CD19 mutation or expression loss, as well as tumor cell-intrinsic CART19 resistance that we validated in vivo. SIGNIFICANCE: Here, we describe X-LYMPH (Xenografts of Lymphoma), a publicly available and molecularly annotated PDX repository that captures the heterogeneity of LBCL. X-LYMPH includes models of CAR T-cell resistance, providing a shared foundation for mechanistic research and therapeutic development for lymphomas. See related commentary by Evgin and Steidl, p. 655.

Humans↗

PubMed2026

Genomic and phenotypic characterization of Klebsiella pneumoniae phage KP Ø1: a novel lytic Slopekvirus targeting uropathogenic multidrug-resistant Klebsiella pneumoniae.

The rise of multidrug-resistant (MDR) uropathogenic gram-negative bacteria (GNB) necessitates the development of alternative therapeutic strategies. This study aimed to isolate, phenotypically characterize, and perform whole-genome sequencing of the bacteriophage demonstrating the broadest host range against MDR uropathogens. Fifty MDR GNB isolates were screened for lytic phages. The most promising candidate, Klebsiella pneumoniae phage KP Ø1, was characterized using plaque assay, Transmission Electron Microscopy (TEM), and pH/thermal stability testing. Genomic characterization was performed via whole-genome sequencing (WGS), with functional annotation and lifestyle prediction using PhaBOX and PhageScope software. Klebsiella pneumoniae was the most prevalent MDR uropathogen. Klebsiella pneumoniae phage KP Ø1 exhibited a 50% host range and high lytic titer (10⁸ PFU/mL). TEM revealed an icosahedral head and short contractile tail. Genomic characterization by WGS revealed that Klebsiella pneumoniae phage KP Ø1 possesses a 174,591 bp double-stranded deoxyribonucleic acid (dsDNA) genome containing 274 predicted open reading frames (ORFs). No lysogeny-related genes, toxins, or antibiotic resistance markers were detected, confirming its strictly lytic nature and supporting its potential as a candidate for phage therapy applications. The phage remained stable (10⁸ PFU/mL) across temperatures of - 20 °C to 50 °C; supporting its suitability for long-term biobanking and suggesting potential activity at physiological temperature, and across a pH range of 7-9. Klebsiella pneumoniae phage KP Ø1 is a novel, obligately lytic Slopekvirus whose genomic architecture, stability profile, and absence of lysogeny-associated, virulence, and antimicrobial resistance genes ( AMR) collectively support its candidacy for further preclinical evaluation as a phage therapy agent against uropathogenic MDR Klebsiella pneumoniae.

Klebsiella pneumoniae↗

PubMed2026

Oncogene activation mechanism determines the limits of targeted protein degradation.

Protein degrader drugs such as PROTACs are being advanced as therapeutics targeted against oncogenic proteins. During tumorigenesis, oncogenic proteins can become constitutively activated via mechanisms including gene amplification, which increases protein production, and point mutations, which can extend protein half-life. Few experimental studies have addressed how disease-associated changes in target protein homeostasis influence PROTAC activity. We developed orthogonal methods to increase production or enhance stability of β-catenin, an important oncoprotein and target for degrader therapeutics, and used the dTAG system to evaluate the consequences for PROTAC activity. Stabilizing oncogenic missense mutations increase protein expression up to 5-fold but do not alter the PROTAC-imposed minimal steady-state level. In contrast, transcriptional upregulation increases both pre- and post-treatment target levels, revealing a synthesis-dependent ceiling on achievable depletion. Our results highlight distinct constraints on PROTAC activity arising from different mechanisms of oncogene activation, with potential implications for preclinical modeling, drug resistance and personalized medicine.

Humans↗