{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:08:33Z","timestamp":1772168913960,"version":"3.50.1"},"update-to":[{"DOI":"10.12688\/f1000research.157465.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T00:00:00Z","timestamp":1758585600000}}],"reference-count":43,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T00:00:00Z","timestamp":1758585600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"publisher","award":["ICMR-SRF#45\/33\/2019\/PHA\/BMS"],"award-info":[{"award-number":["ICMR-SRF#45\/33\/2019\/PHA\/BMS"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001843","name":"Science and Engineering Research Board","doi-asserted-by":"publisher","award":["EMR\/2016\/007006"],"award-info":[{"award-number":["EMR\/2016\/007006"]}],"id":[{"id":"10.13039\/501100001843","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    Background Non-small-cell lung cancer (NSCLC) is a highly morbid disease. Chemotherapy for NSCLC lacks specificity and efficacy mainly because of drug resistance. The current study aimed to explore computational tools to target allosteric epidermal growth factor receptor (EGFR) sites and screen for the top molecules\n                    <ns3:italic>in vitro<\/ns3:italic>\n                    and\n                    <ns3:italic>in vivo<\/ns3:italic>\n                    xenograft models. Methods Molecular docking, virtual screening, and molecular dynamic studies revealed that acenocoumarol and silodosin are the top two allosteric EGFR inhibitors. They were further tested for cytotoxicity, apoptosis, cell cycle, and gene expression by qPCR, western blotting, A549 cell xenograft anti-proliferative activity, and tumor regression efficacy analysis. Results Acenocoumarol and silodosin exhibited cytotoxicity in A549 and IMR-90 cells at concentrations below 50 and 80 \u03bcM, respectively. Acenocoumarol and silodosin induced S-phase and G2\/M-phase arrest in A549 cells in the cell cycle analysis. Both drugs showed early apoptosis at their IC\n                    <ns3:sub>50<\/ns3:sub>\n                    doses (acenocoumarol 50 \u03bcM and silodosin 25 \u03bcM). KRAS (Kirsten rat sarcoma viral oncogene homolog) and ERK2 (extracellular signal-regulated kinase 2) gene regulation in A549 cells was confirmed using qPCR. KRAS and ERK2 activities were quantified by western blot analysis. In the xenograft study, tumor size, body weight, and organ weight were significantly attenuated by the test drugs compared with the standard cisplatin. Immunoblotting and western blot results of the A549-xenograft tissue indicated downregulation of KRAS and ERK2. Furthermore, the test drugs have upregulated caspase-3 gene expression. Conclusion The drugs acenocoumarol and silodosin downregulate KRAS and ERK2 in both A549 cell lines and Xenograft model. KRAS and ERK2 are components of EGFR-associated signaling pathways. Hence, acenocoumarol and silodosin can be further explored as repurposed candidates in future preclinical and clinical studies.\n                  <\/ns3:p>","DOI":"10.12688\/f1000research.157465.2","type":"journal-article","created":{"date-parts":[[2025,11,11]],"date-time":"2025-11-11T13:45:09Z","timestamp":1762868709000},"page":"1398","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Exploring acenocoumarol and silodosin in non-small cell lung cancer: Insights into EGFR-linked signaling mechanisms"],"prefix":"10.12688","volume":"13","author":[{"given":"Swastika","family":"Maity","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Krishnaprasad","family":"Baby","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1362-2090","authenticated-orcid":false,"given":"Bharath Harohalli","family":"Byregowda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-0186-4906","authenticated-orcid":false,"given":"Megh Pravin","family":"Vithalkar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1995-3114","authenticated-orcid":false,"given":"Usha Y","family":"Nayak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2017-9533","authenticated-orcid":false,"given":"K Sreedhara Ranganath","family":"Pai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0508-1394","authenticated-orcid":false,"given":"Yogendra","family":"Nayak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2025,9,23]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"2130","DOI":"10.1038\/s41467-024-46284-x","article-title":"Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.","volume":"15","author":"R Iyer","year":"2024","journal-title":"Nat. Commun."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"e2206588119","DOI":"10.1073\/pnas.2206588119","article-title":"Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer.","volume":"119","author":"B Brown","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"4655","DOI":"10.1038\/s41467-018-07078-0","article-title":"Overcoming EGFR G724S -mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors.","volume":"9","author":"J Fassunke","year":"2018","journal-title":"Nat. Commun."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"17495-17504","DOI":"10.1074\/jbc.M115.660498","article-title":"Signalling through the phosphatidylinositol 3-kinase (PI3K)\/mammalian target of rapamycin (mTOR) axis is responsible for aerobic glycolysis mediated by glucose transporter in epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma.","volume":"290","author":"H Makinoshima","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"9","DOI":"10.3389\/fmolb.2022.922839","article-title":"Uncovering the Mechanism of Drug Resistance Caused by the T790M Mutation in EGFR Kinase From Absolute Binding Free Energy Calculations.","volume":"9","author":"H Zhou","year":"2022","journal-title":"Front. Mol. Biosci."},{"key":"ref6","doi-asserted-by":"publisher","DOI":"10.3390\/cancers14215307","article-title":"Afatinib and Dacomitinib Efficacy, Safety, Progression Patterns, and Resistance Mechanisms in Patients with Non-Small Cell Lung Cancer Carrying Uncommon EGFR Mutations: A Comparative Cohort Study in China (AFANDA Study).","volume":"14","author":"H Li","year":"2022","journal-title":"Cancers (Basel)."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"1008-1046","DOI":"10.1021\/acs.jmedchem.1c00876","article-title":"Emerging Approaches to Overcome Acquired Drug Resistance Obstacles to Osimertinib in Non-Small-Cell Lung Cancer.","volume":"65","author":"M Shaikh","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"114452","DOI":"10.1016\/j.biopha.2023.114452","article-title":"Investigating regulated signalling pathways in therapeutic targeting of non-small cell lung carcinoma.","volume":"161","author":"M Alam","year":"2023","journal-title":"Biomed. Pharmacother."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1038\/s41392-023-01705-z","article-title":"Targeting the RAS\/RAF\/MAPK pathway for cancer therapy: from mechanism to clinical studies.","volume":"8","author":"M Bahar","year":"2023","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"725-737","DOI":"10.1038\/s41416-019-0573-8","article-title":"Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer.","volume":"121","author":"A Leonetti","year":"2019","journal-title":"Br. J. Cancer."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"402-417","DOI":"10.1038\/s43018-022-00351-8","article-title":"An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer.","volume":"3","author":"C To","year":"2022","journal-title":"Nat. Can."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"799-813","DOI":"10.1007\/s43440-020-00131-0","article-title":"Advances in targeting EGFR allosteric site as anti-NSCLC therapy to overcome the drug resistance.","volume":"72","author":"S Maity","year":"2020","journal-title":"Pharmacol. Rep."},{"key":"ref13","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.24587592.v5","article-title":"Targeting EGFR Allosteric Site - An In Silico, in vitro and in vivo Drug Repurposing Study for Treating Non-Small Cell Lung Cancer.","author":"S Maity","year":"2024"},{"key":"ref14","doi-asserted-by":"publisher","DOI":"10.3390\/ijms222312827","article-title":"The mtt assay: Utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis.","volume":"22","author":"M Ghasemi","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"ref15","first-page":"1-25","author":"T Riss","year":"2004. 2013 May 1 updated 2016 Jul 1","journal-title":"Assay Guidance Manual."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"e374","DOI":"10.21769\/BioProtoc.374","article-title":"Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method.","volume":"3","author":"I Lakshmanan","year":"2013","journal-title":"Bio Protoc."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"89-114","DOI":"10.1007\/978-1-0716-2740-2_5","article-title":"Principles of Advanced Flow Cytometry: A Practical Guide.","volume":"2580","author":"S Siddiqui","year":"2023","journal-title":"Methods Mol. Biol."},{"key":"ref18","first-page":"1-15","article-title":"Activated MEK\/ERK Pathway Drives Widespread and Coordinated Overexpression of UHRF1 and DNMT1 in Cancer cells.","volume":"9","author":"J Li","year":"2019","journal-title":"Sci. Rep."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"1-13","DOI":"10.1371\/journal.pone.0163070","article-title":"Optimized multiplex detection of 7 KRAS Mutations by Taqman Allele-SpecificqPCR.","volume":"11","author":"A Orue","year":"2016","journal-title":"PLoS One."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"4821-4837","DOI":"10.1038\/s41388-018-0298-9","article-title":"C5a induces A549 cell proliferation of non-small cell lung cancer via GDF15 gene activation mediated by GCN5-dependent KLF5 acetylation.","volume":"37","author":"C Zhao","year":"2018","journal-title":"Oncogene."},{"key":"ref21","first-page":"3218-3222","article-title":"Research on the A549 cell apoptosis mechanism of the nude mouse model using MenSC-sTRAIL.","volume":"21","author":"H Ding","year":"2017","journal-title":"Eur. Rev. Med. Pharmacol. Sci."},{"key":"ref22","doi-asserted-by":"crossref","first-page":"1-11","DOI":"10.1038\/s41598-020-65767-7","article-title":"Statistical analysis of longitudinal data on tumour growth in mice experiments.","volume":"10","author":"I Zavrakidis","year":"2020","journal-title":"Sci. Rep."},{"key":"ref23","first-page":"1-10","article-title":"The prodomain of caspase-3 regulates its own removal and caspase activation.","volume":"5","author":"K Ponder","year":"2019","journal-title":"Cell Death Dis."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"206-218","DOI":"10.1002\/elps.202000199","article-title":"A comparative study of CE-SDS, SDS-PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins.","volume":"42","author":"R Wiesner","year":"2021","journal-title":"Electrophoresis."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"1-20","DOI":"10.1371\/journal.pone.0164438","article-title":"Long term culture of the a549 cancer cell line promotes multilamellar body formation and differentiation towards an alveolar type II Pneumocyte phenotype.","volume":"11","author":"J Cooper","year":"2016","journal-title":"PLoS One."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1-16","DOI":"10.3390\/ph13100273","article-title":"Epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC).","volume":"13","author":"C O\u2019Leary","year":"2020","journal-title":"Pharmaceuticals."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"926-943","DOI":"10.1158\/2159-8290.CD-18-0903","article-title":"Single and dual targeting of mutant EGFR with an allosteric inhibitor.","volume":"9","author":"C To","year":"2019","journal-title":"Cancer Discov."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1038\/s42004-024-01108-3","article-title":"Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.","volume":"7","author":"F Wittlinger","year":"2024","journal-title":"Commun. Chem."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"503-525","DOI":"10.1358\/dot.2014.50.07.2185913","article-title":"Current status of targeted therapy in non-small cell lung cancer.","volume":"50","author":"D Parums","year":"2014 Jul","journal-title":"Drugs Today (Barc.)."},{"key":"ref30","first-page":"4-5","article-title":"Acenocoumarol or warfarin: Which is the clinician\u2019s ally?","volume":"9","author":"N Karthik Rao","year":"2016","journal-title":"Asian J. Pharm. Clin. Res."},{"key":"ref31","first-page":"2110-2121","article-title":"\u03b11-Blockers in Men with Lower Urinary Tract Symptoms Suggestive of Benign Prostatic Obstruction: Is Silodosin Different?","volume":"33","author":"C Roehrborn","year":"2017","journal-title":"Adv. Ther."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1-24","DOI":"10.3390\/molecules25061465","article-title":"and New Synthetic and Natural Coumarin Derivatives.","volume":"25","author":"K Kasperkiewicz","year":"2020","journal-title":"Molecules."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"626-641","DOI":"10.1111\/bcp.12220","article-title":"Pharmacogenetic-guided dosing of coumarin anticoagulants: Algorithms for warfarin, acenocoumarol and phenprocoumon.","volume":"77","author":"T Verhoef","year":"2014","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref34","doi-asserted-by":"publisher","DOI":"10.3390\/ph16040604","article-title":"Acenocoumarol, an Anticoagulant Drug, Prevents Melanogenesis in B16F10 Melanoma Cells.","volume":"16","author":"H Han","year":"2023","journal-title":"Pharmaceuticals."},{"key":"ref35","first-page":"2959-2968","article-title":"Silodosin inhibits the growth of bladder cancer cells and enhances the cytotoxic activity of cisplatin via ELK1 inactivation.","volume":"5","author":"T Kawahara","year":"2015","journal-title":"Am. J. Cancer Res."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"2861-2884","DOI":"10.2147\/DDDT.S373659","article-title":"Evolving Role of Silodosin for the Treatment of Urological Disorders \u2013 A Narrative Review.","volume":"16","author":"L Jindan","year":"2022","journal-title":"Drug Des. Devel. Ther."},{"key":"ref37","doi-asserted-by":"crossref","first-page":"1-11","DOI":"10.3389\/fnins.2011.00035","article-title":"Elk-1 a transcription factor with multiple facets in the brain.","volume":"5","author":"A Besnard","year":"2011","journal-title":"Front. Neurosci."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"1-10","DOI":"10.1016\/j.jpba.2016.11.013","article-title":"LC\u2013ESI\u2013MS\/MS evaluation of forced degradation behaviour of silodosin: in vitro anti cancer activity evaluation of silodosin and major degradation products.","volume":"134","author":"C Vishnuvardhan","year":"2017","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref39","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2022.840241","article-title":"Identification of Synergistic Drug Combinations to Target KRAS-Driven Chemoradioresistant Cancers Utilizing Tumoroid Models of Colorectal Adenocarcinoma and Recurrent Glioblastoma.","volume":"12","author":"K Gupta","year":"2022","journal-title":"Front. Oncol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"753-765","DOI":"10.1142\/S0192415X2150035X","article-title":"20(S)-Ginsenoside Rg3 Inhibits Lung Cancer Cell Proliferation by Targeting EGFR-Mediated Ras\/Raf\/MEK\/ERK Pathway.","volume":"49","author":"Y Liang","year":"2021","journal-title":"Am. J. Chin. Med."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"129-132","DOI":"10.1038\/nature17960","article-title":"Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors.","volume":"534","author":"Y Jia","year":"2016","journal-title":"Nature."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"4","DOI":"10.4172\/2329-6607.1000157","article-title":"Acenocoumarol in Thromboembolic Disorders.","volume":"4","author":"A Trailokya","year":"2015","journal-title":"Cardiovasc. Pharm. Open Access"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"302-310","DOI":"10.1345\/aph.1M320","article-title":"New drug approvals: silodosin for benign prostatic hyperplasia.","volume":"44","author":"M Cantrell","year":"2010","journal-title":"Ann. 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The claim that acenocoumarol and silodosin are allosteric EGFR inhibitors was primarily based on in silico docking studies and prior literature suggesting possible interaction with the allosteric site. However, we recognize that docking studies alone do not suffice to label them as &quot;allosteric inhibitors.&quot; We have accordingly:  Revised the manuscript title, abstract, conclusion, and discussion to reflect a more evidence-aligned interpretation of the findings. The drugs are now presented as repurposed candidates with predicted interactions at EGFR-associated signaling levels, based on computational docking and preclinical evaluation. The title has been changed to: \u201c\n<b>Exploring acenocoumarol and silodosin in non-small cell lung cancer: Insights into EGFR-linked signaling mechanisms<\/b>\u201d. This updated title avoids asserting mechanistic inhibition, emphasizes pathway-level relevance, and accurately reflects the scope and limitations of the study. Removed definitive language such as &quot;allosteric inhibitor&quot; and replaced it with &quot;computationally predicted to bind to allosteric sites of EGFR.&quot; Cited the lack of direct evidence as a limitation and proposed further biochemical studies (e.g., kinase assays, phospho-EGFR western blots) in the future.   \n<b>Major Comment 2: No direct evidence that cytotoxicity is related to EGFR inhibition.<\/b> \n<b>Response: <\/b>We agree. While we observed apoptosis and downregulation of KRAS and ERK2 components downstream of EGFR signaling, our current dataset does not establish a causal link between drug activity and EGFR inhibition. We have removed statements that imply a direct mechanistic link and instead suggest that the observed effects \n<b>may be associated<\/b> with EGFR signaling pathways, pending further validation. This clarification is incorporated into the discussion.  \n<b>Major Comment 3: Missing replicate (n) numbers and statistical clarity.<\/b> \n<b>Response: <\/b>We apologize for the oversight. The manuscript has been revised to:  Explicitly mention the number of \n<b>biological replicates (n=3)<\/b> for in vitro studies and \n<b>n=4 mice per group<\/b> for xenograft experiments. Clearly indicate \n<b>technical duplicates<\/b> where applicable. Ensure all SEM values are tied to biological replicates, not technical ones.   \n<b>Major Comment 4: Use of inappropriate controls in EGFR assays.<\/b> \n<b>Response: <\/b>We acknowledge the point regarding the use of cisplatin instead of a known EGFR inhibitor. Our intention was to benchmark general anticancer activity rather than EGFR-specific inhibition, given the exploratory nature of this study. This rationale is now mentioned in the Methods and Limitations sections. We agree that future studies must incorporate controls like \n<b>afatinib or osimertinib<\/b> to provide a valid comparative frame.  \n<b>Major Comment 5: Concerns with IC50 calculations and molecule selection.<\/b> \n<b>Response: <\/b>The IC50 values were calculated using nonlinear regression via GraphPad Prism. While sigmoidal fitting may not be ideal for all curves due to cell line-specific response variability, the curve fitting and R&sup2; values are considered. Regarding molecule selection, \n<b>danusertib<\/b> was excluded from further testing due to \n<b>known off-target cytotoxicity and poor pharmacokinetic properties<\/b> in pilot studies, though it showed high potency. This is now clarified in the Results section.  \n<b>Major Comment 6: Mislabeling of Western blot data as enzyme inhibition.<\/b> \n<b>Response: <\/b>We regret the terminology used. The sections titled \u201cenzymatic inhibition\u201d now read \n<b>\u201cprotein expression levels\u201d<\/b> via Western blot. No enzymatic activity assay was performed. All conclusions based on these blots have been modified accordingly to describe expression rather than inhibition.  \n<b>Major Comment 7: Inaccurate claims of signaling pathway inhibition.<\/b> \n<b>Response: <\/b>We have removed all claims of direct inhibition of KRAS and ERK2 enzymatic activity or pathway signaling. All references to &quot;significant inhibition&quot; have been qualified to reflect \n<b>expression changes<\/b>, not enzyme activity or pathway suppression, and we note the need for \n<b>phospho-ERK or GTP-RAS<\/b> assays in future studies.  \n<b>Major Comment 8: Incomplete statistical test descriptions.<\/b> \n<b>Response: <\/b>Statistical tests have now been specified as \n<b>ANOVA with Tukey\u2019s post hoc test<\/b> or \n<b>unpaired t-test<\/b>, as appropriate. Significance values and p-values have been revised throughout the figure legends. The p&lt;0.5 error has been corrected to \n<b>p&lt;0.05<\/b>.  \n<b>Major Comment 9: Disconnect between mRNA and protein levels.<\/b> \n<b>Response: <\/b>This is a valid observation. We have acknowledged in the discussion that \n<b>post-transcriptional or translational regulation<\/b> may cause such discrepancies, and that one-time point assessments without time-course validation may limit interpretability. These are now added to the limitations.  \n<b>Major Comment 10: Clarification in Figures 9\u201311 and statistical interpretations.<\/b> \n<b>Response: <\/b>Clarifications have been added regarding Statistical tests in tumor volume measurements (Figure 9), Significance at \n<b>multiple time points<\/b> (but analyzed at day 21), The error in p-value notation has been corrected. Organ weight data (Figure 11) now includes the statistical method and indicates whether post hoc corrections were applied.  \n<b>Minor Comments (combined response):<\/b>  All antibody sources and catalog numbers have now been added to the Methods section. Typos including T750M\/L858M (corrected to T790M\/L858R) have been fixed. All red underlines and formatting errors in figures have been corrected. Raw data files now include individual replicates and are available on Figshare [https:\/\/doi.org\/10.6084\/m9.figshare.24587592.v5].","URL":"https:\/\/f1000research.com\/articles\/13-1398\/v2#referee-comment-14591","order":1,"name":"referee-comment-14591","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.172916.r347134, Chandan Shivamallu, Department of Biotechnology and Bioinformatics, JSS Academy of Higher Education and Research, Mysuru, 03 Jan 2025, version 1, 1 approved with reservations, 1 not approved","URL":"https:\/\/f1000research.com\/articles\/13-1398\/v1#referee-response-347134","order":2,"name":"referee-response-347134","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Yogendra Nayak<\/b>; \n<i>Posted: 15 Sep 2025<\/i>; We thank the reviewer for the encouraging and insightful comments. The following revisions have been made based on the suggestions:  \n<b>Figures and Tables<\/b>: All figures have been relabeled for clarity. Figure legends are now more detailed, and axis labels\/treatments are standardized. \n<b>Comparison with existing EGFR inhibitors<\/b>: A paragraph comparing our findings with allosteric inhibitors like \n<b>EAI045<\/b> and \n<b>JBJ-09-063<\/b> has been added to the discussion. \n<b>Discussion on clinical translation<\/b>: We now include a note on challenges related to pharmacokinetics, metabolism, and dosage limitations of repurposed drugs, particularly acenocoumarol (bleeding risk) and silodosin (hypotension). \n<b>Language revision<\/b>: The manuscript has undergone thorough proofreading to remove redundancies and grammatical inconsistencies.","URL":"https:\/\/f1000research.com\/articles\/13-1398\/v2#referee-comment-14592","order":3,"name":"referee-comment-14592","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.188258.r423555, Jigna Samir Shah, Nirma University, Ahmedabad, Gujarat, 11 Nov 2025, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/13-1398\/v2#referee-response-423555","order":4,"name":"referee-response-423555","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.188258.r434405, Navjot Kanwar, Maharaja Ranjit Singh Technical University, Bathinda, Punjab, 23 Dec 2025, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/13-1398\/v2#referee-response-434405","order":5,"name":"referee-response-434405","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Swastika Maity received an ICMR Senior Research Fellowship (ICMR-SRF #45\/33\/2019\/PHA\/BMS). The authors are thankful to the Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, for facilitating the computer simulations. Schrodinger's Software and Computers were procured under a grant from DST-SERB, New Delhi, to Usha YN (EMR\/2016\/007006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":6,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}