Bioorganic & Medicinal Chemistry最新文献

{"title":"Investigation of the histone deacetylase inhibitor potential of phorbazole analogues","authors":"Ida Amalie Nystad Reierth ,&nbsp;Yngve Guttormsen ,&nbsp;Jeanette H. Andersen ,&nbsp;Espen H. Hansen ,&nbsp;Ahmed Mossad Abdelhady ,&nbsp;Manuel K. Langer ,&nbsp;Bengt Erik Haug ,&nbsp;Annette Bayer ,&nbsp;Guillaume A. Petit","doi":"10.1016/j.bmc.2025.118489","DOIUrl":"10.1016/j.bmc.2025.118489","url":null,"abstract":"<div><div>Histone deacetylases (HDACs) are enzymes that remove acyl groups from histones and other proteins. This process leads to condensation of DNA and subsequent downregulation in expression of specific genes altering the activity of essential cellular pathways and changing the cell's behaviour. As a result, HDACs have emerged as potential therapeutic targets to treat different forms of cancer. In this study we synthesized and investigated compounds built around a phorbazole scaffold to characterize their histone deacetylase inhibitor (HDACi) activity. Eighteen phorbazole analogues were tested against class I and II HDACs in cell lysate, seven showed moderate activity, of which, three had IC₅₀ values below 50 μM. The five best candidates were evaluated more extensively against HDAC1-11 (except for HDAC10). The best candidate, compound <strong>9</strong>, initially appeared to reduce the activity of HDAC9 and HDAC11 by more than 50 % at 10 μM concentration. The binding mode of compound <strong>9</strong> to HDACs was explored via computational docking, where two poses stood out. These were explored further by molecular dynamic simulation. We found that <strong>9</strong> likely binds HDAC9 with the pyrrole group buried in the active site and forming H-bonds with the backbone oxygen of one of the glycine residues lining the cavity. But neither of the poses explored offered convincing arguments to describe the mode of action of the phorbazole, especially when comparing <strong>9</strong> to other analogues tested in this study. Additional experiments found that <strong>9</strong> interfered with the cell lysate assays by inhibiting luciferase in a dose-dependent manner (IC₅₀ &lt; 1 μM) and by exhibiting autofluorescence when tested on purified HDAC proteins, thereby confounding the obtained results during both the pan-HDAC screening and the single point HDAC inhibition assay. To address this, we employed fluorophores with excitation and emission wavelengths outside of the emission range of <strong>9</strong> and found that the HDAC inhibition potential of <strong>9</strong> was weaker than first observed. Finally, Compound <strong>9</strong> was found to be very soluble in water (418 μM) and membrane permeable (&gt;48 % flux). This study highlights the need for rigorous validation of results. In our case, two orthogonal testing methods were not sufficient to catch all the confounding factors involved in measurement of HDAC inhibition, and a third approach was required to identify the actual inhibition of <strong>9</strong> against HDAC9 and 11.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"133 ","pages":"Article 118489"},"PeriodicalIF":3.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145622400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Repurposing the USFDA-approved small molecules for their affinity against the colchicine binding site (CBS) in the tubulin: Corroborating the in-silico findings through biological assessment","authors":"Kumar Rohit,&nbsp;Bibekananda Sarkar","doi":"10.1016/j.bmc.2025.118488","DOIUrl":"10.1016/j.bmc.2025.118488","url":null,"abstract":"<div><div>The microtubules are known to possess inimitable characteristics termed ‘exquisite dynamic behavior,’ which allows cells to maintain their shape and assist in cellular division. This exquisite dynamicity is lost in the case of cancer, and considering their pivotal role, they are considered as central drug targets in cancer chemotherapy. To target them, microtubule-targeting agents (MTAs) are unique small molecules that inhibit one of the seven unique druggable pockets of microtubules, impacting their dynamicity and leading to cell cycle arrest and apoptosis. In the present work, we employ a drug repurposing strategy to identify plausible leads targeting the colchicine binding site (CBS) from a pool of 2787 FDA-approved drugs. CBS is a unique and flexible druggable pocket site in microtubules that has been targeted for both cancer and non-cancer diseases. Additionally, the inhibition of this site is reported to reverse drug resistance and exhibit synergistic anti-inflammatory and anti-angiogenic properties, further enhancing the anticancer effects. The cumulative analysis led to the identification of Tafenoquine, a well-known antimalarial FDA-approved molecule, as a potential tubulin-targeting agent with predicted binding to the colchicine site. The in-silico analysis was further corroborated using in vitro investigation on breast (MCF-7 and MDA-MB-231), lung (A549), and colon (HCT-116) cancer cells. Tafenoquine was found to exert broad-spectrum antiproliferative effects, with greatest potency against MDA-MB-231 cells (IC₅₀ = 4.75 ± 0.18 μM), a model for aggressive and treatment-resistant triple-negative breast cancer (TNBC). Considering the tubulin polymerization assay, the identified hit was found to inhibit microtubule assembly (low Vmax) in a manner similar to colchicine, and exhibited a similar G2/M arrest, indicating a mode of inhibition similar to that of colchicine.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"133 ","pages":"Article 118488"},"PeriodicalIF":3.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145675992","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Benzothiazole sulfones as a tool for peptide modification and cleavage","authors":"Phoebe R. Rubio ,&nbsp;Gavin A. Clausen ,&nbsp;Zachary D. Ruighaver ,&nbsp;Abigail F. Dalton,&nbsp;John P. Blobe,&nbsp;Amanda L. Moore,&nbsp;Christopher R. Shugrue","doi":"10.1016/j.bmc.2025.118500","DOIUrl":"10.1016/j.bmc.2025.118500","url":null,"abstract":"<div><div>We report the development of a new benzothiazole sulfone (BTS) tool for peptide modification and cleavage. Benzothiazole sulfones are electrophiles that rapidly react with thiols in nucleophilic aromatic substitution (S_<em>N</em>Ar) reactions. Alkyne-containing BTS compounds were incorporated onto azidolysine-containing peptides through copper-catalyzed azide-alkyne cycloadditions (CuAACs). BTS-containing peptides were found to react rapidly with two equivalents of thiol nucleophiles at room temperature in 30 min or less. BTS-containing peptides were conjugated to a variety of different compounds, including small molecules, fluorophores, and peptides. Alternatively, a BTS-based cleavable linker could also be synthesized with small molecule cargo, such as biotin, and attached to peptides through CuAAC. This linker enabled the capture and release of a peptide with immobilized streptavidin. The BTS motif is a versatile tool for various applications in peptide science.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"133 ","pages":"Article 118500"},"PeriodicalIF":3.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145666512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Natural products that suppress 2-deoxy-d-glucose-induced cell death as potential drug seeds for mitochondrial diseases","authors":"Koyo Honda ,&nbsp;Yuki Hitora ,&nbsp;Yusaku Sadahiro ,&nbsp;Masahiro Wato ,&nbsp;Yuriko Nagano ,&nbsp;Sachiko Tsukamoto","doi":"10.1016/j.bmc.2025.118478","DOIUrl":"10.1016/j.bmc.2025.118478","url":null,"abstract":"<div><div>Mitochondrial dysfunction is not only a known cause of mitochondrial disease but has also been implicated in diabetes and neurodegenerative diseases. Therefore, modulating mitochondrial function may provide new insights into the treatment of these diseases. In this study, we used a cancer cell-based energy metabolism restriction model with a glycolytic inhibitor and screened a natural product library and a fungal extract library to identify natural products that improve mitochondrial function. Seven compounds, including quillaic acid (<strong>1</strong>) and 20-deoxyingenol (<strong>2</strong>), were identified as mitochondrial function modulators from the Selleck natural product library. Furthermore, an extract of a fungus, <em>Trichoderma</em> sp., from an in-house fungal extract library suppressed 2-deoxy-<span>d</span>-glucose-induced cell death. Bioassay-guided fractionation of the extract afforded seven known sorbicillinoids. Among them, bisvertinol (<strong>8</strong>) did not directly affect cellular energy metabolism but appeared to protect mitochondria from oxidative stress through its antioxidant properties.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118478"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145450276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design, synthesis, and evaluation of antipyrine and nicotinic acid derivatives as anti-inflammatory agents: in vitro and in vivo studies","authors":"Rasha A. Hassan ,&nbsp;Soha H. Emam ,&nbsp;Demiana S. Mikhail ,&nbsp;Soha O. Hassanin ,&nbsp;Mona G. Khalil ,&nbsp;Amr M. Abdou ,&nbsp;Eman O. Osman","doi":"10.1016/j.bmc.2025.118439","DOIUrl":"10.1016/j.bmc.2025.118439","url":null,"abstract":"<div><div>A series of new antipyrine and nicotinic acid-based scaffolds were synthesized and evaluated for their inhibitory activity against cyclooxygenase-1 and cyclooxygenase-2, using indomethacin and celecoxib as reference drugs. Among the synthesized compounds, <strong>4d</strong> and <strong>6b</strong> demonstrated the most potent COX-2 inhibition, exhibiting IC₅₀ values of 0.940 ± 0.05 μM and 0.614 ± 0.03 μM, respectively, in comparison to celecoxib (IC₅₀ = 0.844 ± 0.04 μM). Compound <strong>4d</strong> displayed the highest COX-2 selectivity index (SI = 13.57), surpassing that of celecoxib (SI = 7.92), thereby indicating superior COX-2 selectivity. Consequently, the <em>in vivo</em> anti-inflammatory activity of compound <strong>4d</strong> was comparable to that of indomethacin. Compound <strong>4d</strong> significantly reduced paw edema, achieving edema inhibition percentages of 14.75 %, 30.77 %, and 45.33 % at the first, second, and third hours, respectively. In contrast, celecoxib exhibited the most pronounced anti-inflammatory effect, achieving edema inhibition percentages of 59.02 %, 69.23 %, and 78.67 % at the corresponding time points. Histopathological examination confirmed the absence of gastric ulceration following oral administration of compound <strong>4d</strong>. Furthermore, it effectively lowered serum levels of prostaglandin E2 and interleukin-1β. To elucidate its selectivity and binding affinity, molecular docking of compound <strong>4d</strong> into the COX-2 active site was conducted, revealing favorable interactions consistent with its biological activity. Collectively, these findings identify compound <strong>4d</strong>, an antipyrine–urea hybrid with para-Cl and meta-CF₃ substitutions, as a useful lead scaffold for effective, safe, and selective COX-2 inhibitors.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118439"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145297737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Helix stabilization by i,i + 7 amine-containing hydrocarbon Staples: Effects of length, stereochemistry, and orientation","authors":"Ha T.N. Nguyen ,&nbsp;Su-Yeon Lee ,&nbsp;Duc V.H. Tran ,&nbsp;Young-Woo Kim","doi":"10.1016/j.bmc.2025.118443","DOIUrl":"10.1016/j.bmc.2025.118443","url":null,"abstract":"<div><div>We previously introduced the <em>i</em>,<em>i</em> + 4 amine-containing hydrocarbon (ACH) stapling system as a helix-stabilizing alternative to conventional all-hydrocarbon (AHC) staples, offering improved aqueous compatibility and chemical tunability. Here, we extend this approach to the <em>i</em>,<em>i</em> + 7 topology, which spans two helical turns and enables long-range conformational control. Systematic variation of cross-link length, stereochemistry, and orientation identified a 13-atom butylaminoalkenyl tether with <em>S</em><img><em>S</em> configuration as the most effective helix-stabilizing <em>i</em>,<em>i</em> + 7 ACH staple. Notably, orientation reversal substantially enhanced helicity relative to the canonical arrangement, and this effect proved transferable across helical registers. Furthermore, the orientation-optimized staple not only reinforced α-helical conformation but also conferred significant proteolytic resistance, thereby revealing a direct link between structural preorganization and biochemical resilience. Taken together with earlier <em>i</em>,<em>i</em> + 4 variants, <em>i</em>,<em>i</em> + 7 ACH staples constitute a coherent design platform that expands the chemical and functional space of peptide stapling, with broad implications for structurally reinforced, therapeutically relevant α-helical peptides.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118443"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145311930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel trifluoromethylquinoline derivatives as potent tubulin polymerization inhibitors with antitumor activity","authors":"Mengsha Mao ,&nbsp;Liangliang Hu ,&nbsp;Xiangkai Kong ,&nbsp;Shijiao Wei ,&nbsp;Ping Yi ,&nbsp;Gang Yu ,&nbsp;Guangcan Xu ,&nbsp;Jia Yu ,&nbsp;Sha Cheng ,&nbsp;Xiaoping Zeng ,&nbsp;Kun Liu ,&nbsp;Bixue Xu ,&nbsp;Heng Luo ,&nbsp;Xueling Meng","doi":"10.1016/j.bmc.2025.118472","DOIUrl":"10.1016/j.bmc.2025.118472","url":null,"abstract":"<div><div>Herein, two series of <em>N</em>-aryl-2-trifluoromethyl-quinoline-4-amine derivatives as tubulin polymerization inhibitors were designed and synthesized. Compounds <strong>14b</strong> exhibited potency inhibitory activity against LNCaP cells (IC₅₀ = 0.0027 μM and Selectivity Index = 4.07) which was more effective than the positive control colchicine, while compounds <strong>12</strong>, <strong>16a</strong>, and <strong>14e</strong> exhibited moderate activity against LNCaP cells with IC₅₀ values of 0.28, 0.0413, and 0.43 μM, respectively. Moreover, mechanism studies showed that <strong>14b</strong> could disrupt tubulin polymerization effectively, block cell cycle in G2/M phase, and induced cellular apoptosis. <strong>14b</strong> significantly inhibited LNCaP cell migration and invasion. Meanwhile, SwissADME predictions showed that <strong>14b</strong> has good pharmacokinetic properties and molecular docking confirmed that it may bind to the colchicine binding site. In brief, these data suggested that <strong>14b</strong> was a promising lead compound with antitumor activity for discovering novel tubulin polymerization inhibitors.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118472"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145494235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis and degradation effect of PROTACs targeting EGFR triple mutants","authors":"Xiao-Xiao Xi,&nbsp;Hong-Yi Zhao,&nbsp;Minhang Xin,&nbsp;Shuai Mao,&nbsp;San-Qi Zhang","doi":"10.1016/j.bmc.2025.118427","DOIUrl":"10.1016/j.bmc.2025.118427","url":null,"abstract":"<div><div>Drug resistance caused by epidermal growth factor receptor (EGFR) mutation has significantly limited the clinical efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study, we selected fourth-generation EGFR-TKIs (compounds <strong>A</strong>, <strong>B</strong>, and <strong>C</strong>) and EGFR-TKI <strong>D</strong> as ligands for EGFR, along with VHL-L1 and VHL-L2 as ligands for the E3 ligase, using acyl linkers to design novel PROTACs targeting EGFR triple mutants. We synthesized and evaluated compounds EP1–EP12. Among them, <strong>EP7</strong>, <strong>EP8</strong>, <strong>EP9</strong>, <strong>EP11</strong> and <strong>EP12</strong> exhibited potent antiproliferative activities against cells expressing EGFR mutants. Notably, <strong>EP9</strong> and <strong>EP12</strong> effectively induced the degradation of EGFR triple mutants (EGFR^{Del19/T790M/C797S} and EGFR^{L858R/T790M/C797S}) and significantly suppressed EGFR pathway signal transduction. Mechanism studies revealed that the degradation process requires the formation of ternary complexes and the ubiquitination of the EGFR protein, with the degradation being associated with lysosomal activity. In conclusion, compounds EP9 and EP12 were identified as potent degraders of EGFR triple mutants, offering new insights into the development of PROTACs for targeting these mutations.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118427"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145264923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of novel 20S proteasome activators for hypoxic cardiomyocyte protection via endoplasmic reticulum stress alleviation","authors":"Dan Wu ,&nbsp;Yu Cao ,&nbsp;Yubing Han ,&nbsp;Linhao Xu ,&nbsp;Han Han ,&nbsp;Yaoli Shi ,&nbsp;Jiayi Ma ,&nbsp;Qingyi Wang ,&nbsp;Lixin Gao ,&nbsp;Mingzhi Wang ,&nbsp;Jianjun Xi ,&nbsp;Jingjing Sun ,&nbsp;Huajian Zhu ,&nbsp;Jiaan Shao ,&nbsp;Bin Ju ,&nbsp;Yizhou Xu ,&nbsp;Yubo Zhou ,&nbsp;Jiankang Zhang","doi":"10.1016/j.bmc.2025.118452","DOIUrl":"10.1016/j.bmc.2025.118452","url":null,"abstract":"<div><div>Under hypoxic stress, cardiomyocytes predominantly depend on 20S proteasome-mediated degradation to clear accumulated and misfolded proteins. The resultant proteotoxic stress from impaired protein homeostasis contributes significantly to the pathogenesis of cardiovascular disorders. Pharmacological enhancement of 20S proteasome activity thus represents a novel therapeutic paradigm for ischemic cardiomyopathy by restoring proteostasis in myocardial cells. In this study, the hit compound <strong>X-1</strong> was identified through screening of proteasome activation profile. Subsequent structure-activity relationship optimization yielded a series of highly potent activators. Intracellular protein degradation assessment revealed that these compounds possessed abilities to alleviate endoplasmic reticulum stress, as demonstrated by the luciferase reporter system. Additionally, selected compound <strong>B3</strong> exhibited superior cytoprotection, increasing viability of hypoxia-injured cardiomyocytes while downregulating ER stress markers CHOP and Grp78 at the protein level. AlphaFold3-predicted binding modes and 100 ns molecular dynamics simulations revealing its stabilization of the <em>α</em> region to induce proteasome gate opening, thereby establishing a structure-function rationale for its 20S activation mechanism.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118452"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145407808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"D-DARTS: an alternative method for NaV1.5 affinity molecules identification based on dual-drug affinity responsive target stability","authors":"Zirui Lü,&nbsp;Xiandong Dai,&nbsp;Huixia Li,&nbsp;Cunlin Wang,&nbsp;Fanhua Meng","doi":"10.1016/j.bmc.2025.118436","DOIUrl":"10.1016/j.bmc.2025.118436","url":null,"abstract":"<div><div>Drug affinity responsive target stability (DARTS) is a powerful label-free technique for detecting target engagement by measuring the increased resistance of a protein to proteolytic degradation upon ligand binding. However, its application to multi-transmembrane channel proteins has been limited due to their intrinsic structural instability and general resistance to protease digestion. To address this challenge, we developed a novel strategy termed Dual-DARTS (D-DARTS), which employs controlled proteolysis in an SDS-containing denaturing buffer to evaluate a target protein's dual stability—namely, resistance to both chemical denaturation and enzymatic degradation. Using this approach, we successfully identified affinity ligands for the NaV1.5 channel, including both inhibitors and agonists. Notably, D-DARTS enabled the discovery of poneratoxin, a peptide derived from bullet ants, as a high-affinity binder to NaV1.5, a finding validated through electrophysiological assays. Preliminary molecular docking analyses suggested that poneratoxin binds to an epitope analogous to that of the alkaloid agonist BTX<img>B. The general utility of the D-DARTS method was further corroborated by its successful application to the mitochondrial membrane protein VDAC1. By enabling target profiling based on binding affinity rather than functional activity, D-DARTS provides a complementary alternative to electrophysiology-based screening for NaV1.5 channel binders, with advantages including operational simplicity, cost-efficiency, and reduced reliance on specialized equipment. This strategy is anticipated to be applicable to target identification and active molecule screening for a wide range of channel proteins and other multi-transmembrane proteins.</div></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"132 ","pages":"Article 118436"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145290492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}