Archives of biochemistry and biophysics最新文献

{"title":"Understanding the mechanism involved in lung epithelial potassium current modulation by ozone","authors":"Rita Canella ,&nbsp;Mascia Benedusi ,&nbsp;Giulia Trinchera ,&nbsp;Angela Pignatelli ,&nbsp;Giuseppe Valacchi","doi":"10.1016/j.abb.2025.110522","DOIUrl":"10.1016/j.abb.2025.110522","url":null,"abstract":"<div><div>Given that the ionic balance of the pulmonary alveolar lining fluid is essential for correct gas exchange, our previous studies analyzed the effects of ozone (O₃), one of the most harmful pollutants for the respiratory system, on K⁺ current (I_K)in human cultured lung epithelial cells (A549). O₃ exposure significantly alters the flow of K⁺ ions, reducing the outward rectifier current component. O₃ does not act directly, but through its byproducts, among which the main ones are 4-hydroxy-2-nonenal (4HNE) and hydrogen peroxide (H₂O₂).</div><div>In this study we analyzed the action of 4HNE and H₂O₂ on I_K in human lung cells and verified the protective effect of catalase.</div></div><div><h3>Material</h3><div>A549 cellular line. 30’ exposure: O₃: 0.1 ppm; 4HNE: 5, 10 and 20 μM; H₂O₂: 20 μM. Glucose-oxidase: 10 mU/l, 1.00h exposure, analyzed after 24h. Catalase: 1000 U, 1.30h exposure.</div></div><div><h3>Methods</h3><div>Western blot, immunofluorescence and patch clamp techniques to study the action of the bio-products on I_K.</div><div>4HNE was able to significantly decrease the I_K, but it was less effective than O₃. H₂O₂ produced by the cells after GO administration completely reproduced the O₃ effect. Catalase showed its ability in preserving the outward rectifier component depressed by O₃, bringing back the current to the control level.</div><div>We can conclude that 4HNE and H₂O₂ are responsible for the O₃ action on potassium channels. The protective role of catalase confirms the ability of O₃ bio-product to modify the cellular redox homeostasis. In perspective it will be interesting to analyze their interaction, and the bio-molecular pathways activated.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110522"},"PeriodicalIF":3.8,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526229","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Instability and resilience at the lipid membrane interface under ultrasound: composition matters","authors":"Alexandre Blanco-González ,&nbsp;Ángel Piñeiro ,&nbsp;Rebeca García-Fandiño","doi":"10.1016/j.abb.2025.110523","DOIUrl":"10.1016/j.abb.2025.110523","url":null,"abstract":"<div><div>Lipid membranes play a crucial role in cellular function, acting not only as structural barriers but also facilitating key biological processes such as selective permeability, signaling, and mechanical stability. The composition of these membranes varies significantly across different cell types, species, and disease states, influencing their mechanical properties and susceptibility to disruption. This variability presents an opportunity to selectively target pathological cells based on their unique lipid profiles, potentially allowing for the precise disruption of diseased cells while sparing healthy ones. Additionally, focused ultrasound (FUS) has emerged as a promising tool for modulating membrane integrity, with applications in targeted drug delivery and cancer therapy. However, the precise interactions between FUS waves and different lipid compositions remain insufficiently understood. This study systematically investigates the effects of varying ultrasound frequencies (5–50 MHz) and overpressures (5–50 bar) on the mechanical responses of four distinct lipid bilayers—POPC, POPE, POPG, and POPS—using molecular dynamics simulations. These lipids are commonly found in mammalian, bacterial, and cancerous cell membranes. Key structural parameters, including area per lipid, curvature, thickness, and lipid tail order, were analyzed to determine how different ultrasound conditions affect membrane integrity. The results reveal that lipid composition critically determines membrane vulnerability to mechanical perturbations. For instance, POPC membranes are more prone to deformation under certain ultrasound conditions, while POPG and POPS exhibit abrupt transitions to instability at extreme pressures and frequencies. These findings offer valuable insights into the selective tuning of ultrasound parameters for therapeutic applications and highlight the critical role of membrane composition in determining mechanical responses to ultrasound-induced stress.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110523"},"PeriodicalIF":3.8,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of active site residues involved in substrate binding and cofactor specificity in a putrescine N-monooxygenase","authors":"Noah S. Lyons ,&nbsp;Robert A. Zalenski II ,&nbsp;Pablo Sobrado","doi":"10.1016/j.abb.2025.110519","DOIUrl":"10.1016/j.abb.2025.110519","url":null,"abstract":"<div><div>The putrescine <em>N</em>-monooxygenase (NMO) FbsI from <em>Acinetobacter baumannii</em> is a flavin-dependent enzyme that catalyzes the NADPH-dependent hydroxylation of putrescine to <em>N</em>-hydroxyputrescine, an important component of the siderophore fimsbactin A. Here, we probe the roles of T240, D390, and K223 in substrate binding and cofactor recognition. Site-directed mutagenesis and biochemical characterization showed that mutation of T240 to alanine resulted in a &gt;500-fold increase in the <em>K</em>_M for putrescine, with little effect on the <em>k</em>_cat value, highlighting the importance of this residue in binding. Mutation of D390 to alanine and asparagine rendered insoluble or inactive protein, respectively, suggesting this residue is essential for catalysis. Specificity for NAD(P)H was probed by mutating K223 to alanine and arginine. The K223R mutant had a 9-fold lower <em>K</em>_M with NADPH, while K223A had a 2-fold lower <em>k</em>_cat value and minimal change to the <em>K</em>_M value when compared to wild-type (WT) enzyme. However, rapid-reaction kinetics showed that K223R had a &gt;15-fold lower <em>K</em>_D with NADPH while K223A had a 3-fold higher <em>K</em>_D and 7.5-fold lower <em>k</em>_red compared to WT. These results demonstrate that mutation of K223 to arginine increases the specificity and efficiency of the enzyme for NADPH, identifying a key residue in cofactor recognition in FbsI.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110519"},"PeriodicalIF":3.8,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144517873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antidiabetic properties of dihydrooxazole Derivatives: In vitro and in silico evaluation as potential aldose reductase and α-glucosidase inhibitors","authors":"Busra Ozturk Aydin ,&nbsp;Baris Anil ,&nbsp;Yeliz Demir ,&nbsp;Aleksandra Rakić ,&nbsp;Dušan Dimić ,&nbsp;Derya Aktas Anil","doi":"10.1016/j.abb.2025.110521","DOIUrl":"10.1016/j.abb.2025.110521","url":null,"abstract":"<div><div>Proteins included in type 2 diabetes mellitus are potential targets for minimizing the disease progression. In this contribution, fourteen cinnamoyl compounds were synthesized and characterized, leading to five new oxazole derivatives. Their structures were optimized at the B3LYP/6–311++G(d,p) level of theory, and the global and local reactivity parameters were calculated. Based on these parameters, the reactive sites were determined. The experimental inhibitory effect towards aldose reductase (ALR2) and α-glucosidase (α-Glu) was followed, with some of the compounds showing higher activity than standard compounds, epalrestat, and acarbose. The interactions at the molecular level were investigated by molecular docking simulation, and the specific binding explained the relative reactivity order. The toxicity of compounds was assessed through ecotoxicology examination.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110521"},"PeriodicalIF":3.8,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526226","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular interactions of chiral ionic liquids with fungal membranes: thermodynamic and molecular dynamics simulation insights","authors":"Anita Wnętrzak ,&nbsp;Joanna Feder-Kubis ,&nbsp;Anna Chachaj-Brekiesz ,&nbsp;Krzysztof Łukawski ,&nbsp;Jan Kobierski ,&nbsp;Aneta D. Petelska ,&nbsp;Patrycja Dynarowicz-Latka","doi":"10.1016/j.abb.2025.110524","DOIUrl":"10.1016/j.abb.2025.110524","url":null,"abstract":"<div><div>The development of selective antifungal agents is crucial to improve therapeutic options while minimizing side effects. This study assessed the potential antimicrobial efficacy of ionic liquids, particularly against fungal pathogens. For this, a functionalized chiral ionic liquid (FCIL) with a naturally occurring (1<em>R</em>,2<em>S</em>,5<em>R</em>)-(−)-menthol moiety and a long alkyl chain was synthesized and characterized using spectral and thermal methods. The antifungal potential of this FCIL was evaluated by examining interactions with artificial fungal and mammalian membranes modeled as Langmuir monolayers. Thermodynamic analyses, complemented by adsorption and penetration experiments, Brewster angle microscopy, polarization modulation infrared reflection absorption spectroscopy, and molecular dynamics simulations, showed that FCIL incorporated into membranes and caused fungal membrane disintegration. This can be related to <em>π-π</em> interactions with ergosterol, a primary fungal membrane sterol, and favorable assimilation into membranes containing dioleoylphosphatidylcholine, an unsaturated phospholipid abundant in fungal cells. Conversely, interactions with mammalian membranes modeled using dipalmitoylphosphatidylcholine and cholesterol were thermodynamically unfavorable due to their tighter packing. These findings underline the FCIL's ability to selectively disrupt fungal membranes and suggest its potential use as a targeted antifungal agent with reduced mammalian cell toxicity. This research highlights the benefit of integrating experimental and computational methods to understand the molecular mechanics driving selective antifungal activity.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110524"},"PeriodicalIF":3.8,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144526228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of residues mediating homotropic regulation in human hexokinase 3 reveals a common allosteric interface shared with hexokinase 1","authors":"Carolin Freye,&nbsp;Brian G. Miller","doi":"10.1016/j.abb.2025.110517","DOIUrl":"10.1016/j.abb.2025.110517","url":null,"abstract":"<div><div>Vertebrate hexokinases (HKs) display a variety of allosteric phenomena, including activation and inhibition by both homotropic and heterotropic ligands. The extent to which these homologs share a common allosteric mechanism is unknown. A unique trait of the vertebrate hexokinase 3 (HK3) orthologs is substrate inhibition by glucose. Here, we demonstrate that the isolated, regulatory N-terminal domain of human HK3 contains a low affinity glucose binding site whose dissociation constant (3.2 ± 0.4 mM) approximates the <em>K</em>_<em>i</em> value for glucose (11 ± 2 mM) observed in assays of the full-length enzyme. The isolated, catalytic C-terminal domain harbors a high affinity glucose binding site whose dissociation constant (6.2 ± 0.6 μM) resembles the <em>K</em>_<em>m</em> value for glucose (53 ± 1 μM). Substitution of Asn221 in full-length HK3, which lies within the N-terminal glucose binding site, reduces substrate inhibition by 30-fold while leaving other steady-state kinetic parameters unchanged. Homotropic inhibition of HK3 is largely independent of ATP concentrations, in contrast to heterotropic inhibition of hexokinase 1 (HK1) by glucose 6-phosphate, which is competitive with respect to ATP. Adding a 10-fold molar excess of the N-terminal domain to the C-terminal domain fails to alter substrate inhibition, suggesting that interdomain communication in HK3 requires their physical connection. Disrupting a coulombic interaction between N-terminal residue Asp264 and C-terminal residue Arg807, two conserved residues previously shown to participate in HK1 regulation, attenuates glucose inhibition of HK3. Our data support a common allosteric interface in HK1 and HK3, wherein effector binding at spatially distinct sites within the regulatory N-terminus is communicated to the catalytic C-terminus via conserved coulombic residues.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110517"},"PeriodicalIF":3.8,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144511476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Shear-induced fibronectin fibrillogenesis: Differential mechanisms under high and low shear stress conditions","authors":"Quang Phuc Le ,&nbsp;Hoang Huy Mai ,&nbsp;Thao Nghi Nguyen Hoang ,&nbsp;Hanh Thu Thi Tran ,&nbsp;Duy Phuoc Tran ,&nbsp;Khon Huynh","doi":"10.1016/j.abb.2025.110518","DOIUrl":"10.1016/j.abb.2025.110518","url":null,"abstract":"<div><div>Fibrillogenesis of plasma fibronectin (FN) can be triggered by shear stress, even in the absence of cells. While high shear stress is known to promote FN fibril formation, recent evidence suggests that low shear conditions also contribute to FN assembly. It is likely that distinct molecular mechanisms govern fibril formation under different flow regimes. In this study, we investigated FN fibril formation under high shear (2000-5000 s⁻¹) and low shear (50 s⁻¹) conditions which correspond to 7–17.5 Pa and 0.175 Pa, respectively. Morphological analysis showed that high shear rapidly induced thick, interconnected fibrils, whereas low shear led to the gradual formation of thin, sparse fibrils. Molecular dynamic simulations indicated that shear stress alone did not unfold FN in suspension; however, surface-adsorbed FN enabled shear-driven interaction with soluble FN. Under low shear, fibrillogenesis proceeded via slow FN accumulation and weak intermolecular binding, while high shear promoted strong domain-domain interactions. Molecular docking identified top-ranked <em>trans</em>-binding interfaces between FNI1–5 and FNIII1–3 regions, stabilized by salt bridges (e.g., Glu92-Arg694) and hydrogen bonds involving residues such as Lys149-Gly713 and Tyr265-Ser864. These interdomain interactions provide a structural basis for shear-induced fibril assembly.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110518"},"PeriodicalIF":3.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The role of COX-2 and its use as a therapeutic target in IBD and related colorectal cancer","authors":"Lulu Yang ,&nbsp;Francis Atim Akanyibah ,&nbsp;Dongming Yao ,&nbsp;Tao Jin ,&nbsp;Fei Mao","doi":"10.1016/j.abb.2025.110516","DOIUrl":"10.1016/j.abb.2025.110516","url":null,"abstract":"<div><div>Inflammatory bowel disease (IBD) is a persistent inflammation of the gastrointestinal tract with a significant risk of progression to colorectal cancer (CRC). IBD and CRC have increased cyclooxygenase-2 (COX-2) levels, which is important in these conditions. In IBD, COX-2 contributes to intestinal inflammation, mucosal injury, and immune response dysregulation, while in CRC, it contributes to carcinogenesis and influences tumor microenvironment and angiogenesis. COX-2 may also facilitate its harmful function via prostanoids. Research suggests that COX-2 polymorphisms in IBD and CRC elevate the chance of developing both conditions. Researchers have recognized COX-2 as a biomarker for CRC. Consequently, inhibiting COX-2 may aid in the prevention of IBD and CRC. Non-selective and selective COX-2 inhibitors have demonstrated the ability to mitigate IBD and CRC; however, their efficacy and safety diminish. Nanoparticles, prodrugs, small molecules, and polymeric substrates can deliver COX-2 inhibitors to specific tissues, which makes therapy more effective and safer. Consequently, their application for administering COX-2 inhibitors may herald a new era of improved effectiveness and reduced side effects. Molecular investigations have identified new compounds as potential COX-2 inhibitors. Extracts, microRNAs, and mesenchymal stem cells also help target COX-2 to prevent IBD and CRC.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110516"},"PeriodicalIF":3.8,"publicationDate":"2025-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144366844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New 1,2,3-triazole derivatives as acetylcholinesterase and carbonic anhydrase inhibitors: Synthesis, molecular docking, and solubility","authors":"Cagla Efeoglu ,&nbsp;Yeliz Demir ,&nbsp;Cüneyt Türkeş ,&nbsp;Erdal Yabalak ,&nbsp;Zeynel Seferoglu ,&nbsp;Yahya Nural","doi":"10.1016/j.abb.2025.110515","DOIUrl":"10.1016/j.abb.2025.110515","url":null,"abstract":"<div><div>In this study, a series of new 1,2,3-triazole derivatives were synthesized in 84–93 % yield using copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry and characterized by ¹H/¹³C NMR, FT-IR, and HRMS analyses. The synthesized compounds (<strong>4a–h</strong>) were evaluated for their inhibitory activities against human carbonic anhydrase isoforms I and II (<em>h</em>CA I and <em>h</em>CA II) and acetylcholinesterase (AChE), which are clinically relevant targets in neurological and metabolic disorders. Among them, compounds <strong>4f</strong> and <strong>4g</strong> exhibited the most potent dual inhibitory activities. Compound <strong>4f</strong> showed <em>K</em>_I values of 144.30 nM for <em>h</em>CA II and 205.10 nM for AChE, while compound <strong>4g</strong> exhibited <em>K</em>_I values of 239.10 nM for <em>h</em>CA II and 125.90 nM for AChE. These values demonstrate that <strong>4f</strong> and <strong>4g</strong> are more effective than the reference drugs acetazolamide (<em>h</em>CA II, <em>K</em>_I = 381.44 nM) and tacrine (AChE, <em>K</em>_I = 255.44 nM). Structure-activity relationship (SAR) analysis revealed that hydrophobicity, steric bulk, and aromaticity significantly influenced enzyme affinity. <em>In silico</em> docking confirmed strong interactions with key active site residues. Furthermore, ethanol solubility profiling revealed that polar and hydrogen-bonding groups significantly improved solubility, while bulky or aromatic hydrophobic substituents reduced it. The combined biological activity and solubility data emphasize the potential of these triazole derivatives particularly <strong>4f</strong> and <strong>4g</strong> as promising candidates for multitarget drug design and further preclinical development.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110515"},"PeriodicalIF":3.8,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144366845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Organosilicon molecules bind to the intrinsically disordered protein NUPR1 by clamping its hot-spots","authors":"Laura F. Peña ,&nbsp;Matías Estaras ,&nbsp;Paula González-Andrés ,&nbsp;Carlos Díez-Poza ,&nbsp;Bruno Rizzuti ,&nbsp;Olga Abian ,&nbsp;Adrian Velazquez-Campoy ,&nbsp;Juan L. Iovanna ,&nbsp;Patricia Santofimia-Castaño ,&nbsp;José L. Neira ,&nbsp;Asunción Barbero","doi":"10.1016/j.abb.2025.110513","DOIUrl":"10.1016/j.abb.2025.110513","url":null,"abstract":"<div><div>The nuclear protein 1, or NUPR1, is an intrinsically disordered protein (IDP) involved in the development and progression of pancreatic ductal adenocarcinoma (PDAC). We have previously developed drugs capable of binding at the two hot-spot regions of NUPR1, around residues Ala33 and Thr68, hampering its interactions <em>in cellulo</em>. In this work, we synthesized new organosilicon molecules targeting those key hot-spots. The compounds were obtained by an acid-catalyzed intramolecular cyclization of a starting alkenol that contains a silyl group attached to the double bond. Binding between the silyl compounds and NUPR1 involved the two hot-spots, as shown by 2D ¹H–¹⁵N HSQC NMR. Molecular simulations clarified that the binding relies on a loose clamp mechanism of the ligands towards the hot-spots. The dissociation constants (<em>K</em>_d) were around 20 μM, as measured by several biophysical techniques. However, studies <em>in cellulo</em> with PDAC cells did not show a decrease of cell viability upon treatment with the compounds; furthermore, proximity ligation assays <em>in cellulo</em> with a natural partner protein of NUPR1, G3BP, did not show a significant level of interfering in such interaction when silyl compounds were present, probably due to the high hydrophobicity of the designed compounds. Thus, in the case of NUPR1, moderate-to-high drug binding affinities (<em>K</em>_d &lt; 10 μM) <em>in vitro</em> and a higher hydrophilicity are necessary to hamper protein-protein interactions <em>in cellulo</em>. As a more general conclusion, <em>in vitro</em> binding of ligands to the protein hot-spots is a necessary condition in the drug design targeting IDPs, but it is not enough to guarantee inhibition of their interactions <em>in cellulo</em>.</div></div>","PeriodicalId":8174,"journal":{"name":"Archives of biochemistry and biophysics","volume":"771 ","pages":"Article 110513"},"PeriodicalIF":3.8,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}