The glycopeptide antibiotics (GPAs) remain clinical antibiotics used against drug-resistant Gram-positive infections. The discovery of GPAs continues, with new type IIa and V GPAs, the kineomicins, the rimomycins, and corbomycin, being recently identified. Despite this larger repertoire of GPA scaffolds and crosslinking types, limitations remain with the in vitro exploration of the cytochrome P450 enzymes that install the essential crosslinks between the aromatic side chain residues of GPAs. While the chemoenzymatic synthesis of the more hydrophilic type I GPAs like vancomycin has aided our understanding of GPA crosslinking pathways, type II-V GPAs remain underexplored. This is due to the hydrophobic nature of these GPAs that makes access to peptidyl-CoA substrates difficult and reduces in vitro crosslinking activity. Here, we explore the use of modified Knorr-pyrazole chemistry to provide access to hydrophobic peptidyl-CoA substrates of the type II/IIa GPAs, kineomicin and actinoidin, and the type IV GPA, teicoplanin. Yields of peptidyl-CoAs were improved 3-7-fold through careful tuning of reaction solvents and the arylthiol used for displacement of the pyrazole. The type II/IIa GPA scaffolds explored in this study displayed the highest in vitro OxyC activity seen to date, likely due to both improved synthesis and their structural properties.
Breast cancer is one of the most common cancers worldwide. Approximately 30%-40% of breast cancers harbor mutations in the TP53 gene, leading to structural and functional alterations in the p53 protein. These changes result in mutant proteins that are unable to perform their canonical tumor suppressor functions and, in many cases, exhibit gain-of-function properties, making mutant p53 a highly attractive therapeutic target. Among these mutations, R280K and R273H are two clinically relevant DNA-binding mutations. In this study, we extended our investigation of the (R)-tryptophanol isoindolinone derivative RVJB59 to these two p53 mutants. Differential scanning fluorimetry showed that RVJB59 thermally stabilized the R280K mutant p53 in a dose-dependent manner. Furthermore, the mechanism of action of RVJB59 was investigated by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) using the DNA‑binding domains of the R280K and R273H mutants, confirming covalent binding of the compound to Cys141 in both proteins. Computational studies with both p53 mutants suggested that RVJB59 can stably bind in proximity to Cys141, further reinforcing the results obtained by LC-HRMS/MS. Our findings further underscore the potential of RVJB59 for the development of novel therapies for the treatment of breast cancers harboring these mutations.
Given the reluctance of pharmaceutical companies to invest in new antileishmanial drugs, due to high costs and low perspectives of financial return, there is growing interest in repurposing existing drugs, often a more cost-effective and quicker strategy to launch effective therapeutic solutions. Artemisinins, used as first-line malaria treatments, have attracted attention for their potential activity against Leishmania. The mechanisms by which artemisinins treat malaria were deeply investigated and are thought to involve peroxide cleavage with formation of radicals, alkylating molecular targets within Plasmodium. However, the action of endoperoxides on Leishmania spp. remains poorly understood. To gain insight, we undertook the synthesis and structural characterization of 1,2,4-trioxolanes, 1,2,4,5-tetraoxanes, and respective deoxygenated controls. The compounds were evaluated in vitro against axenic and intracellular forms of Leishmania donovani and Leishmania major. Their selectivity indexes (SIs) were determined. Among 29 compounds, tetraoxanes 27, 29, 30, 32, and 33 exhibited the highest potency against both Leishmania species, but most showed limited selectivity (SI < 10) toward the parasite. Nevertheless, 33 demonstrated strong efficacy against L. major (SI of 13.7). Comparing peroxides versus their ether controls often revealed similar activities, suggesting involvement of other mechanisms beyond the iron-dependent peroxide activation, unlike what is described for Plasmodium.
Abstract Commercial nonionic surfactants are multicomponent mixtures, typically derived from ethoxylation of a primary alcohol that may be a mixture of homologues and isomers with a range of degrees of ethoxylation. Daphnia magna and Vibrio fischeri toxicity data have been obtained for several nonionic surfactants, covering a wide range of average degrees of ethoxylation and parent alcohol structure, including both narrow-range and broad-range ethoxamer distributions. These data are analyzed by means of a general narcosis quantitative structure–activity relationship (QSAR) based on calculated log P values together with mixture toxicity equations. In some cases not all components are sufficiently soluble to make their full toxic contributions. An approach for identifying such components and for taking their reduced toxic contributions into account is derived from previously published structure-solubility relationships and validated against the experimental toxicity data.
Abstract Aflatoxin B1 (AFB1) is a potent foodborne hepatocarcinogen that contributes substantially to the global burden of hepatocellular carcinoma (HCC), yet the molecular mechanisms underlying AFB1-driven HCC progression remain incompletely understood. In this study, we identified thyroid hormone receptor-interacting protein 13 (TRIP13) as an important contributor to AFB1-related HCC progression and investigated its involvement in ferroptosis regulation. TCGA-LIHC, GSE25844, and single-cell RNA sequencing data (GSE166635) revealed that TRIP13 was significantly upregulated in AFB1-related HCC, enriched in malignant cell populations, and associated with poor clinical outcomes. In Huh7 cells, AFB1 treatment markedly increased TRIP13 expression, while TRIP13 knockdown significantly suppressed AFB1-induced cell proliferation, colony formation, migration, and epithelial-mesenchymal transition (EMT). Bulk RNA sequencing and pathway enrichment analyses indicated that TRIP13 overexpression was strongly associated with ferroptosis-related signaling. Mechanistically, TRIP13 was associated with ferroptosis resistance through modulation of the KEAP1/NRF2/xCT/GPX4 pathway, thereby contributing to redox homeostasis during AFB1 exposure. Consistently, TRIP13 knockdown restored ferroptotic sensitivity, as evidenced by increased Fe2+ and malondialdehyde levels and altered glutathione metabolism. Importantly, ferroptosis induction with RSL3 effectively reversed the pro-tumor effects of TRIP13 overexpression both in vitro and in vivo, including enhanced proliferation, EMT, migration, and xenograft tumor growth. Collectively, these findings suggest that TRIP13 contributes to AFB1-associated HCC progression through ferroptosis-related regulation and highlight the AFB1/TRIP13/ferroptosis axis as a potential molecular mechanism and candidate target for further investigation in AFB1-related HCC.
Autotaxin (ATX), a lysophospholipase D playing an important role in several inflammatory diseases, as well as in tumor invasion, progression, and metastasis, is an attractive therapeutic target. Herein, we report a virtual screening study of an in-house molecular database. In silico simulations highlighted five putative ATX inhibitors. The enzyme assay showed that compound 3 achieves inhibition potency in the submicromolar range (IC50 = 0.534 µM), whereas all the other test compounds proved to be moderate (2) or weak (1, 4, and 5) inhibitors of ATX at 10 µM concentration. These compounds, evaluated for the in vitro antiproliferative activity in four ATX-expressing cancer cell lines, namely the ovarian cancer cell lines A2780 and SK-OV-3, the breast cancer cells MCF-7, and immortalized mouse embryonic fibroblast cell line NIH/3T3, demonstrated preliminary phenotypic effects, inhibiting cell proliferation with IC50 values mostly in the low micromolar range. Compound 4 (IC50 = 11.5 µM against SK-OV-3) significantly reduced the motility of SK-OV-3 ovarian carcinoma cells in the wound-healing assay, suggesting that it may represent a hit structure for the development of potential antimetastatic agents.
Pyrazolone and its keto-enol tautomers constitute a privileged class of scaffolds in contemporary medicinal chemistry, characterized by remarkable structural plasticity. This review comprehensively summarizes recent advancements in the structural optimization of pyrazolone-based compounds, highlighting how molecular hybridization drives polypharmacology against complex conditions, including neurodegenerative disorders, inflammation, metabolic syndromes, cancer, and infectious diseases. By detailing structure-activity relationships, the review elucidates how the strategic pharmacophore merging at the N-1, C-3, and C-4 positions transforms the pyrazolone core into highly potent therapeutics. Mechanistically, these hybrid molecules exhibit diverse capabilities, such as inhibiting key survival kinases, blocking pathogenic protein aggregation, antagonizing immune checkpoints, and restoring cellular redox homeostasis via robust reactive oxygen species scavenging. Ultimately, this review underscores the critical role of molecular hybridization in overcoming drug resistance, minimizing systemic toxicity, and driving the future discovery of pyrazolone-based precision therapeutics.
In this study, a series of novel angiotensin II type 1 receptor (AT1R) antagonist candidates containing the 1,2,4-oxadiazol-5-one nucleus and a 1,2,3-triazole moiety was designed, synthesized, and evaluated. Compounds 7a-j were obtained via a semiconvergent synthetic route employing copper-catalyzed azide-alkyne cycloaddition (click chemistry), yielding the target molecules in moderate to good yields. Biological activity was assessed through vascular reactivity assays using rat aortic rings in the presence of angiotensin II. Most compounds reduced angiotensin II-induced contraction, consistent with a putative AT1 receptor antagonist profile. Among them, compound 7f, bearing a methylenedioxyphenyl substituent, exhibited the most favorable biological profile within the series. The results indicate that electron-donating groups favor activity, while strongly electron-withdrawing substituents, such as nitro, are detrimental. Molecular docking studies predicted consistent interactions with key residues, particularly Arg167 and Tyr87, providing a plausible structural rationale for the observed biological activity. In silico ADME predictions indicated acceptable drug-like properties, including compliance with drug-likeness filters and a predicted bioavailability score, and no blood-brain barrier permeation.
Oxidative stress and inflammation are key players in central nervous system (CNS) diseases and in neurodegeneration. In this field, the search for novel targets and therapeutic tools is wide open. In this study, the identification of potential inhibitors of phosphodiesterase 9 (PDE9), a target of growing interest in CNS diseases, was done by using a rational virtual screening approach that employed ligand- and structure-based methodologies. Furthermore, a novel method to evaluate PDE9A activity based on high-performance liquid chromatography (HPLC) was developed to address the need for a time- and cost-effective assay to evaluate inhibitors. One of the candidates identified by virtual screening demonstrated potent enzymatic inhibition, and subsequent in vitro tests proved its significant PDE9A-dependent anti-neuroinflammatory effects as it acted on pro-inflammatory mediators such as COX-2, IL-1β, TNFα, and IL-6. The results of this multidisciplinary study underscore the potential of developing PDE9A inhibitors to modulate cyclic guanosine monophosphate (cGMP) signaling pathways implicated in neuroinflammation and also potentially in synaptic plasticity and cognitive functions, paving the way for novel PDE9A-targeting inhibitors addressing neurodegenerative diseases.
Spirooxindoles are a class of spirocyclic scaffolds in drug discovery, as the rigid frame and well-defined substitution pattern can interact with various biological targets. Recent advances in synthetic methodologies, including multicomponent 1,3-dipolar cycloadditions, organocatalytic cascades, and one-pot protocols, have facilitated the efficient synthesis of a wide variety of derivatives for SAR studies. A diverse range of classes, including pyrrolidine, cyclopropyl carboxamide, carbamate, oxadiazoline, pyrrolizidine, and furan derivatives, show cytotoxicity in the submicromolar-to-low micromolar range with good selectivity. The SAR studies suggest that substitution on the isatin core generally enhances biological activity in individual series, especially with electron-withdrawing groups, but these effects are scaffold dependent. Furthermore, spirooxindole derivatives have been reported to show antibacterial activity, including TrpRS inhibition, antimicrobial, acetylcholinesterase inhibition, α-glucosidase inhibition, antiviral, and anticonvulsant activities. The recent patents, especially on MDM2 inhibitors and other anticancer agents with nanomolar potency, provide further evidence for the continued interest in spirooxindole-based drug discovery. This review highlights the recent advancements in spirooxindole chemistry involving synthetic strategies, SAR, biological activities, and recent patents.
The ongoing emergence and reemergence of RNA viruses from multiple viral families highlights the urgent need for broad-spectrum antiviral therapeutics. To address this issue, a series of 4'-azido fleximer nucleoside analogs were synthesized and evaluated for broad-spectrum antiviral activity against pathogens of epidemic/pandemic concern. One compound, KAD-039, featuring a 2', 3'-diisobutyrate-5'-ProTide prodrug, exhibited potent antiviral activity across flaviviruses, filoviruses, and coronaviruses, with EC50 values ranging from 1 to 10 µM and minimal cytotoxicity (CC50 > 50 µM). Pharmacokinetic evaluation of KAD-039 demonstrated high plasma stability in dog, monkey, and human plasma, with half-lives ranging from 19.9 to 28 h but exhibited poor stability in human liver microsomes (t1/2 = 6.1 min). To help elucidate the mechanism of action of KAD-039 against these viral families, computational molecular docking studies were performed using the expected triphosphate active form within the cap/GTP-binding pockets of several viral methyltransferases and showed that KAD-039TP docked into all of the viral MTases with similar or higher predicted affinity compared to the natural substrate. In addition, KAD-039TP displayed reduced predicted affinity for the human N7 methyltransferase, suggesting low host toxicity. These results highlight 4'-azido fleximer analogs as promising broad-spectrum antivirals, potentially functioning through viral methyltransferase inhibition, and support further investigation.
The Na+/K+-ATPase (NKA) is a central regulator of cardiac ion homeostasis and a validated target for heart failure. Yet, cardiotonic steroids used in clinical practice are limited by a narrow therapeutic window and pro-arrhythmic effects. Here, we identify Ro 41-0960 as a non-steroidal inhibitor of NKA with a distinct mechanistic profile. The compound inhibits NKA activity with IC50 values of 17.9 ± 1.1 µM for purified enzyme and 10.3 ± 1.1 µM in microsomal preparations. ATP-dependent activity measurements revealed a non-monotonic response in which inhibition was most pronounced at ATP concentrations below 4 mM and diminished at the highest ATP concentrations tested. Docking and molecular dynamics simulations suggest that Ro 41-0960 can access both the cardiotonic steroid-binding pocket and the nucleotide-binding site; however, the ATP-dependence data are not consistent with a simple ATP-competitive mechanism. Furthermore, the compound shows only weak inhibition of SERCA (IC50 > 100 µM) and does not alter electrophysiological parameters in human iPSC-derived cardiomyocytes at concentrations producing near-maximal NKA inhibition, providing initial evidence of a favorable cardiac safety profile. Together, these findings identify Ro 41-0960 as an NKA inhibitor with a distinct chemical scaffold and provide a framework for the development of non-steroidal NKA modulators.
The S-phase kinase-associated protein 2 (Skp2)-cyclin-dependent kinase subunit 1 (Cks1) protein-protein interaction (PPI) plays a central role in recognition of phosphorylated p27 and therefore represents an attractive target for anticancer drug discovery. Herein, we sought to identify new small-molecule disruptors of the Skp2-Cks1 interface from a focused fumiquinazoline-scaffold library by combining virtual screening, molecular simulation, and experimental validation. Docking showed that all selected compounds occupied the Skp2-Cks1 interfacial hotspot. However, longer 1000-ns MD simulations, comparative MM/PBSA calculations, and interface-related metrics revealed distinct structural, energetic, and dynamic binding profiles among the selected compounds. Consistent with these analyses, comparative MM/PBSA evaluation of PPI stability showed that Fumiquinazoline D produced a positive Δ Δ G PPI , indicating weakening of the Skp2-Cks1 interface relative to the apo complex, whereas Ardeemin, Fiscalin A, and Fumiquinazoline F generally yielded negative Δ Δ G PPI values consistent with interfacial stabilization. Free energy landscape analysis further supported a more weakly confined and dynamically disruptive binding mode for Fumiquinazoline- D. In vitro homogeneous time-resolved fluorescence assays validated these predictions: Fumiquinazoline D inhibited the Skp2-Cks1 interaction with an IC50 of 6.33 ± 0.44 µM, whereas Ardeemin, Fiscalin A, and Fumiquinazoline F were substantially weaker. Although ADMET profiling identified substantial absorption and toxicity liabilities requiring future optimization, these findings identify Fumiquinazoline D as an early-stage biochemical hit for disruption of the Skp2-Cks1 interaction and as a potential scaffold for further mechanistic validation and medicinal-chemistry investigation.
Photodynamic therapy (PDT) is a clinically established approach that relies on the localized generation of reactive oxygen species (ROS) mediated by a photosensitizer (PS), light, and molecular oxygen. Protoporphyrin IX (PpIX) is an approved PS; however, its intracellular retention is limited, factor that can alter its overall photodynamic efficiency. In this study, we report how the design of PpIX derivatives bearing piperazine (PpIX-Pip) or morpholine (PpIX-Morp) substituents may modulate lipophilicity, cellular uptake, oxidative stress, and phototoxicity. The photodynamic activity of these derivatives was evaluated in the aggressive triple-negative human breast cancer cell line MDA-MB-231. Both derivatives showed greater time-dependent cellular retention and enhanced photodynamic activity compared to PpIX, promoting oxidative stress, and triggering loss of mitochondrial membrane potential and caspase-3 activation. These results highlight the impact of rational structural modification of PpIX on photodynamic efficiency and provide valuable structure-activity relationship insights for the development of improved porphyrin-based PS for PDT.
The tandem Tudor domain (TTD) of UHRF1 is a compelling epigenetic target for novel cancer therapeutics. Here, we integrate theoretical simulations, sophisticated biophysical evaluations and cellular assays to characterize potent TTD binders. Screening of small drug- and fragment-like collections identifies several TTD ligands, with the most promising hit being the local anesthetic hydroxyprocaine. The ligand is characterized in terms of its binding requisites by calorimetry, affording a Kd of 1.46 μM and a well-balanced thermodynamic profile. Molecular dynamics simulations combined with heat capacity measurements and osmotic stress titrations confirm that hydroxyprocaine binds stably to the TTD by displacing approximately 26 interfacial water molecules upon complexation. A targeted follow-up screen focusing on sodium channel blockers yields two additional, although less promising hits, mexiletine and triamterene. In the DU145 prostate cancer cell line, hydroxyprocaine treatment significantly up-regulates key downstream targets including the tumor-suppressor p53 and, to a lesser degree, the stress and inflammation regulators p38 and p65, respectively, while exhibiting very low cytotoxicity. Finally, a previously undocumented interdomain interaction between TTD and its N-terminal adjacent Ubiquitin-like domain is reported, introducing a novel, potentially druggable UHRF1 regulatory feature. Together, these findings establish hydroxyprocaine as a highly viable chemical scaffold for TTD-targeted drug development.
Chagas disease and leishmaniasis are neglected tropical diseases caused by the trypanosomatid protozoa Trypanosoma cruzi and Leishmania spp., respectively. Currently available treatments are often insufficiently effective and lack the characteristics of ideal therapeutics. Here we report the design, synthesis, and antitrypanosomatid evaluation of 20 novel hybrid compounds incorporating pyridine, thiazole, and thiosemicarbazone scaffolds. All compounds were tested against trypomastigote and amastigote forms of T. cruzi, as well as promastigote and amastigote forms of Leishmania infantum and L. amazonensis. Cytotoxicity was assessed in L929 fibroblasts and RAW 264.7 macrophages. Compound 8 emerged as the lead, displaying an IC50 of 1.0 µM against T. cruzi and a selectivity index of 52.8. Despite its potent in vitro activity, Compound 8 exhibited poor in vivo efficacy when administered orally at 150 mg/kg/day. In silico analyses predicted strong binding to cruzain, alongside favorable bioavailability, drug-likeness, and chemical stability. Against Leishmania species, Compound 8 demonstrated selectivity comparable to miltefosine against L. amazonensis amastigotes. Collectively, these findings identify a novel chemical scaffold with promising antitrypanosomatid potential that warrants further optimization to improve in vivo performance.
Acylthiourea ligands, featuring hard (N, O) and soft (S) donor atoms, enable diverse coordination modes in half-sandwich Ru(II)-p-cymene complexes. Herein, we report the synthesis and full characterization of eight half-sandwich Ru(II)-p-cymene complexes, comprising four monodentate (1m-4m) and four bidentate (1b-4b) species prepared under distinct synthetic conditions. The structures of the complexes 1m, 2m, 3m, 3b, and 4b were confirmed by single-crystal X-ray diffraction, while FT-IR, UV-vis, ESI-MS, and 1H/13C NMR spectroscopy unambiguously distinguish the two binding modes. The reversible coordination mode was monitored by 1H NMR and UV-vis spectroscopy, with base-induced deprotonation favoring bidentate binding and HCl addition regenerating the monodentate form. Cytotoxic assays revealed that metal coordination markedly enhanced the antiproliferative activity of the ligands. Most bidentate complexes exhibited higher cytotoxicity than their monodentate analogues. In particular, 2b displayed a sixfold increase in potency (IC50 = 6.05 μM) compared to 2m (36.4 μM) and showed much lower toxicity toward normal ARPE-19 cells (IC50 = 55.0 μM), indicating greater selectivity toward cancer cells. Molecular docking suggests DNA groove binding as the anticancer mechanism, highlighting the role of bidentate coordination, with 2b identified as the most promising Ru(II)-p-cymene complex.
Abstract Per/polyfluoroalkyl substances (PFASs) have been confirmed to have negative health effects in animal models. As a novel alternative to legacy PFASs, sodium ρ-perfluorous nonenoxybenzenesulfonate (OBS) raises growing concerns over environmental exposure and biological toxicity. Herein, colitis models were constructed for exploring the toxicity of OBS in intestinal homeostasis during the recovery period. The results verified the gut toxicity of OBS, which increased serum interleukin-1β/6/17α/22 (IL-1β, IL-6, IL-17α, IL-22) levels, decreased colonic mucus secretion, and changed gut microbiota. During colitis recovery, no statistically significant difference was observed in colonic mucus secretion and related gene expression in dextran sulfate sodium-OBS (DSS-OBS) compared with the DSS; compared with the CON group, the unchanged mucus secretion and gene expression in DSS-OBS ghad still a statistically significant difference. It indicated that the intestinal barrier disruption did not worsen after the OBS exposure, but the recovery might be delayed. Moreover, OBS exposure significantly changed gut microbiota during the recovery period, suggesting that disrupted gut microbiota might be associated with the delay of OBS-mediated inhibition of intestinal barrier repair. Additionally, network toxicology analysis identified the action targets of OBS (including Nuclear Receptor Subfamily 1 Group I Member 2 (NR1I2), Solute Carrier Family 22 Member 5 (SLC22A5), Glutathione S-Transferase Pi 1 (GSTP1), etc.), which mediate gut microbiota disorder and intestinal barrier dysfunction, and serve as potential biomarkers for OBS-induced intestinal-related diseases. Collectively, OBS exhibits definitive gut toxicity. Although short-term OBS exposure did not exacerbate the progression of colitis, it delayed the recovery of intestinal function. Among, multiple target sites, coupled with the gut microbiota, commonly mediate the gut toxicity of OBS. It suggested that the potential health risks of OBS cannot be overlooked, and the relevant departments should strengthen the management and develop next-generation alternatives.
Abstract Oxaliplatin (OXA), a platinum-based chemotherapeutic agent, frequently induces acute peripheral neuropathy characterized by mechanical and cold allodynia, for which effective therapeutic options remain limited. Here, we investigated the acute antinociceptive and biochemical effects of 4-amino-3-(phenylselanyl)benzenesulfonamide (4-APSB), a compound with antioxidant and antinociceptive properties, in female Wistar rats with OXA-induced acute neuropathy. Animals received OXA (4 mg/kg, i.p.) on two consecutive days and were subsequently treated with 4-APSB (1 mg/kg, i.g.) or duloxetine (30 mg/kg, i.g.) as a reference drug. Nociceptive behaviors were assessed using mechanical and cold sensitivity assays, followed by biochemical analyses in nervous tissues. OXA administration induced mechanical and cold hypersensitivity, elevated oxidative stress markers, and disrupted membrane enzyme function, reducing Na+/K+-ATPase activity and increasing acetylcholinesterase (AChE) activity. Treatment with 4-APSB significantly attenuated hypersensitivity, increased Na+/K+-ATPase and catalase activities, reduced reactive species generation, and attenuated the OXA-induced increase in AChE activity in the cerebral cortex, without affecting locomotor behavior. Collectively, these findings show that 4-APSB produces acute antinociceptive effects accompanied by the modulation of redox homeostasis, Na+/K+-ATPase activity, and AChE activity following OXA exposure. The coordinated improvement in behavioral and biochemical parameters supports the potential relevance of redox balance and membrane-associated enzyme function to the pharmacological effects of 4-APSB. These results identify 4-APSB as a promising compound for further investigation as a therapeutic strategy for OXA-induced peripheral neuropathy.
Abstract DNA is continuously exposed to endogenous and exogenous agents, including chemotherapeutic agents, environmental pollutants, and food-borne toxicants, resulting in covalent modifications of DNA bases known as DNA adducts. The biological impact of these adducts is primarily determined by their interaction with cellular proteins, which dictates whether the damage results in DNA repair, tolerance via translesion synthesis with potential mutagenesis, or cell death if the damage persists. Consequently, investigation of DNA adduct–protein interactions is essential for a deeper understanding of their biological processing. With this review, we provide a comprehensive overview of methodologies used to identify proteins that interact with covalent DNA modifications, with a focus on monovalent DNA adducts and a particular emphasis on proteins involved in DNA repair pathways. We introduce and critically discuss functional and biochemical assays used to assess repair pathways and isolate DNA adduct–binding proteins, as well as analytical strategies used to identify these proteins. We also provide a summary of approaches used for the further characterization of the DNA adduct–protein interactions and discuss how these insights can inform our understanding of the cellular impact of specific DNA adducts. Finally, we give an overview of the preparations necessary to perform the assays reviewed here. This work provides a roadmap to identify and characterize DNA adduct–binding proteins, establishing a foundation to elucidate their roles in DNA repair and cellular responses to damage.
