Abstract The pathogenic yeast Candida glabrata is intrinsically resistant to azole antifungals through the overexpression of the multidrug transporter Cdr1. CgCdr1 detoxifies the yeast by expelling azoles out of the cell, thereby decreasing their intracellular concentration. Tacrolimus (FK506), one of the most widely used immunosuppressant medications used world-wide, has been identified as a broad-spectrum inhibitor of Cdr1 homologs in several Candida species. However, its mechanism of action remains unknown. We solved the cryoEM structure of CgCdr1 in complex with FK506, with or without ATP. The structure revealed that FK506 binds within the drug-binding site of CgCdr1, occupying the space occupied by Itraconazole. The hydrophobic face of FK506 stacks against the TMD1 and forms hydrogen bonds with TMD2, stabilizing a different conformation from the one adopted in FK-binding-proteins. FK506 binding triggered structural rearrangements bringing the nucleotide-binding-domains closer to the trans-membrane-domains, while stabilizing the inward-facing conformation. While ATP can still bind to the catalytic nucleotide-binding site, FK506 prevents the conformational transition required for ATP hydrolysis, thereby effectively blocking azole transport. Inter-particle variability analysis (3DVA) revealed significant conformational flexibility of FK506 within the binding pocket, with minimal transporter mobility. It allowed to visualize the conformational space occupied by the inhibitor within its binding-pocket, serving as a useful tool for inhibitor rational design. Overall, these findings demonstrate that FK506’s inhibition extends beyond competitive binding, involving allosteric modulation of the ATPase cycle. Significance statement The pathogenic yeast Candida glabrata exhibits intrinsic resistance to azole antifungals via the multidrug transporter Cdr1, which expels azoles from the cell. Tacrolimus (FK506), a widely used immunosuppressant, inhibits Cdr1 homologs across Candida species, yet its mechanism remained unknown. Here, we resolved the cryoEM structure of Cg Cdr1 in complex with FK506, revealing that FK506 binds to the drug-binding site, like itraconazole. Its hydrophobic face interacts with TMD1, while hydrogen bonds form with TMD2. FK506 stabilizes the inward-facing conformation, preventing ATP hydrolysis despite ATP binding, thereby blocking azole transport. Variability analysis highlighted FK506’s conformational flexibility within the pocket, offering insights for rational inhibitor design. These findings demonstrate that FK506’s inhibition involves both competitive binding and allosteric modulation of the ATPase cycle.

ABSTRACT Isoprenoid quinones are ubiquitous redox lipids that mediate electron transfer in various cellular processes across all domains of life. These molecules also serve as taxonomic and metabolic markers, facilitating the characterisation of microbial communities. However, their structural diversity and extreme hydrophobicity are challenging for comprehensive detection and quantification in complex biological matrices. In this study, we present a semi-quantitative HPLC-MS/MS method that enables the sensitive analysis of the widest range of quinones reported to date. Using a 16-quinone standard mixture, we optimised separation within a 14-minute HPLC gradient and achieved femtomole-level sensitivity in targeted analyses. When applied to sewage sludges sampled weekly over three weeks, our method detected 57 distinct quinones, revealing stage-specific quinone profiles that reflect shifts in bacterial communities during wastewater treatment. This rapid and sensitive workflow provides a robust tool for accurate quinone profiling in complex samples, opening avenues for the discovery of novel quinones through untargeted approaches. By pushing the boundaries of quinone profiling, our method holds significant promise for advancing microbial ecology, environmental monitoring, and biotechnological applications. Highlights uHPLC-Orbitrap method for the semi-quantitative profiling of isoprenoid quinones Analysis of the widest range of isoprenoid quinones to date Femtomole-level sensitivity in just 14 minutes of chromatographic separation Detection of 57 quinones in complex wastewater sludge matrices Most comprehensive set of quinone standards including purified microbial quinones

Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters remains a major obstacle to cancer chemotherapy, particularly at later disease stages with metastases. Among the 48 human ABC proteins, P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are the most studied ABC transporters associated with MDR. Inhibition of ABC transporters has been considered as one possible strategy to overcome MDR. In this study, twelve N-methylpyrazole derivatives were evaluated as inhibitors of ABCB1, ABCC1, and ABCG2. Several compounds selectively inhibited ABCG2 while showing no activity against ABCB1 or ABCC1. The most potent derivative, 1l, inhibited more than 50% of ABCG2 activity at 10 μM and displayed substrate-independent inhibition, with IC50 values ranging from 1.6 to 3.8 μM depending on the fluorescent probe used. Compound 1l exhibited only mild cytotoxicity and was not transported itself by ABCG2. Mechanistic studies revealed that 1l induced conformational changes in ABCG2, as evidenced by increased binding of the 5D3 conformational antibody. Combination assays with established ABCG2 inhibitors, including chromone 4a and indeno-[1,2-b]indole 5e, showed neither synergistic nor antagonistic effects. Induced-fit docking simulations supported the experimental data, indicating that 1l binds within the central transmembrane cavity of ABCG2, engaging key residues such as Phe439 and stabilizing inward-facing conformations. Finally, co-treatment with 1l restored sensitivity of ABCG2-overexpressing cells to the anticancer drug SN38, effectively reversing the MDR phenotype. Collectively, these results identify N-methylpyrazole derivatives as promising selective inhibitors of ABCG2.

Myocardial infarction (MI) is the leading cause of death worldwide. The extent of infarct size, determined by the degree of cardiomyocyte death, is a major factor in post-MI functional recovery. Therefore, cardioprotective strategies focus on limiting infarct size. Among the mechanisms involved in the MI response, Hypoxia Inducible Factor-1 (HIF-1) plays a complex role that can be either beneficial or detrimental depending on the nature of its activation (i.e., acute or chronic). Given the potential cardioprotective effects of curcumin through HIF-1 modulation, we investigated the impact of hydroxylated aurone derivatives on cardiomyoblasts (H9C2) incubated with cobalt chloride (CoCl2, 1 mM for 2 h), which mimics hypoxia by stabilizing HIF-1 alpha. Cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay, and HIF-1 alpha expression was assessed by western blot. Interestingly, only the three 7-hydroxyaurones enhanced cell viability under CoCl2-induced stress, showing efficiencies comparable to curcumin (120-140 % improvement at 100 & micro;M). However, they exhibited divergent effects on HIF-1 alpha expression: 2 '- and 3 '-hydroxyaurones led to a reduction, whereas 4 '-hydroxyaurone caused an increase. This study thus identifies a new class of molecules capable of modulating HIF-1 activity and preventing cardiac cell death. These findings open new perspectives, as these aurones could potentially be developed for strategies aiming to reduce infarct size by regulating HIF-1 expression, either increasing HIF-1 alpha in the context of MI alone or suppressing its expression under chronic conditions such as obstructive sleep apnea syndrome (OSA).

The ABCG2 transporter is an efflux pump that can transport various anticancer drugs and is strongly associated with multidrug resistance (MDR) in cancer. A promising strategy to combat MDR mediated by this transporter is through functional inhibition. However, there are currently no potent selective ABCG2 inhibitors in clinical trials. Chalcone, a privileged scaffold, can be easily synthesized to produce a wide array of derivatives with diverse biological applications. Notably, chalcone derivatives have been identified as inhibitors of ABC transporters, including ABCG2. In this study, we synthesized and evaluated a series of twenty chalcone derivatives as potential ABCG2 inhibitors. Out of these, ten compounds were found to completely inhibit ABCG2 transport activity. Among them, compounds 10, 19, and 20 demonstrated particularly high potency, with EC50 values (compound concentrations giving a half-maximal inhibition) of 0.34, 0.83, and 0.94 μM, respectively. These three promising chalcone derivatives selectively inhibited ABCG2, enhanced the binding of the 5D3 conformational antibody, and exhibited low cytotoxicity, with estimated IG50 values (compound concentrations giving a half-maximal cell viability) exceeding 50 μM. The therapeutic ratio (TR), calculated as the ratio of IG50 to EC50 values, revealed that 10 had a TR greater than 147, nearly 3-fold higher than those of 19 (greater than 60) and 20 (greater than 53). Modeling studies, while biased towards the conformation of the selected starting compound, successfully reproduced relevant interactions with residues Phe439 and Asn436, which are consistent with ABCG2 inhibition. Finally, 10 was shown to chemosensitize cell lines that overexpress ABCG2, effectively overcoming the MDR phenotype mediated by this transporter.

The structural knowledge of membrane proteins (MPs) is crucial for the structure-based drug design. The stabilization of MPs during extraction processes is essential for structural and functional maintenance. In this regard, detergents are used to achieve extractions of MPs in their functional form. Based on previous work showing the importance of adjacent dicarboxylate groups for the detergency properties, the synthesis of a new generation of detergents bearing more dicarboxylate groups is reported. The molecular structure of the new generation is characterized by the presence of four chemical entities: a DOTA or NOTA scaffold, three or four pairs of carboxylates, a fatty acid, and PEG chains. The preliminary biochemical evaluation reveals promising features of this novel generation of surfactants for the aqueous extraction of MPs.

Natural peptides from animal venoms effectively modulate ion channel activity. While photoswitches regulate small compound pharmacology, their application to natural peptides rich in disulfide bridges and active on ion channels is novel due to larger pharmacophores. A pilot study integrating azobenzene photoswitches into charybdotoxin (ChTx), known for blocking potassium channels is initiated. Two click-chemistry-compatible azobenzene are synthesized differing in length and amide orientation (Az1 & Az2). Az1 is grafted onto ChTx at various amino acid positions using L-azidohomoalanine mutation. ChTx monomers outperformed dimers, particularly with azobenzene at position 14, by exhibiting optimal photoswitching activity. In the cis configuration, Az1 altered ChTx's pharmacophore, reducing potassium channel blockage, while conversely, Az2 increased ChTx potency. This study pioneers photoswitch application to complex peptides, leveraging structure-activity relationships. Successful integration depends on precise azobenzene positioning and chemical grafting guided by SAR insights. This advancement underscores the adaptability of photoswitch technology to intricate peptide structures, offering new avenues for pharmacological modulation.

Azole resistance in Candida species often results from overexpression of the ABC transporter Cdr1, which expels drugs via ATP-driven conformational changes. Despite its clinical relevance, the structural basis of Cdr1 function has remained elusive. Here, we present four high-resolution cryo-EM structures of Candida glabrata Cdr1 under active turnover with ATP-Mg2+, itraconazole, and vanadate. Itraconazole is seen to curve deeply into the drug-binding pocket. A deep exploration of the variability within these particle datasets uncovered 80 transient conformations, including two nucleotide-bound states— one closed and one open—that capture, for the first time, the conformational transitions triggered by ATP hydrolysis in both nucleotide-binding and transmembrane domains. At the catalytic site, motion initiates in the C-helix adjacent to the ABC signature motif, retracting 4 Å from the γ-phosphate/vanadate position. This movement, transmitted to nearby TMH-1, opens the drug-binding site via its lateral displacement. A second set of conformations reveals coordinated rearrangements of transmembrane helices that drive substrate extrusion. These findings provide a direct structural and dynamic framework for understanding Cdr1-mediated azole resistance, and, more broadly, illuminate the conserved chemo-mechanical cycle of nucleotide-binding domains across the ABC superfamily, including non-membranous members. ### Competing Interest Statement The authors have declared no competing interest.

The dominant organisms in modern oxic ecosystems rely on respiratory quinones with high redox potential (HPQs) for electron transport in aerobic respiration and photosynthesis. The diversification of quinones, from low redox potential (LPQ) in anaerobes to HPQs in aerobes, is assumed to have followed Earth's surface oxygenation ~2.3 billion years ago. However, the evolutionary origins of HPQs remain unresolved. Here, we characterize the structure and biosynthetic pathway of an ancestral HPQ, methyl-plastoquinone (mPQ), that is unique to bacteria of the phylum Nitrospirota. mPQ is structurally related to the two previously known HPQs, plastoquinone from Cyanobacteriota/chloroplasts and ubiquinone from Pseudomonadota/mitochondria, respectively. We demonstrate a common origin of the three HPQ biosynthetic pathways that predates the emergence of Nitrospirota, Cyanobacteriota, and Pseudomonadota. An ancestral HPQ biosynthetic pathway evolved ≥ 3.4 billion years ago in an extinct lineage and was laterally transferred to these three phyla ~2.5 to 3.2 billion years ago. We show that Cyanobacteriota and Pseudomonadota were ancestrally aerobic and thus propose that aerobic metabolism using HPQs significantly predates Earth's surface oxygenation. Two of the three HPQ pathways were later obtained by eukaryotes through endosymbiosis forming chloroplasts and mitochondria, enabling their rise to dominance in modern oxic ecosystems.

Artificial intelligence (AI) is revolutionizing drug discovery with unprecedented speed and efficiency. In computer-aided drug design, structure-based and ligand-based methodologies are the main driving forces for innovation. In cases where no experimental structure or high-confidence homology/AlphaFold-predicted model of the target is available in 3D, ligand-based strategies are generally preferable. Here, we aim to develop and evaluate new predictive AI models for ligand-based drug discovery. To illustrate our workflow, we propose, as an example, an ensemble classification model for Cdr1 inhibitor prediction. We leverage target-specific experimental data from different sources, various molecular feature types, and multiple state-of-the-art machine learning (ML) algorithms alongside a multi-instance 3D graph neural network (multiple conformations of a single molecule are considered). Bayesian hyperparameter tuning, stacked generalization, and soft voting are involved in our workflow. The final target-specific ensemble model benefits from the classification and screening power of those constituting it. On an external test set structurally dissimilar to the training data, its average precision is 0.755, its F1-score is 0.714, the area under the receiver operating characteristic curve is 0.884, and the balanced accuracy is 0.799. It gives a low false positive rate of 0.1236 on another test set outside the training chemical space, indicating its ability to avoid false positives. The present work highlights the potential of stacking ensemble ML and offers a rigorous general workflow to build ligand-based predictive AI models for other targets.

Azole resistance in Candida species is often caused by the overexpression of Cdr1. Despite its clinical relevance, the structural basis for its ATP-driven efflux pump function remains elusive. We present four high-resolution cryo-EM structures for Candida glabrata Cdr1 under active turnover conditions in the absence and presence of ATP-Mg²⁺, itraconazole, and vanadate. Additional transient cryo-EM structures were unveiled by 3D variability analysis offering a detailed view of the step-by-step transitions triggered by ATP-hydrolysis. The motion cascade starts with a 4 Å piston-like retraction of the C-helix from the γ-phosphate/vanadate of the hydrolyzed ATP. This causes the nearby transmembrane helix-1 (TMH-1) to open the drug-binding site via lateral displacement and unwinding of the inner-leaflet region of TMH-2. A reverse ‘squeeze-and-push’ motion of TMH-2 possibly drives substrate extrusion. High resolution structures also reveal how itraconazole adapts its shape to fit into the drug-binding site. Our findings provide a dynamic structural framework for Cdr1-mediated azole resistance and the conserved chemo-mechanical cycle of ABC proteins, including non-membranous members.

The breast cancer resistance protein (BCRP/ABCG2) plays a major role in the multidrug resistance of cancers toward chemotherapeutic treatments. It was demonstrated that cholesterol regulates the ABCG2 activity, suggesting that lower levels of membrane cholesterol decrease the ABCG2 activity in mammalian cells. However, the precise mechanism remains unclear. To better understand the role of cholesterol in the ABCG2 activity, we studied the ABCG2-mediated efflux of different substrates in the presence of different concentrations of cholesterol. Moreover, we synthetized derivatives of cholesterol linked either to known ABCG2 inhibitors or fluorescents probes. A chalcone-cholesterol was synthetized to investigate the influence of cholesterol on ABCG2 inhibition, and a BODIPY-cholesterol was developed to track cholesterol trafficking on mammalian cells and investigate the behavior of cholesterol as an ABCG2 substrate. The obtained results with three different substrates of ABCG2 showed that cholesterol did not affect the intracellular amount of substrates nor the transport activity.

Garcinia kola is an emblematic tree used in traditional medicine in many regions in Africa. In particular, its nuts are commonly used for the management of various diseases. However, to the best of our knowledge, G. kola nuts have never been investigated as potential sources of active ingredients in dermocosmetics. In this paper, nuts from G. kola were investigated to shed light on the extraction, purification and characterization of three components with potential dermocosmetic applications. The nuts were subjected to extraction with different solvents, including cyclohexane, dichloromethane, ethyl acetate, and methanol. Each extract was purified by column chromatography on silica gel. Pure compounds were characterized by NMR and mass spectrometry and comparison with reported literature data. Unsaturated fatty acids were found in the cyclohexane and dichloromethane extract, garcinoic acid (a vitamin E derivative) in the dichloromethane extract, and the biflavanone GB1 in the methanol extract. The presence of unsaturated fatty acids, garcinoic acid, and biflavanone in the nuts of G. kola as dominant compounds suggests that this plant material holds potential to be used for the development of active compounds for skin care and well-being.

Over the past few decades, many current uses for cannabinoids have been described, ranging from controlling epilepsy to neuropathic pain and anxiety treatment. Medicines containing cannabinoids have been approved by both the FDA and the EMA for the control of specific diseases for which there are few alternatives. However, the molecular-level mechanism of action of cannabinoids is still poorly understood. Recently, cannabinoids have been shown to interact with autotaxin (ATX), a secreted lysophospholipase D enzyme responsible for catalyzing lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a pleiotropic growth factor that interacts with LPA receptors. In addition, a high-resolution structure of ATX in complex with THC has recently been published, accompanied by biochemical studies investigating this interaction. Due to their LPA-like structure, endocannabinoids have been shown to interact with ATX in a less potent manner. This finding opens new areas of research regarding cannabinoids and endocannabinoids, as it could establish the effect of these compounds at the molecular level, particularly in relation to inflammation, which cannot be explained by the interaction with CB1 and CB2 receptors alone. Further research is needed to elucidate the mechanism behind the interaction between cannabinoids and endocannabinoids in humans and to fully explore the therapeutic potential of such approaches.

Multiple drugs administration is a common practice in hospitals and clinics. This is the case when heavy pathologies are treated. In this context, frequently, antivirals and antibiotics are co-administrated either simultaneously or sequentially ignoring potential incompatibilities. In this study, we focused on an antiviral (Acyclovir) and an antibiotic (Ciprofloxacin) compatibility/incompatibility when they co-administrated with special interest on the effect of drugs ratio, pH and delay. By implementing robust HPLC method, we determined the main factors leading to potential modification of concentration of one of the two drugs in the mixture. The results showed that acyclovir is quantitatively recovered in all conditions related to pH, concentration and time (up to 24 hours). However, ciprofloxacin is strongly altered in acidic and basic conditions, the quantity of ciprofloxacin and time after mixing the two drugs. The most valuable piece of information is that the ratio of acyclovir/ciprofloxacin is crucial. The latter should be calculated and managed to provide at least two fold quantity of acyclovir versus ciprofloxacin in order to maintain the administrated doses and warrant efficient therapeutic effect.

Aim: BCRP plays a major role in the efflux of cytotoxic molecules, limiting their antiproliferative activity. We aimed to design and synthesize new BCRP inhibitors to render cancerous tumors more sensitive toward anticancer agents. Materials & methods: Based on our previous work, we conceived potential BCRP inhibitors derived from 1,3,4-oxadiazoles bearing two substituted phenyl rings. Results: Evaluating 19 derivatives, we found that 2,5-diaryl-1,3,4-oxadiazoles possessing methoxy groups were the most active. The highest activity was recorded with derivatives bearing three methoxy groups. The most active compound (3j) was selective in inhibiting BCRP and nontoxic as evidenced by cellular tests. Conclusion: 3j is a promising BCRP inhibitor thanks to its synthetic accessibility and biological profile.

Autotaxin (ATX) is an enzyme primarily known for the production of lysophosphatidic acid. Being involved in the development of major human diseases, such as cancer and neurodegenerative diseases, the enzyme has been featured in multiple studies as a pharmacological target. We previously found that the cannabinoid tetrahydrocannabinol (THC) could bind and act as an excellent inhibitor of ATX. This study aims to use the cannabinoid scaffold as a starting point to find cannabinoid-unrelated ATX inhibitors, following a funnel down approach in which large chemical libraries sharing chemical similarities with THC were screened to identify lead scaffold types for optimization. This approach allowed us to identify compounds bearing chromone and indole scaffolds as promising ATX inhibitors. Further optimization led to MEY-003, which is characterized by the direct linkage of an N-pentyl indole to the 5,7-dihydroxychromone moiety. This molecule has potent inhibitory activity towards ATX-β and ATX-ɣ as evidenced by enzymatic studies and its mode of action was rationalized by structural biology studies using macromolecular X-ray crystallography.

In this study, six vacuum liquid chromatography (VLC) fractions (F1-F6) of the n-BuOH extract of L. numidicum Murb. (BELN) were examined for their anticancer capacity. The composition of secondary metabolites was analyzed by LC-HRMS/MS. The antiproliferative effect against PC3 and MDA-MB-231 lines was evaluated by MTT assay. Apoptosis of PC3 cells was detected by annexin V-FITC/PI staining using a flow cytometer. The results showed that only fractions 1 and 6 inhibited PC3 and MDA-MB 231 cell proliferation in a dose-dependent manner and induced dose-dependent apoptosis of PC3 cells, evidenced by the accumulation of early and late apoptotic cells, and by the decrease in viable cells. LC-HRMS/MS profiling of fractions 1 and 6 revealed the presence of known compounds that may be responsible for the observed anticancer activity. F1 and F6 may be an excellent source of active phytochemicals for cancer treatment.

The bioavailability of cosmetic, pharmaceutical, nutraceutical, and food preparations depends, among other factors, on the galenic form and the control of the granulometric structure of powders. The present study aimed to evaluate the effect of argan pulp powder particle size on functional, physicochemical properties, and antioxidant bioactivity. The particle size study revealed a unimodal particle volume distribution, explaining the regular particle shape. The results relating to functional properties indicated that the critical fraction was in the range of 50–125 µm. However, the study of the particles in each class, evaluated via SEM, showed that the morphology of the pulp powder was strongly dependent on the degree of grinding. The classes in the range of 50–125 µm had the highest polyphenol content, while those of <25 µm had the highest flavonoid content (893.33 mg GAE/100 g DW and 128.67 mg CE/100 g DW, respectively). Molecular analysis via LC and GC-MS showed that particle size had a significant effect on the release of bioactive molecules. ABTS, DPPH, and TAC tests showed that the fraction, ‘‘50–125 µm’’, had the highest antioxidant activity. However, the FRAP test showed highest antioxidant activity for particles of <25 µm. The analysis of the bioactive compounds of the argan pulp powder confirmed a differential distribution, depending on the size of the particles.

In human, Tyrosinase enzyme (TyH) is involved in the key steps of protective pigments biosynthesis (in skin, eyes and hair). The use of molecules targeting its binuclear copper active site represents a relevant strategy to regulate TyH activities. In this work, we targeted 2-Hydroxypyridine-N-oxide analogs (HOPNO, an established chelating group for the tyrosinase dicopper active site) with the aim to combine effects induced by combination with a reference inhibitor (kojic acid) or natural substrate (tyrosine). The HOPNO-MeOH (3) and the racemic amino acid HOPNO-AA compounds (11) were tested on purified tyrosinases from different sources (fungal, bacterial and human) for comparison purposes. Both compounds have more potent inhibitory activities than the parent HOPNO moiety and display strictly competitive inhibition constant, in particular with human tyrosinase. Furthermore, 11 appears to be the most active on the B16-F1 mammal melanoma cells. The investigations were completed by stereospecificity analysis. Racemic mixture of the fully protected amino acid 10 was separated by chiral HPLC into the corresponding enantiomers. Assignment of the absolute configuration of the deprotected compounds was completed, based on X-ray crystallography. The inhibition activities on melanin production were tested on lysates and whole human melanoma MNT-1 cells. Results showed significant enhancement of the inhibitory effects for the (S) enantiomer compared to the (R) enantiomer. Computational studies led to an explanation of this difference of activity based for both enantiomers on the respective position of the amino acid group versus the HOPNO plane.