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Red kidney beans (RKB) serve as a powerhouse packed with a plethora of largely unexplored extraordinary chemical entities with potential significance. However, their nutraceutical applications as a functional hypoglycemic food still lag behind and warrant further investigation. With a scope to optimize chemical and biological traits of RKB, green modification approaches (processing methods) seem inevitable. Accordingly, the current study offered the first integrative workflow to scrutinize dynamic changes in chemical profiles of differently processed RKB and their potential entanglements on diabetes mitigation using Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS/MS) coupled with chemometrics. Different physical and biological processing treatments namely germination, fermentation, cooking and dehulling were preliminarily implemented on RKB. Complementarily, the concomitant metabolite alterations among differently processed RKB were monitored and interpreted. Next, an in-vitro α-amylase and α-glycosidase inhibitory testing of the differently processed samples was conducted and integrated with orthogonal projection to latent structures (OPLS) analysis to pinpoint the possible efficacy compounds. A total of 72 compounds spanning fatty acids and their glycerides, flavonoids, phenolic acids, amino acids, dipeptides, phytosterols and betaxanthins were profiled. Given this analysis and compared with raw unprocessed samples, it was found that flavonoids experienced notable accumulation during germination while both fermentation and dehulling approaches sharply intensified the content of amino acids and dipeptides. Comparably, Fatty acids, phytosterols and betaxanthins were unevenly distributed among the comparable samples. Admittedly, OPLS-DA revealed an evident discrimination among the processed samples assuring their quite compositional discrepancies. In a more targeted approach, kaempferol-O-sophoroside, quercetin, carlinoside and betavulgarin emerged as focal discriminators of sprouted samples while citrulline, linoleic acid, linolenoyl-glycerol and stigmasterol were the determining metabolites in cooked samples. Our efficacy experimental findings emphasized that the different RKB samples exerted profound inhibitory actions against both α-amylase and α-glycosidase enzymes with the most promising observations in the case of sprouted and cooked samples. Coincidently, OPLS analysis revealed selective enhancement of possible efficacy constituents primarily citrulline, formononetin, gamabufotalin, kaempferol-O-sophoroside, carlinoside, oleic acid and ergosterol in sprouted and cooked samples rationalizing their noteworthy α-amylase and α-glucosidase inhibitory activities. Taken together, this integrated work provides insightful perspectives beyond the positive impact of different processing protocols on bioactives accumulation and pharmacological traits of RKB expanding their utilization as functional hypoglycemic food to rectify diabetes.

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To convert ginsenosides Rb1, Rb2, Rb3, and Rc into Rd by a single enzyme, a putative ß-glycosidase (Pxbgl) from the xylan-degrading bacterium Petroclostridium xylanilyticum was identified and used. The kcat/Km value of Pxbgl for Rb3 was 18.18 ± 0.07 mM-1/s, which was significantly higher than those of Pxbgl for other ginsenosides. Pxbgl converted almost all Rb3 to Rd with a productivity of 5884 µM/h, which was 346-fold higher than that of only ß-xylosidase from Thermoascus aurantiacus. The productivity of Rd from the Panax ginseng root and Panax notoginseng leaf was 146 and 995 µM/h, respectively. Mutants N293 K and I447L from site-directed mutagenesis based on bioinformatics analysis showed an increase in specific activity of 29 and 7% toward Rb3, respectively. This is the first report of a ß-glycosidase that can simultaneously remove four different glycosyls at the C-20 position of natural PPD-type ginsenosides and produce Rd as the sole product from P. notoginseng leaf extracts with the highest productivity.

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Saccharide mapping was a promising scheme to unveil the mystery of polysaccharide structure by analysis of the fragments generated from polysaccharide decomposition process. However, saccharide mapping was not widely applied in the polysaccharide analysis for lacking of systematic introduction. In this review, a detailed description of the establishment process of saccharide mapping, the pros and cons of downstream technologies, an overview of the application of saccharide mapping, and practical strategies were summarized. With the updating of the available downstream technologies, saccharide mapping had been expanding its scope of application to various kinds of polysaccharides. The process of saccharide mapping analysis included polysaccharides degradation and hydrolysates analysis, and the degradation process was no longer limited to acid hydrolysis. Some downstream technologies were convenient for rapid qualitative analysis, while others could achieve quantitative analysis. For the more detailed structure information could be provided by saccharide mapping, it was possible to improve the quality control of polysaccharides during preparation and application. This review filled the blank of basic information about saccharide mapping and was helpful for the establishment of a professional workflow for the saccharide mapping application to promote the deep study of polysaccharide structure.

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Trihydroxypiperidines are a therapeutically valuable class of iminosugar. We applied a one-pot amination-cyclisation cascade reaction to synthesise 3,4,5-trihydroxypiperidine stereoisomers in three steps from commercially available pentoses and in excellent overall yields. Using our methodology, the yields of the syntheses of meso-1, meso-2 and 3L are the highest reported to date. The synthetic methodology was readily extended to the three-step synthesis of N-alkyl derivatives by replacing the ammonia nitrogen source with a primary amine. The trihydroxypiperidines and N-alkyl analogues were screened for enzyme inhibitory activity using Fabrazyme (Fabry disease), GCase (Gaucher's disease), Agrobacterium sp. ß-glucosidase, and Escherichia coli ß-galactosidase. N-Phenylethyl 3,4,5-trihydroxypiperidine (N-phenylethyl-1-(3R,4R,5S)-piperidine-3,4,5-triol) showed good inhibitory activity of Fabrazyme (Ki = 46 µM). This activity was abolished when the N-phenylethyl group was removed or replaced with a non-aromatic alkyl chain.

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In contemporary medicinal chemistry, employing a singular small molecule to concurrently multi-target disparate molecular entities is emerging as a potent strategy in the ongoing battle against metabolic disease. In this study, we present the meticulous design, synthesis, and comprehensive biological evaluation of a novel series of 1,2,3-triazolylmethylthio-1,3,4-oxadiazolylbenzenesulfonamide derivatives (8a-m) as potential multi-target inhibitors against human carbonic anhydrase (EC.4.2.1.1, hCA I/II), α-glycosidase (EC.3.2.1.20, α-GLY), and α-amylase (EC.3.2.1.1, α-AMY). Each synthesized sulfonamide underwent rigorous assessment for inhibitory effects against four distinct enzymes, revealing varying degrees of hCA I/II, a-GLY, and a-AMY inhibition across the tested compounds. hCA I was notably susceptible to inhibition by all compounds, demonstrating remarkably low inhibition constants (KI) ranging from 42.20 ± 3.90 nM to 217.90 ± 11.81 nM compared to the reference standard AAZ (KI of 439.17 ± 9.30 nM). The evaluation against hCA II showed that most of the synthesized compounds exhibited potent inhibition effects with KI values spanning the nanomolar range 16.44 ± 1.53-70.82 ± 4.51 nM, while three specific compounds, namely 8a-b and 8d, showcased lower inhibitory potency than other derivatives that did not exceed that of the reference drug AAZ (with a KI of 98.28 ± 1.69 nM). Moreover, across the spectrum of synthesized compounds, potent inhibition profiles were observed against diabetes mellitus-associated α-GLY (KI values spanning from 0.54 ± 0.06 µM to 5.48 ± 0.50 µM), while significant inhibition effects were noted against α-AMY, with IC50 values ranging between 0.16 ± 0.04 µM and 7.81 ± 0.51 µM) compared to reference standard ACR (KI of 23.53 ± 2.72 µM and IC50 of 48.17 ± 2.34 µM, respectively). Subsequently, these inhibitors were evaluated for their DPPH· and ABTS+· radical scavenging activity. Moreover, molecular docking investigations were meticulously conducted within the active sites of hCA I/II, α-GLY, and α-AMY to provide comprehensive elucidation and rationale for the observed inhibitory outcomes.

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Diabetic cardiomyopathy (DCM) is a heart failure syndrome, and is one of the major causes of morbidity and mortality in diabetes. DCM is mainly characterized by ventricular dilation, myocardial hypertrophy, myocardial fibrosis and cardiac dysfunction. Clinical studies have found that insulin resistance is an independent risk factor for DCM. However, its specific mechanism of DCM remains unclear. 8-hydroxyguanine DNA glycosylase 1(OGG1)is involved in DNA base repair and the regulation of inflammatory genes. In this study, we show that OGG1 was associated with the occurrence of DCM. for the first time. The expression of OGG1 was increased in the heart tissue of DCM mice, and OGG1 deficiency aggravated the cardiac dysfunction of DCM mice. Metabolomics show that OGG1 deficiency resulted in obstruction of glycolytic pathway. At the molecular level, OGG1 regulated glucose uptake and insulin resistance by interacting with PPAR-γ in vitro. In order to explore the protective effect of exogenous OGG1 on DCM, OGG1 adeno-associated virus was injected into DCM mice through tail vein in the middle stage of the disease. We found that the overexpression of OGG1 could improve cardiac dysfunction of DCM mice, indicating that OGG1 had a certain therapeutic effect on DCM. These results demonstrate that OGG1 is a new molecular target for the treatment of DCM and has certain clinical significance.

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BACKGROUND: Effective control of postprandial blood glucose (PBG) level is essential for the prevention and treatment of diabetes and its complications. Several flavonoids have attracted much attention due to their significant PBG-lowering effects. However, there is still a certain gap in the in vivo hypoglycemic activity of most flavonoids compared to first-line drugs available on the market, and are still lack of the PBG-lowering effects of 8-hydroxyflavones and their structure-activity relationship. PURPOSE: Evaluate hypoglycemic effects of 8-hydroxyflavones from Rhodiola crenulata in vitro and in vivo, especially comparatively analyze the relationship between hypoglycemic effects and flavonoid configuration and reveal the possible mechanism of 8-hydroxyflavones in lowering hyperglycemia. METHODS: Starch, maltose, sucrose, and glucose tolerance tests in both diabetic and normal mice were used to evaluate and compare the hypoglycemic effects of 8-hydroxyflavones rhodiosin (RHS), rhodionin (RHN), and herbacetin (HBT). Molecular docking, enzyme kinetics, and immunofluorescence analysis were used to research the possible hypoglycemic mechanisms of 8-hydroxyflavones. RESULTS: RHS (5 and 10 mg/kg) could efficiently decrease PBG levels in both normal and diabetes mice. Moreover, RHS, RHN, and HBT all had significant PBG-lowering effects in transgenic diabetes mice, and the effects were equivalent to or stronger than acarbose. Further mechanism research indicated that 8-hydroxyflavones achieved PBG-lowering effects by inhibiting both the activity and production of glycosidase. Notably, we have innovatively discovered that inhibiting the expression of glycosidases rather than just their activities may be a new target for hypoglycemic drugs. CONCLUSION: We have firstly comprehensively and systematically clarified PBG-lowering effects of 8-hydroxyflavones from Rhodiola crenulata, and revealed their structure-activity relationships and hypoglycemic mechanisms. The study demonstrated that the substitution of 8-hydroxy groups in flavonoids could significantly enhance their hypoglycemic effects, which were equivalent to or stronger than commercially available drug acarbose. 8-Hydroxyflavones could be used as therapeutic or health drugs with significant potential to reduce postprandial hyperglycemia.

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Ginsenoside, the principal active constituent of ginseng, exhibits enhanced bioavailability and medicinal efficacy in rare ginsenosides compared to major ginsenosides. Current research is focused on efficiently and selectively removing sugar groups attached to the major ginsenoside sugar chains to convert them into rare ginsenosides that meet the demands of medical industry and functional foods. The methods for preparing rare ginsenosides encompass chemical, microbial, and enzymatic approaches. Among these, the enzyme conversion method is highly favored by researchers due to its exceptional specificity and robust efficiency. This review summarizes the biological activities of different rare ginsenosides, explores the various glycosidases used in the biotransformation of different major ginsenosides as substrates, and elucidates their respective corresponding biotransformation pathways. These findings will provide valuable references for the development, utilization, and industrial production of ginsenosides.

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The conventional approach to developing light-sensitive glycosidase activity regulators, involving the combination of a glycomimetic moiety and a photoactive azobenzene module, results in conjugates with differences in glycosidase inhibitory activity between the interchangeable E and Z-isomers at the azo group that are generally below one-order of magnitude. In this study, we have exploited the chemical mimic character of sp2-iminosugars to access photoswitchable p- and o-azobenzene α-O-glycosides based on the gluco-configured representative ONJ. Notably, we achieved remarkably high switching factors for glycosidase inhibition, favoring either the E- or Z-isomer depending on the aglycone structure. Our data also indicate a correlation between the isomeric state of the azobenzene module and the selectivity towards α- and ß-glucosidase isoenzymes. The most effective derivative reached over a 103-fold higher inhibitory potency towards human ß-glucocerebrosidase in the Z as compared with the E isomeric form. This sharp contrast is compatible with ex-vivo activation and programmed self-deactivation at physiological temperatures, positioning it as a prime candidate for pharmacological chaperone therapy in Gaucher disease. Additionally, our results illustrate that chemical tailoring enables the engineering of photocommutators with the ability to toggle inhibition between α- and ß-glucosidase enzymes in a reversible manner, thus expanding the versatility and potential therapeutic applications of this approach.

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A new class of compounds, namely highly substituted diaminocyclopentane-l-lysine adducts, have been discovered as potent inhibitors of O-GlcNAcase, an enzyme crucial for protein de-O-glycosylation. These inhibitors exhibit exceptional selectivity and reversibility and are the first example of human O-GlcNAcase inhibitors that are structurally related to the transition state of the rate-limiting step with the "aglycon" still in bond-length proximity. The ease of their preparation, remarkable biological activities, stability, and non-toxicity make them promising candidates for the development of anti-tau-phosphorylation agents holding significant potential for the treatment of Alzheimer's disease.

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When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

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Propolis, a natural product collected by honeybees from various plant sources, has gained significant attention due to its diverse bioactive compounds and potential therapeutic properties. To further explore its contents and biological activities, this study aimed to analyze the phenolic compounds in Siirt propolis extracts obtained using different solvents, namely ethanol, water, and ethanol-water mixtures. The primary objective of this research was to investigate the phenolic profile, as well as the antidiabetic and antioxidant activities of the propolis extracts. Chemical profiling of extracts was performed using LC-MS/MS. The antioxidant potential of the propolis extracts was evaluated through free radical scavenging methods, including DPPH and ABTS assays. As a result of these analyses, propolis extracts showed moderate radical scavenging potential with 13.86%-35.72% for DPPH and 33.62%-62.50% for ABTS at a concentration of 30 µg mL-1, respectively. This radical scavenging potential of the extracts sheds light on its ability to combat oxidative stress, which is implicated in the development of diabetes, and its potential effects on cellular health. Additionally, the study assessed the antidiabetic properties of the propolis extracts by examining their inhibition effects on α-amylase and α-glycosidase enzymes. Extracts with high phenolic content showed a high inhibitory effect against α-glucosidase with an IC50 of 5.72 ± 0.83 µg mL-1. This research provided significant findings regarding the potential use of propolis in the treatment of diabetes and related metabolic disorders.

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The ß-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The ß-glucosidase was obtained from T. koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 °C and 6 days. The purification efficiency of the obtained ß-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of ß-glucosidase activity, p-nitrophenyl-ß-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which ß-glucosidase showed high activity were determined to be pH 3.0 and 75 °C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K m and V max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. It was also found that d-(+)-glucose and δ-gluconolactone inhibitors exhibited competitive inhibition of ß-glucosidase in the presence of pNPG.

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α-Glycosidase inhibition is one of the main approaches to treat Diabetes mellitus. Polyphenolic moieties are known to be responsible for yielding exhibit potent α-glycosidase inhibitory effects. In addition, compounds containing benzothiazole and Schiff base functionalities were previously reported to show α-glycosidase inhibition. In this paper, the synthesis of seven new phloroglucinol-containing benzothiazole Schiff base derivatives through the reaction of 6-substituted-2-aminobenzothiazole compounds with 2,4,6-trihydroxybenzaldehyde using acetic acid as a catalyst was reported. The synthesized compounds were characterized using spectroscopic methods such as FT-IR, 1H NMR, 13C NMR, and elemental analysis. The synthesized compounds were evaluated for their inhibitory effects on α-glycosidase, compounds 3f and 3g were found to show significant inhibitory properties when compared to the positive control. The IC50 values of 3f and 3g were calculated as 24.05 ± 2.28 and 18.51 ± 1.19 µM, respectively. Kinetic studies revealed that compounds 3f and 3g exhibited uncompetitive mode of inhibition against α-glycosidase. Molecular modeling predicted druglikeness for the title compounds and underpinned the importance of phloroglucinol hydroxyls for interacting with the key residues of α-glycosidase.

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Glycogen Storage Disease (GSD) IXd, caused by PHKA1 gene mutations, is an X-linked rare disorder that can be asymptomatic or associated with exercise intolerance. GSD type II is an autosomal recessive disorder caused by mutations in the GAA gene that lead to severe cardiac and skeletal muscle myopathy. We report the first case of co-occurrence of type IXd and type II GSDs in a 53-year-old man with an atypical glycogen storage disease presentation consisting in myalgia in the lower limbs at both rest and after exercise and increased levels of transaminases from the age of 16. At the age of 43, the patient presented a steppage gait, inability to run and walk on his heels, hypotrophy of the pectoral and proximal muscles, reflexes not elicitable, and CK levels 3.6 times the upper reference limit. Next Generation Sequencing (NGS) identified one variant in the PHKA1 gene, c.1360A > G p.Ile454Val (exon 14) inherited by his mother, and two heterozygous variants in the GAA gene, c.784G > A (exon 4) and c.956-6T > C (exon 6). A review of GSD IXd cases reported to date in the literature is also provided.

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Anthocyanins are susceptible to degradation by ß-glycosidase, resulting in color loss. This study analyzed the impact of ß-glycosidase on carboxylpyranocyanidin-3-O-glucoside (Carboxyl-pycy-3-gluc) and its precursor cyanidin-3-O-glucoside (Cy-3-gluc). Carboxyl-pycy-3-gluc exhibited enhanced stability upon treatment with ß-glucosidase. Ultraviolet-visible and circular dichroism spectroscopy revealed slight changes in the microenvironment and secondary structure of ß-glycosidase when carboxyl-pycy-3-gluc was present. The fluorescence experiment indicated that anthocyanins quench the fluorescence of ß-glycosidase through static quenching via hydrophobic interactions. Molecular docking of six types of carboxylpyranoanthocyanins and their precursors with ß-glycosidase revealed that carboxylpyranoanthocyanins exhibited lower binding affinity than their precursors, consistent with the enzyme kinetic experiment results. The incorporation carboxyl-pycy-3-gluc into Sanhua Plum Juice and Wine endowed them with vivid and stable coloration. The study illustrated that carboxyl-pycy-3-gluc exhibits low binding affinity with ß-glycosidase, thereby maintaining stability and confirming its potential as a colorant.

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Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli, enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.

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Gut microbiota are significant to the host's nutrition and provide a flexible way for the host to adapt to extreme environments. However, whether gut microbiota help the host to colonize caves, a resource-limited environment, remains unknown. The nonobligate cave frog Oreolalax rhodostigmatus completes its metamorphosis within caves for 3-5 years before foraging outside. Their tadpoles are occasionally removed from the caves by floods and utilize outside resources, providing a contrast to the cave-dwelling population. For both cave and outside tadpoles, the development-related reduction in their growth rate and gut length during prometamorphosis coincided with a shift in their gut microbiota, which was characterized by decreased Lactobacillus and Cellulosilyticum and Proteocatella in the cave and outside individuals, respectively. The proportion of these three genera was significantly higher in the gut microbiota of cave-dwelling individuals compared with those outside. The cave-dwellers' gut microbiota harbored more abundant fibrolytic, glycolytic, and fermentative enzymes and yielded more short-chain fatty acids, potentially benefitting the host's nutrition. Experimentally depriving the animals of food resulted in gut atrophy for the individuals collected outside the cave, but not for those from inside the cave. Imitating food scarcity reproduced some major microbial features (e.g. abundant Proteocatella and fermentative genes) of the field-collected cave individuals, indicating an association between the cave-associated gut microbiota and resource scarcity. Overall, the gut microbiota may reflect the adaptation of O. rhodostigmatus tadpoles to resource-limited environments. This extends our understanding of the role of gut microbiota in the adaptation of animals to extreme environments.

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Calystegines are potent glycosidase inhibitors with therapeutic potential and are constituents of food and feed with potential toxic effects. This study aims to target calystegines and other nitrogenous substances in food plants. Hydroalcoholic extracts from Solanum tuberosum, Ipomoea batatas, S. lycocarpum, and fruit from S. lycopersicum, S. aethiopicum, S. paniculatum, S. crinitum, and S. acanthodes were analyzed by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using an acidic HILIC column. The dereplication approach included data processing using MZMine2, FBMN-GNPS, and structure elucidation and interpretation of the organized data. The calystegines A3, A5, B2, and C1 were identified, and several potential new calystegine analogues: three may correspond to new calystegines of the A-group, one glycosyl derivative of calystegine A3, and two glycosyl derivatives of the B-group. These findings help to direct the search for new calystegines. In addition, the dereplication approach enabled the annotation of 22 other nitrogen compounds.

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Multivalency represents an appealing option to modulate selectivity in enzyme inhibition and transform moderate glycosidase inhibitors into highly potent ones. The rational design of multivalent inhibitors is however challenging because global affinity enhancement relies on several interconnected local mechanistic events, whose relative impact is unknown. So far, the largest multivalent effects ever reported for a non-polymeric glycosidase inhibitor have been obtained with cyclopeptoid-based inhibitors of Jack bean α-mannosidase (JBα-man). Here, we report a structure-activity relationship (SAR) study based on the top-down deconstruction of best-in-class multivalent inhibitors. This approach provides a valuable tool to understand the complex interdependent mechanisms underpinning the inhibitory multivalent effect. Combining SAR experiments, binding stoichiometry assessments, thermodynamic modelling and atomistic simulations allowed us to establish the significant contribution of statistical rebinding mechanisms and the importance of several key parameters, including inhitope accessibility, topological restrictions, and electrostatic interactions. Our findings indicate that strong chelate-binding, resulting from the formation of a cross-linked complex between a multivalent inhibitor and two dimeric JBα-man molecules, is not a sufficient condition to reach high levels of affinity enhancements. The deconstruction approach thus offers unique opportunities to better understand multivalent binding and provides important guidelines for the design of potent and selective multiheaded inhibitors.

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