BUB1B monoallelic germline variants contribute to prostate cancer predisposition by triggering chromosomal instability.

BACKGROUND: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. METHODS: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. RESULTS: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. CONCLUSIONS: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making.

Comparing the Chemistry of Malvidin-3-O-glucoside and Malvidin-3,5-O-diglucoside Networks: A Holistic Approach to the Acidic and Basic Paradigms with Implications in Biological Studies.

The kinetics, thermodynamics, and degradation of malvidin mono- and diglucosides were studied following a holistic approach by extending to the basic medium. In acidic conditions, the reversible kinetics of the flavylium cation toward the equilibrium is controlled by the hydration and cis-trans isomerization steps, while in the basic medium, the OH- nucleophilic addition to the anionic quinoidal bases is the slowest step. There is a pH range (transition pHs), between the acidic and basic paradigms, that includes physiological pH (7.4), where degradation reactions occur faster, preventing the system from reaching the equilibrium. The transition pH of the diglucoside is narrower, and in contrast with the monoglucoside, there is no evidence for the formation of colored oligomers among the degradation products. Noteworthy, OH- addition in position 4 to form B42-, a kinetic product that decreases the overall equilibration rate, was observed only for the diglucoside.

Supramolecular Study of the Interactions between Malvidin-3-O-Glucoside and Wine Phenolic Compounds: Influence on Color.

Supramolecular study of the interactions between the major wine anthocyanin, malvidin-3-O-glucoside (Mv3G) and different wine phenolic compounds (quercetin 3-O-ß-glucopyranoside (QG), caffeic acid, (-)-epicatechin, (+)-catechin, and gallic acid) has been performed at two different molar ratios (1:1 and 1:2) in acidic medium where flavylium cation predominates (pH ≤ 2). Color variations have been evaluated by differential colorimetry using CIELAB color space. These studies have been complemented with isothermal titration calorimetry assays and molecular dynamics simulations. The color of Mv3G flavylium cation is modified by the interaction with QG toward more bluish and intense colors. Interaction constants between the anthocyanin and the different phenolic compounds were obtained, ranging from 9.72 × 108 M-1 for QG to 1.50 × 102 M-1 for catechin. Hydrophobic interactions and H-bonds are the main driving forces in the pigment/copigment aggregation, except for the interactions where caffeic acid is involved, in which hydrophobic interactions acquire greater preponderance.

Redox-Mediated Amination of Pyrogallol-Based Polyphenols.

Flavonoids are known to covalently modify amyloidogenic peptides by amination reactions. The underlying coupling process between polyphenols and N-nucleophiles is assessed by several in vitro and in silico approaches. The coupling reaction involves a sequence of oxidative dearomatization, amination, and reductive amination (ODARA) reaction steps. The C6-regioselectivity of the product is confirmed by crystallographic analysis. Under aqueous conditions, the reaction of baicalein with lysine derivatives yields C-N coupling as well as hydrolysis products of transient imine intermediates. The observed C-N coupling reactions work best for flavonoids combining a pyrogallol substructure with an electron-withdrawing group attached to the C4a-position. Thermodynamic properties such as bond dissociation energies also highlight the key role of pyrogallol units for the antioxidant ability. Combining the computed electronic properties and in vitro antioxidant assays suggests that the studied pyrogallol-containing flavonoids act by various radical-scavenging mechanisms working in synergy. Multivariate analysis indicates that a small number of descriptors for transient intermediates of the ODARA process generates a model with excellent performance (r=0.93) for the prediction of cross-coupling yields. The same model has been employed to predict novel antioxidant flavonoid-based molecules as potential covalent inhibitors, opening a new avenue to the design of therapeutically relevant anti-amyloid compounds.

Design, synthesis, and anticancer activity of some novel 1H-benzo[d]imidazole-5-carboxamide derivatives as fatty acid synthase inhibitors.

Multiple malignancies exhibit aberrant FASN expression, associated with enhanced de novo lipogenesis to meet the metabolic demands of rapidly proliferating tumour cells. Furthermore, elevated FASN expression has been linked to tumour aggressiveness and poor prognosis in a variety of malignant tumours, making FASN is an attractive target for anticancer drug discovery. Herein, we report the de novo design and synthesis of (2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperazin-1-yl)methanone derivatives as novel FASN inhibitors with potential therapeutic applications in breast and colorectal cancers. Twelve (2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperazin-1-yl)methanone derivatives (CTL) were synthesized and evaluated for FASN inhibition and cytotoxicity against colon cancer (HCT-116, Caco-2 cell lines), breast cancer (MCF-7 cell line) and normal cell line (HEK-293). Compounds CTL-06 and CTL-12 were chosen as the most promising lead molecules based on FASN inhibition and selective cytotoxicity profiles against colon and breast cancer cell lines. Compounds CTL-06 and CTL-12 demonstrate promising FASN inhibitory activity at IC50 of 3 ± 0.25 µM and 2.5 ± 0.25 µM when compared to the FASN inhibitor orlistat, which has an IC50 of 13.5 ± 1.0 µM. Mechanistic investigations on HCT-116 revealed that CTL-06 and CTL-12 treatment led to cell cycle arrest in Sub-G1/S phase along with apoptosis induction. Western blot studies indicated that CTL-06 and CTL-12 inhibited FASN expression in a dose-dependent manner. CTL-06 and CTL-12 treatment of HCT-116 cells enhanced caspase-9 expression in a dose-dependent manner, while upregulating proapoptotic marker Bax and downregulating antiapoptotic Bcl-xL. Molecular docking experiments of CTL-06 and CTL-12 with FASN enzyme revealed the mode of binding of these analogues in the KR domain of the enzyme.

In silico guided designing of optimized benzochalcones derivatives as potent CYP1B1 inhibitors: An integrated in vitro and ONIOM study.

Cytochrome P4501B1 (CYP1B1) is reported to be overexpressed in various malignancies including ovarian, lung, lymph, and breast cancers. The overexpression of this enzyme is accountable for the biotransformation-based inactivation of some anti-cancer drugs i.e. Docetaxel, Paclitaxel, and Cisplatin. To circumvent solutions to this issue, the current study reports some optimized derivatives of benzochalcone as selective CYP1B1 inhibitors. The optimized derivatives were screened using some structure-based drug-designing approaches including molecular docking and molecular dynamics. The implemented approaches revealed that all the designed molecules demonstrated not only essential interactions with key amino acid residues but also maintained stability within the active site of CYP1B1. Furthermore, to validate the in-silico results and develop a SAR, the designed molecules were subsequently synthesized and tested for their ability to selectively inhibit CYP1B1 over CYP1A1 using well established EROD assay. This assay results suggested that compounds 1(c), 1(d), and 1(e) are eightfold more selective CYP1B1 inhibitors over CYP1A1 with IC50 values ranging from 0.06 to 0.09 µM respectively. Among these, compound 1(d) manifested potent inhibitory activity i.e. IC50 of 0.06 µM with 24 folds selectivity over 1A1. To have a better insight into the binding pattern of 1(d) within CYP1B1 and precisely compute binding affinity for 1(d)-CYP1B1 complex, one of the advanced QM/MM approaches i.e. ONIOM has been implemented. Where 1(d)-CYP1B1 complex conferred comparable binding affinity in terms of ΔG (kcal/mol) with that of ANF-CYP1B1 complex. This research could provide a suitable starting point for the development of more potent multi-functional compounds with CYP1B1 inhibitory activity.

Intermolecular Copigmentation Between Delphinidin 3-O-Glucoside and Chlorogenic Acid: Taking into Account the Existence of Neutral and Negatively Charged Forms of the Copigment.

Stopped flow corroborated by UV-vis measurements allowed for the calculation of the copigmentation constants of delphinidin 3-O-glucoside with the neutral (CP) and negatively charged CP(-) forms of chlorogenic acid. Solutions of delphinidin 3-O-glucoside in the absence and presence of the copigment were equilibrated at several pH values in the acidic region, pH < 6, and reverse pH jumps monitored by stopped flow were carried out by adding sufficient acid to give flavylium cation at pH ≤ 1. This procedure allows for the separation of three contributions: (i) all flavylium cation and quinoidal base species, (ii) all hemiketal species, and (iii) all cis-chalcone species. Reverse pH jumps can also be performed at fixed pH versus copigment addition. The contribution of trans-chalcone, minor species in the present system, requires reverse pH jumps from the equilibrium followed by a common spectrophotometer. The system was also studied by UV-vis as a function of the copigment addition at different pH values. A global fitting of all experimental data allowed for determination of the copigmentation constants with flavylium cation, KAH+CP = 167 M-1, KAH+CP(-) = 338 M-1; and quinoidal base, KACP = 1041 M-1, KACP(-)= 221 M-1. No significant copigmentation was observed for hemiketal and chalcones. Computational calculations confirm different geometries for the interactions of flavylium cation and quinoidal base with the neutral or the negatively charged forms of the copigment as well as predict identical relative order for the binding energies of the four adducts.

Epigenetic Anti-Cancer Treatment With a Stabilized Carbocyclic Decitabine Analogue.

5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

Combined in Silico and in Vitro Approaches To Uncover the Oxidation and Schiff Base Reaction of Baicalein as an Inhibitor of Amyloid Protein Aggregation.

The oxidized form of baicalein (BA) leads to covalent binding with human amyloid proteins. Such adducts hamper the aggregation and deposition of fibrils. A novel reaction of BA with pentylamine (PA) as a model for the lysine side chain is described. This is the first study addressing the atomistic details of a Schiff base reaction with the trihydroxylated moiety of BA. Nuclear magnetic resonance and mass spectrometry approaches clearly indicate the formation of dehydrobaicalein in solution as well as its condensation with PA under aerobic conditions, yielding regioselectively C6-substituted products. The combined results suggest initial ion pair formation between BA and PA, followed by a redox chain reaction: the initiation by oxygen/air; an o-quinone-based chain involving oxidation and reduction steps; and extra off-chain formation of a doubly oxidized product. These mechanistic details support the anti-amyloid activity of BA and endorse its trihydroxyphenyl moiety as a pharmacophore for drug-design studies.

Plant derived active compounds as potential anti SARS-CoV-2 agents: an in-silico study.

Plants are a valued potential source of drugs for a variety of diseases and are often considered less toxic to humans. We investigated antiviral compounds that may potentially target SARS-CoV-2 antigenic spike (S) and host proteins; angiotensin-converting enzyme2 (ACE2), and transmembrane serine protease2 (TMPRSS2). We scrutinized 36 phytochemicals from 15 Indian medicinal plants known to be effective against RNA viruses via molecular docking. Besides, the TMPRSS2 structure was modeled and validated using the SWISS-MODEL. Docking was performed using Autodock Vina and 4.2 followed by visualization of the docking poses on Pymol version 2.4.0 and Discovery Studio Visualizer. Molecular docking showed that 12 out of 36 active compounds interacted efficiently with S, ACE2, and TMPRSS2 proteins. The ADMET profile generated using the swissADME and pkCSM server revealed that these compounds were possessed druggable properties. The Amber 12 simulation package was used to carry out energy minimizations and molecular dynamics (MD) simulations. The total simulation time for both S protein: WFA and S protein: WND complexes was 300 ns (100 ns per replica). A total of 120 structures were extracted from the last 60 ns of each MD simulation for further analysis. MM-PBSA and MM-GBSA were employed to assess the binding energy of each ligand and the receptor-binding domain of the viral S-protein. The methods suggested that WND and WFA showed thermodynamically favorable binding energies, and the S protein had a higher affinity with WND. Interestingly, Leu455 hotspot residue in the S protein, also predicted to participate in binding with ACE2, was engaged by WND and WFA. HighlightsPlants' natural active compounds may aid in the development of COVID-19 therapeutics.MD simulation study revealed stable binding of withanolide D and withaferin A with spike proteinWithanolide D and withaferin A could be effective against SARS-CoV-2 spike protein.Discovery of druggable agents that have less or lack of binding affinity with ACE2 to avoid the organs associated with comorbidities.According to ADMET selected phytochemicals may be used as druggable compounds.Communicated by Ramaswamy H. Sarma.

Structure based virtual screening of natural product molecules as glycosidase inhibitors.

Objective of the present investigation comprised of the application of in silico methods to discover novel natural product (NP) based potential inhibitors for carbohydrate mediated diseases. Structure based drug design studies (molecular docking and structure based pharmacophore analysis)  were carried out on a series of natural product compounds to identify significant bioactive molecules to inhibit α-mannosidase (I and II) and ß-galactosidase enzymes. Furthermore, protein ligand interaction fingerprint analysis, molecular dynamics simulations and molecular access system (MACCS) fingerprint analysis were performed to understand the binding behaviors of the studied molecules. The results derived from these analyses showed that the identified compounds exhibit significant binding interactions with the active site residues. The compounds, NP-51, NP-81 and NP-165 have shown significant docking score against the studied enzymes (α-mannosidases-I, α-mannosidases-II and ß-galactosidases). The fingerprint studies showed that the presence of rings (aromatic or aliphatic) with sulfur atoms, nitrogen atoms, methyl groups, etc. have favorable effects on the α-mannosidase II inhibitory activity. However, the presence of halogen atoms substituted in the molecules have reduced inhibitory ability against α-mannosidase II. The compound, NP-165 has significant activity against both enzymes (α-mannosidases and ß-galactosidases). These studies accomplished that the compounds identified through in silico methodologies can be used to develop semisynthetic derivatives of the glycosidase inhibitors and can be screened for the treatment of different carbohydrate mediated diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-021-00115-9.

The Role of Anthocyanins, Deoxyanthocyanins and Pyranoanthocyanins on the Modulation of Tyrosinase Activity: An In Vitro and In Silico Approach.

Tyrosinase is the central enzyme involved in the highly complex process of melanin formation, catalyzing the rate-limiting steps of this biosynthetic pathway. Due to such a preponderant role, it has become a major target in the treatment of undesired skin pigmentation conditions and also in the prevention of enzymatic food browning. Numerous phenolic-based structures from natural sources have been pointed out as potential tyrosinase inhibitors, including anthocyanins. The aim of the present study was to individually assess the tyrosinase inhibitory activity of eight purified compounds with a variable degree of structural complexity: native anthocyanins, deoxyanthocyanins, and pyranoanthocyanins. The latter two, the groups of anthocyanin-related compounds with enhanced stability, were tested for the first time. Compounds 1 to 4 (luteolinidin, deoxymalvidin, cyanidin-, and malvidin-3-O-glucoside) revealed to be the most effective inhibitors, and further kinetic studies suggested their inhibition mechanism to be of a competitive nature. Structure-activity relationships were proposed based on molecular docking studies conducted with mushroom tyrosinase (mTYR) and human tyrosinase-related protein 1 (hTYRP1) crystal structures, providing information about the binding affinity and the different types of interactions established with the enzyme's active center which corroborated the findings of the inhibition and kinetic studies. Overall, these results support the applicability of these compounds as pigmentation modulators.

Anthocyanin-Related Pigments: Natural Allies for Skin Health Maintenance and Protection.

Human skin is commonly described as a particularly dynamic and complex environment, with a physiological balance continuously orchestrated by numerous internal and external factors. Intrinsic aging, exposure to UV radiation and skin pathogens are some of the key players that account for dermatological alterations and ailments. In this regard, this study intended to explore the potential skin-health beneficial properties of a group of molecules belonging to the anthocyanin family: cyanidin- and malvidin-3-O-glucosides and some of their structurally related pigments, resulting in a library of compounds with different structural properties and color hues. The inclusion of both purified compounds and crude extracts provided some insights into their distinctive effects when tested as individual agents or as part of multicomponent mixtures. Overall, most of the compounds were found to reduce biofilm production by S. aureus and P. aeruginosa reference strains, exhibit UV-filter capacity, attenuate the production of reactive oxygen species in human skin keratinocytes and fibroblasts and also showed inhibitory activity of skin-degrading enzymes, in the absence of cytotoxic effects. Carboxypyranocyanidin-3-O-glucoside stood out for its global performance which, combined with its greater structural stability, makes this a particular interesting compound for potential incorporation in topical formulations. Results provide strong evidence of the skin protective effects of these pigments, supporting their further application for cosmeceutical purposes.

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

Cell Wall Mannoproteins from Yeast Affect Salivary Protein-Flavanol Interactions through Different Molecular Mechanisms.

It is known that interactions between wine flavanols and salivary proline-rich proteins (PRPs) are one of the main factors responsible for wine astringency. The addition of commercial yeast mannoproteins (MPs) to wines has been pointed to as a possible tool to modulate the excessive astringency due to a lack of phenolic maturity at harvest time that might occur as a consequence of global climate change. The aim of this work was to study by isothermal titration calorimetry and molecular dynamics simulation the molecular mechanisms by which mannoproteins could modulate astringency elicited by tannins and if it can be influenced by mannoprotein composition. Results obtained indicate that the MPs assayed had an important impact on astringency through the formation of ternary aggregates with different solubilities or by preventing the flavanol-PRP interaction by a competitive mechanism, although in a different strength, depending on the size and the compositional characteristic of the mannoprotein.

In vitro gastrointestinal absorption of red wine anthocyanins - Impact of structural complexity and phase II metabolization.

Malvidin-3-O-glucoside, malvidin-3,5-O-diglucoside, malvidin-3-O-(6-O-coumaroyl)-glucoside-5-O-glucoside from Chinese Vitis davidii red wine were used to investigate the role of glucoside, diglucoside and coumaroylated glucoside moieties on their transport efficiency through MKN-28 gastric and Caco-2 intestinal cells. Due to the already described conversion of 3-O-glucosylated anthocyanins into 3-O-glucuronidated, the 3-O-glucuronidated metabolite of malvidin-3-O-glucoside was also tested. The antiproliferative activity was higher for the glucuronidated metabolite in both cell lines. All anthocyanins were transported through MKN-28 gastric cells and Caco-2 intestinal cells with transport efficiencies ranging from 4% to 9% in MKN-28 and from 3% to 5% in Caco-2. No significant differences on transport efficiencies were observed at 180 min among the different anthocyanins in MKN-28. The transport efficiency of malvidin-3-O-glucuronide at 180 min was about 3-4% in Caco-2 and MKN-28 cells. Computational studies were performed to evaluate the interaction between anthocyanins and glucose gastric transporters GLUT1 and GLUT3, which supported the experimental findings.

A multi-spectroscopic study on the interaction of food polyphenols with a bioactive gluten peptide: From chemistry to biological implications.

This study aims to exploit the molecular and cellular mechanisms concerning the functionality of dietary polyphenols (catechin, procyanidin B3, procyanidin C2, epigallocatechin and epigallocatechin gallate) in a nutritional context to prevent Celiac Disease (CD). In that sense, the interaction between the main CD bioactive peptide (32-mer peptide) and some polyphenols was fully characterized at the intestinal level under near physiological conditions by means of different spectroscopic techniques and dynamic simulations. Accordingly, it is proposed that the primarily polyphenol-binding sites on the 32-mer peptide correspond to leucine, tyrosine and phenylalanine containing domains being this interaction entropy-driven. Although procyanidin B3 and trimer C2 had a similar low-affinity constant at 310 K, both procyanidins were able to reduce the 32-mer peptide apical-to-basolateral translocation in in vitro simulated intestinal epithelial barrier thus prospecting the occurrence of additional and still unexplored regulatory mechanisms by which dietary polyphenols might modulate the transepithelial transport of CD bioactive peptides.

A computational study on the redox properties and binding affinities of iron complexes of hydroxypyridinones.

The potential of hydroxypyridinones for in vivo iron sequestration, in both biological and medical contexts, has been extensively discussed in the literature. Different chelators can be designed, with distinct lipophilicities that should alter their cell permeability, distribution, and rates of metabolism. However, for effective iron scavenging in biological systems, the redox potential and binding affinity of iron must fall within a proper range. Our objective was to assess the impact of different hydroxypyridinone chelators in 3:1 iron(III) complexes through comparison of these thermodynamic properties. For that purpose, we employed a cluster-continuum approach using density functional theory, on a dataset of 25 iron complexes. Whenever possible, our results were compared with experimental stability constants (log ß) and with electrode potentials. We observed a good qualitative agreement between computed free energies of binding and log ß values. In addition, we described which substitutions to the 3-hydroxypyridin-4-one ring should not markedly affect the redox properties and metal ion affinity considering iron. Graphical abstract Iron complexes of hydroxypyridinones.

Understanding the Rate-Limiting Step of Glycogenolysis by Using QM/MM Calculations on Human Glycogen Phosphorylase.

Liver glycogen phosphorylase (GP) is a key enzyme for human health, as its increased activity is associated with type 2 diabetes. The GP catalytic mechanism has been explored by quantum mechanics/molecular mechanics (QM/MM) methods. Herein, we propose a mechanism that proceeds by three steps: 1) it begins with transfer of a hydrogen atom from the phosphate group of the pyridoxal 5'-phosphate (HPO42- -PLP) cofactor to the phosphate substrate; 2) the glycosidic linkage is then cleaved through protonation of the glycosidic oxygen atom by a hydroxy group of the inorganic phosphate group; and 3) an oxygen atom of the phosphate performs a nucleophilic attack on the anomeric carbon atom of glucose, concomitant with the return of a proton from phosphate to PO43- -PLP, which finally leads to formation of the glucose-1-phosphate product and recovers the initial state of the PLP cofactor. The glycosidic bond cleavage and nucleophilic attack from the phosphate group to the glycosyl molecule have the highest activation free energies. The structural properties of the hereby characterized transition states could be very useful in structure-based drug design studies against liver GP.