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The Amaryllidaceae family is distributed mainly in tropical and subtropical areas but also in temperate zones. In Venezuela, there are 6 genera and 13 species distributed around the country. This family is characterized by its ability to biosynthesize alkaloids with an isoquinoline type structure. In addition, a number of investigations have revealed a variety of biological activities for these alkaloids, such as antimalarial, antitumoral, analgesic, antimicrobial, cytotoxic, acetylcholinesterase inhibitory activity, among others. Galanthamine, an alkaloid first obtained from Galanthus woronowii, has been used since 2001 for the palliative therapy of mild-moderate Alzheimer's disease. Therefore, there is great interest on searching for galanthamine sources among the wide range of Amaryllidaceae species distributed around the world. The present investigation, carried out with five species from the Amaryllidaceae family, Crinum amabile, Crinum erubescens, Crinum moorei, Amaryllis belladonna and Zephyranthes carinata, collected from different locations in Merida State-Venezuela and analyzed by GC–MS, showed the alkaloid profile of leaves and bulbs of these species. Results revealed that lycorine and related compounds, such as 11,12-dehydro-anhydrolycorine and 1-O-acetyllycorine, were present in almost all samples analyzed, with lycorine predominant. The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition capacity of these alkaloid extracts showed a strong to moderate inhibitory effect on both enzymes; C. amabile leaves being the extract with the strongest inhibitory activity against AChE at 0.88 µg/mL as well as BuChE at 4.46 µg/mL, followed by Crinum erubescens leaves with values of 1.75 µg/mL and 8.72 µg/mL, respectively, compared to the values observed for galanthamine used as control. Present investigation constitutes the first in silico report about buphanisine interactions with AChE and BuChE active sites.

The dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of FolB protein is required for the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde (GA) in the folate pathway. FolB protein from Mycobacterium tuberculosis (MtFolB) is essential for bacilli survival and represents an important molecular target for drug development. S8-functionalized 8-mercaptoguanine derivatives were synthesised and evaluated for inhibitory activity against MtFolB. The compounds showed IC50 values in the submicromolar range. The inhibition mode and inhibition constants were determined for compounds that exhibited the strongest inhibition. Additionally, molecular docking analyses were performed to suggest enzyme-inhibitor interactions and ligand conformations. To the best of our knowledge, this study describes the first class of MtFolB inhibitors.

Osmotins are multifunctional proteins belonging to the thaumatin-like family related to plant stress responses. To better understand the functions of soybean osmotins in drought stress response, the current study presents the characterisation of four previously described proteins and a novel putative soybean osmotin (GmOLPa-like). Gene and protein structure as well as gene expression analyses were conducted on different tissues and developmental stages of two soybean cultivars with varying dehydration sensitivities (BR16 and EMB48 are highly and slightly sensitive, respectively). The analysed osmotin sequences share the conserved amino acid signature and 3D structure of the thaumatin-like family. Some differences were observed in the conserved regions of protein sequences and in the electrostatic surface potential. P21-like present the most similar electrostatic potential to osmotins previously characterised as promoters of drought tolerance in Nicotiana tabacum and Solanum nigrum. Gene expression analysis indicated that soybean osmotins were differentially expressed in different organs (leaves and roots), developmental stages (R1 and V3), and cultivars in response to dehydration. In addition, under dehydration conditions, the highest level of gene expression was detected for GmOLPa-like and P21-like osmotins in the leaves and roots, respectively, of the less drought sensitive cultivar. Altogether, the results suggest an involvement of these genes in drought stress tolerance.

This study aimed to produce and characterize a recombinant Kluyveromyces sp. β-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl β- d -1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant β-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.

International audience; In this study, we have investigated the enzyme shikimate 5-dehydrogenase from the causative agent of tuberculosis,Mycobacterium tuberculosis. We have employed a mixture of computational techniques, including molecular dynamics, hybrid quantum chemical/molecular mechanical potentials, relaxed surface scans, quantum chemical descriptors and free-energy simulations, to elucidate the enzyme's reaction pathway. Overall, we find a two-step mechanism, with a single transition state, that proceeds by an energetically uphill hydride transfer, followed by an energetically downhill proton transfer. Our mechanism and calculated free energy barrier for the reaction, 64.9 kJ mol(- 1), are in good agreement with those predicted from experiment. An analysis of quantum chemical descriptors along the reaction pathway indicated a possibly important, yet currently unreported, role of the active site threonine residue, Thr65.

Roughly a third of the world’s population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.

Hydrolysis efficiency of β-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial β-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnβGal), Aspergillus oryzae (AoβGal), Bacillus circulans (BcβGal), Bifidobacterium bifidum (BbβGal), and Kluyveromyces lactis (KlβGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnβGal), Asn140Ala and Asn199Ala (AoβGal), Arg111Ala and Glu355Ala (BcβGal), Arg122Ala and Phe358Ala (BbβGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlβGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcβGal, BbβGal, and KlβGal. The double mutations in BcβGal and BbβGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlβGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of β-galactosidases and rendering them promising for industrial applications.

Flexibility is a feature intimately related to protein function, since conformational changes can be used to describe environmental changes, chemical modifications, protein-protein and protein-ligand interactions. In this study, we have investigated the influence of the quaternary structure of 2-trans-enoyl-ACP (CoA) reductase or InhA, from Mycobacterium tuberculosis, to its flexibility. We carried out classical molecular dynamics simulations using monomeric and tetrameric forms to elucidate the enzyme’s flexibility. Overall, we observed statistically significant differences between conformational ensembles of tertiary and quaternary structures. In addition, the enzyme’s binding site is the most affected region, reinforcing the importance of the quaternary structure to evaluate the binding affinity of small molecules, as well as the effect of single point mutations to InhA protein dynamics.

GSTs (Glutathione S-transferases) are known to catalyze the nucleophilic attack of the sulfhydryl group of reduced glutathione (GSH) on electrophilic centers of xenobiotic compounds, including insecticides and acaricides. Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Moreover, we have modeled the structure of the enzyme in apo form, as well as in the form with bound inhibitor. We demonstrated that this protein is a glutathione S-transferase that can conjugate glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). We have tested TuGSTd01 activity with a range of potential substrates such as cinnamic acid, cumene hydroperoxide, and allyl isothiocyanate; however, the enzyme was unable to process these compounds. Using mutagenesis, we showed that putative active site variants S11A, E66A, S67A, and R68A mutants, which were residues predicted to interact directly with GSH, have no measurable activity, and these residues are required for the enzymatic activity of TuGSTd01. There are several reports that associate some T. urticae acaricide resistance with increased activity of GSTs . However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 μM. Therefore, we suggest that the increased GST activity observed in abamectin resistant T. urticae field populations is a part of the compensatory feedback loop. In this case, the increased production of GSTs and relatively high concentration of GSH in cells allow GSTs to maintain physiological functions despite the presence of the acaricide.

Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries with the highest incidence of cutaneous and mucocutaneous forms of leishmaniasis caused mainly by Leishmania braziliensis. The first-line drugs used in the treatment of leishmaniasis have several limitations: parenteral administration, long duration of treatment, and serious toxicity. One key metabolic characteristic of these parasites is the lack of a de novo purine biosynthesis pathway, making them auxotrophic to purines. Accordingly, they rely solely on the purine salvage pathway for nucleotide synthesis. A better understanding of the purine salvage pathway can reveal details of the biology of L. braziliensis that could, in turn, be used to develop new strategies to combat this parasite. The inosine–uridine nucleoside hydrolase from L. braziliensis (LbIU-NH) plays an important role in the salvage process and is an attractive drug target as there is no similar catalytic activity in mammals. Here is described cloning, heterologous protein expression, and a three-step purification protocol that yielded homogenous recombinant protein. The determination of LbIU-NH steady-state kinetic constants for inosine, adenosine, cytidine, uridine and p-nitrophenyl β-D-ribofuranoside is also reported. These data suggest that LbIU-NH displays characteristics of a nonspecific hydrolase. The thermodynamic profile suggests that D-ribose can bind to free enzyme with favorable enthalpic (ΔH) and entropic (ΔS) contributions. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the LbIU-NH-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, and pH-rate profiles are also presented. In addition, the crystal structure of LbIU-NH in complex with β-D-ribose and Ca2+ at 1.5 A resolution is described.

Adenylosuccinate lyase (ASL) is involved in both de novo and salvage pathways of purine biosynthesis. ASL belongs to the argininosuccinate lyase/fumarase C superfamily of enzymes which share a general acid–base catalytic mechanism with β-elimination of fumarate as the common product. Cloning, expression, and a method to obtain homogeneous recombinant ASL from Leishmania braziliensis (LbASL) are described. Mass spectrometry analysis of recombinant LbASL, oligomeric state determination and multiple sequence alignment are presented. Steady-state kinetics of LbASL showed a Michaelis–Menten pattern. Isothermal titration calorimetry binding assays suggested that LbASL follows a Uni-Bi ordered kinetic mechanism, in which release of fumarate is followed by AMP to yield free enzyme. Initial velocity data for the reverse reaction and the Haldane relationship allowed calculation of an unfavorable equilibrium constant for the LbASL-catalyzed chemical reaction. The activation energy and thermodynamic activation parameters were estimated. Solvent kinetic isotope effects V/K and V suggest a modest contribution of solvent proton transference during the rate-limiting step of the reaction. Proton inventory data show that the modest normal effect on V arises from a single protonic site, and the transition state fractionation factor value of 0.74 suggests participation of solvent proton transfer in transition-state vibrations perpendicular to the reaction coordinate. pH-rate profiles for kcat and kcat/KM suggested amino acid residues involved in, respectively, catalysis and substrate binding. A model of LbASL was built to provide a structural basis for the experimental data. A better understanding of the mode of action of LbASL is useful for the rational design of antileishmaniasis agents.

Ethnopharmacological relevance Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) is a plant traditionally used as medicine for anxiety symptoms. This activity was confirmed in preclinical studies. However, its efficacy was never studied in human clinical trials. Aim of the study We aimed to test the hypothesis that the herbal medicine of A. polystachya is superior to placebo for the treatment of anxiety-related symptoms in adults after 8 weeks. Patients and methods This was a randomized, double-blind, placebo-controlled, phase-2 clinical trial. Fifty-four adults with self-reported anxiety symptoms were randomly allocated to receive either capsules containing A. polystachya powdered leaves (300 mg, twice a day) or placebo (maltodextrin), for 8 weeks. The intensity of anxiety symptoms was assessed by the Hamilton Anxiety Ranking Scale (HAM-A) at baseline and after 2, 4 and 8 weeks. All analyses were adjusted for physical activity (assessed by the International Physical Activity Questionnaire [IPAQ], short version) and gender. Results We confirmed the presence of acteoside (chromatographic analysis) and carvone and limonene (gas chromatography) as major constituents in our plant material. Only patients that received A. polystachya experienced a significant decrease in their HAM-A scores, with none or mild side-effects. Conclusion Administration of powdered leaves of A. polystachya, rich in acteoside, carvone and limonene, to adults with anxiety symptoms was significantly superior to placebo in decreasing HAM-A scores after 8 weeks. This finding confirms the ethnopharmacological use of this plant for anxiety symptoms.

HisD-Encoded histidinol dehydrogenase (HisD) catalyzes the two last chemical reactions of the L-histidine biosynthetic pathway, namely the conversion of L-histidinol (L-Hol) to L-histidinaldehyde (L-Hal) and to L-histidine (L-His). The hisD gene product has been shown to be essential for Mycobacterium tuberculosis survival in vitro. Herein, we describe a series of biochemical studies on recombinant Mycobacterium tuberculosis HisD (MtHisD). The synthesis of hydrazones derived from L-histidine yielded inhibitors in the low micromolar range, one of which showed moderate anti-Mtb activity. The compounds described here are, to the best of our knowledge, the first inhibitors of MtHisD activity reported in the literature, and they could become promising candidates for future development.

The PI3K/Akt/mTOR pathway is an important intracellular signaling pathway in cell cycle regulation and its dysregulation is associated with various types of diseases. mTOR (mechanistic or mammalian target of rapamycin) is the main enzyme that performs intermediate control of the signaling pathway through a phosphotransfer process. The classical inhibition of the mTOR pathway is effected by rapamycin and its analogous blocking allosterically the catalytic phosphorylation site, avoiding the deleterious side effects induced by ATP-competitive inhibitors. We employed ligand-based drug design strategies such as pharmacophore searching and analysis, molecular docking, absorption, distribution, metabolism, excretion and toxicity (ADMETox) properties filtering, and molecular dynamics to select potential molecules to become non-ATP competitive inhibitors of the mTOR complex. According to our findings, we propose eight novel potential mTOR inhibitors with similar or better properties than the classic inhibitor complex, rapamycin.

The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H # ), entropy (∆S # ), and Gibbs free energy (∆G # ) of activation were determined. The ∆G # values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES # complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that “soft” interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.

Uridine (Urd) is a promising biochemical modulator to reduce host toxicity caused by 5-fluorouracil (5-FU) without impairing its antitumor activity. Elevated doses of Urd are required to achieve a protective effect against 5-FU toxicity, but exogenous administration of Urd is not well-tolerated. Selective inhibitors of human uridine phosphorylase (hUP) have been proposed as a strategy to increase Urd levels. We describe synthesis and characterization of a new class of ligands that inhibit hUP type 1 (hUP1). The design of ligands was based on a possible SN1 catalytic mechanism and as mimics of the carbocation in the transition state of hUP1. The kinetic and thermodynamic profiles showed that the ligands here presented are the most potent in vitro hUP1 inhibitors developed to date. In addition, a lead compound improved the antiproliferative effects of 5-FU on colon cancer cells, accompanied by a reduction of in vitro 5-FU cytotoxicity in aggressive SW-620 cancer cells.

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease’s causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli’s nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.

Flexibility is involved in a wide range of biological processes, such as protein assembly and binding recognition. EPSP synthase is an enzyme that must undergo a large conformational change to accommodate its ligands into its binding cavity. However, although the structure of EPSP synthase has been determined, its plasticity has not been explored in depth. Therefore, in this work, we extensively examined the influence of the flexibility of Mycobacterium tuberculosis EPSP (MtEPSP) synthase on the function of this protein using classical and replica-exchange metadynamics simulations. We were able to identify five well-populated conformational clusters for MtEPSP synthase: two corresponding to open, one to ajar, and two to closed conformations. We also pinpointed three hydrophobic regions that are responsible for guiding transitions among these states. Taken together, the new findings presented here indicate how the hydrophobic regions modulate the flexibility of MtEPSP synthase, and they highlight the importance of considering these dynamic features in drug design projects employing this enzyme as a target. Graphical abstract The flexibility of EPSP synthase as a function of the pincer angles.

Background and aimsFibromyalgia (FM) is a chronic, rheumatic disease characterized by widespread myofascial pain, of unknown aetiology, having a major impact on quality of life (QOL). Available pharmacotherapy for FM is marginally effective. FM is associated with co-morbidities of gastrointestinal (GI) disorders and Irritable Bowel Syndrome (IBS). There is growing evidence that diets low in FODMAPs, “fermentable oligo-, di- or mono-saccharides and polyols” [Low FODMAP Diet (LFD)], are effective in treating IBS. The aim of this pilot study was to examine the effects of LFDs on symptoms of FM, especially with regard to pain, QOL and GI disorders.MethodsA longitudinal study using LFD intervention was performed on 38, 51±10 year-old, female patients diagnosed with FM for an average of 10 years, based on ACR (American College of Rheumatology) 2010 criteria. The study was conducted from January through May, 2015, using a four-week, repeated-assessment model, as follows: Moment 0 – introduction of the protocol to participants; Moment 1 – first assessment and delivery of individual LFD dietary plans; Moment 2 – second assessment and reintroduction of FODMAPs; Moment 3 – last assessment and final nutritional counselling. Assessment tools used were the following: RFIQ (Revised Fibromyalgia Impact Questionnaire), FSQ (Fibromyalgia Survey Questionnaire), IBS-SSS (Severity Score System), EQ-5D (Euro-QOL quality of life instrument), and VAS (Visual Analogue Scale). Daily consumption of FODMAPs was quantified based on published food content analyses. Statistical analyses included ANOVA, non-parametric Friedman,t-student and Chi-square tests, using SPSS 22 software.ResultsThe mean scores of the 38 participants at the beginning of the study were: FSQ (severity of FM, 0–31) – 22±4.4; RFIQ (0–100) – 65±17; IBS-SSS (0–500) – 275± 101; and EQ-5D (0–100) – 48± 19. Mean adherence to dietary regimens was 86%, confirmed by significant difference in FODMAP intakes (25 g/day vs. 2.5 g/day;p< 0.01). Comparisons between the three moments of assessment showed significant (p< 0.01) declines in scores in VAS, FSQ, and RFIQ scores, in all domains measured. An important improvement was observed with a reduction in the severity of GI symptoms, with 50% reduction in IBS scores to 138±117, following LFD therapy. A significant correlation (r= 0.36;p< 0.05) was found between improvements in FM impact (declined scores) and gastrointestinal scores. There was also a significant correlation (r= 0.65;p< 0.01) between “satisfaction with improvement” after introduction of LFDs and “diet adherence”, with satisfaction of the diet achieving 77% among participants. A significant difference was observed between patients who improved as compared to those that did not improve (Chi-square χ2= 6.16;p< .05), showing that the probability of improvement, depends on the severity of the RFIQ score.ConclusionsImplementation of diet therapy involving FODMAP restrictions, in this cohort of FM patients, resulted in a significant reduction in GI disorders and FM symptoms, including pain scores. These results need to be extended in future larger studies on dietary therapy for treatment of FM.ImplicationsAccording to current scientific knowledge, these are the first relevant results found in an intervention with LFD therapy in FM and must be reproduced looking for a future dietetic approach in FM.

Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO 4 ) at 2.10 A resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO 4 structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active site lost of stability, in agreement with experimental results, which demonstrate a considerable inhibitory activity against MtPNP ( K i = 150 nM). Furthermore, we observed that some residues which are important in the proper ligand’s anchor into the human homologous enzyme do not present the same importance to MtPNP. Therewithal, these findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.

Cytidine Deaminase (CD) is an evolutionarily conserved enzyme that participates in the pyrimidine salvage pathway recycling cytidine and deoxycytidine into uridine and deoxyuridine, respectively. Here, our goal is to apply computational techniques in the pursuit of potential inhibitors of Mycobacterium tuberculosis CD (MtCDA) enzyme activity. Molecular docking simulation was applied to find the possible hit compounds. Molecular dynamics simulations were also carried out to investigate the physically relevant motions involved in the protein-ligand recognition process, aiming at providing estimates for free energy of binding. The proposed approach was capable of identifying a potential inhibitor, which was experimentally confirmed by IC(50) evaluation. Our findings open up the possibility to extend this protocol to different databases in order to find new potential inhibitors for promising targets based on a rational drug design process.

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 A. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP ( K i = 200 μM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.

Bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) catalyses the phosphoryl transfer from ATP to CMP and dCMP, resulting in the formation nucleoside diphosphates. In eukaryotes, CMP/UMP kinase catalyses the conversion of UMP and CMP to, respectively, UDP and CDP with high efficiency. This work describes for the first time a model of bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) from mycobacterium tuberculosis (MtCMPK). We modeled MtPCMPK in apo form and in complex with cytidine 5'-monophosphate (CMP) to try to determine the structural basis for specificity. Comparative analysis of the model of MtCMPK allowed identification of structural features responsible for ligand affinities. Analysis of the molecular dynamics simulations of these two systems indicates the structural features responsible for the stability of the structure, and may help in the identification of new inhibitors for this enzyme.

This study reports a facile and controllable synthetic method for the preparation of both 1,3- and 1,5-isomers of 4-(3(5)-aryl-3(5)-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamides, as well as a new series of 4-(3-aryl-5-hydroxy-5-(trifluoromethyl)-4,5-dihydropyrazol-1-yl)benzenesulfonamides, from the cyclocondensation reaction of 4-aryl-1,1,1-trifluoro-4-methoxybut-3-en-2-ones or 1-aryl-4,4,4-trifluoro-butane-1,3-diones or their enolic forms with 4-hydrazinylbenzenesulfonamide. All compounds of the new series of 3-substituted 1-(4-benzenesulfonamide)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydropyrazoles were tested for their effect on a pathological pain model in mice. The compounds 3a, 3b, 3c, 3e, and 3f presented anti-hyperalgesic action, while the compounds 3a, 3c, 3d, 3f, and 3g exhibited anti-edematogenic effects, without causing locomotive disorders in animals, thus making them comparable to Celecoxib in an arthritic pain model.

Background.Respiratory syncytial virus (RSV) infection is the major cause of respiratory disease in lower respiratory tract in infants and young children. Attempts to develop effective vaccines or pharmacological treatments to inhibit RSV infection without undesired effects on human health have been unsuccessful. However, RSV infection has been reported to be affected by flavonoids. The mechanisms underlying viral inhibition induced by these compounds are largely unknown, making the development of new drugs difficult.Methods.To understand the mechanisms induced by flavonoids to inhibit RSV infection, a systems pharmacology-based study was performed using microarray data from primary culture of human bronchial cells infected by RSV, together with compound-proteomic interaction data available forHomo sapiens.Results.After an initial evaluation of 26 flavonoids, 5 compounds (resveratrol, quercetin, myricetin, apigenin, and tricetin) were identified through topological analysis of a major chemical-protein (CP) and protein-protein interacting (PPI) network. In a nonclustered form, these flavonoids regulate directly the activity of two protein bottlenecks involved in inflammation and apoptosis.Conclusions.Our findings may potentially help uncovering mechanisms of action of early RSV infection and provide chemical backbones and their protein targets in the difficult quest to develop new effective drugs.

Mycobacterium tuberculosis InhA (MtInhA) is an attractive enzyme to drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy. In this work, two different virtual-ligand-screening approaches were applied in order to identify new InhA inhibitors' candidates from a library of ligands selected from the ZINC database. First, a 3-D pharmacophore model was built based on 36 available MtInhA crystal structures. By combining structure-based and ligand-based information, four pharmacophoric points were designed to select molecules able to satisfy the binding features of MtInhA substrate-binding cavity. The second approach consisted of using four well established docking programs, with different search algorithms, to compare the binding mode and score of the selected molecules from the aforementioned library. After detailed analyses of the results, six ligands were selected for in vitro analysis. Three of these molecules presented a satisfactory inhibitory activity with IC50 values ranging from 24 (±2) μM to 83 (±5) μM. The best compound presented an uncompetitive inhibition mode to NADH and 2-trans-dodecenoyl-CoA substrates, with Ki values of 24 (±3) μM and 20 (±2) μM, respectively. These molecules were not yet described as antituberculars or as InhA inhibitors, making its novelty interesting to start efforts on ligand optimization in order to identify new effective drugs against tuberculosis having InhA as a target. More studies are underway to dissect the discovered uncompetitive inhibitor interactions with MtInhA.

Cytidine monophosphate kinase from Mycobacterium tuberculosis (MtCMK) likely plays a role in supplying precursors for nucleic acid synthesis. MtCMK catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Initial velocity studies and Isothermal titration calorimetry (ITC) measurements showed that MtCMK follows a random-order mechanism of substrate (CMP and ATP) binding, and an ordered mechanism for product release, in which ADP is released first followed by CDP. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2′-OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons between CMP/dCMP kinases and UMP/CMP ones.

Molecular recognition process describes the interaction involving two molecules. In the case of biomolecules, these pairs of molecules could be protein-protein, protein-ligand or protein-nucleic acid. The first model to capture the essential features, behind the molecular recognition problem, was the lock-and-key paradigm. The overall analysis protein-protein, protein-nucleic acid and protein-ligand interaction based on the three-dimensional structures and physicochemical parameters, such as binding affinity, opened the possibility to provide further insights in this basic phenomenon. The main ideas behind the molecular recognition are discussed in the present review.

Purine Nucleoside Phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-glycosidic bonds of purine nucleosides and deoxynucleosides, except for adenosine, to generate ribose 1-phosphate and the purine base. PNP has been submitted to intensive structural studies. This work describes for the first time a structural model of PNP from Streptococcus pyogenes (SpPNP). We modeled the complexes of SpPNP with six different ligands in order to determine the structural basis for specificity of these ligands against SpPNP. Molecular dynamics (MD) simulations were performed in order to evaluate the overall stability of SpPNP model. The analysis of the MD simulation was assessed mainly by principal component analysis (PCA) to explore the trimeric structure behavior. Structural comparison, between SpPNP and human PNP, was able to identify the main features responsible for differences in ligand-binding affinities, such as mutation in the purine-binding site and in the second phosphate-binding site. The PCA analysis suggests a different behavior for each subunit in the trimer structure.

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.

Introduction: Fibromyalgia is a chronic rheumatic disease producing widespread pain, associated to a major comorbidity -irritable bowel syndrome. Low FODMAPS diet (low fermentable oligo-di-mono-saccharides and polyols diet) has been effective in controlling irritable bowel syndrome symptoms. Overweight is an aggravating factor for fibromyalgia. We studied effects of low fermentable oligo-di-mono-saccharides and polyols diets on fibromyalgia symptoms and weight status. Methods: A longitudinal study was performed on 38 fibromyalgia patients using a four-week, repeated assessment as follow: M1 = first assessments/presentation of individual low fermentable oligo-di-mono-saccharides and polyols diet; M2 = second assessments/reintroduction of FODMAPs; M3 = final assessments/nutritional counselling. The assessment instruments applied were: Fibromyalgia Survey Questionnaire (FSQ); Severity Score System (IBS-SSS); visual analogic scale (VAS). Body mass-index/composition and waist circumference (WC) were also measured. Daily macro-micronutrients and FODMAP intake were quantified at each moment of the study. Results: The studied cohort was 37% overweight, 34% obese (average body mass-index 27.4 ± 4.6; excess fat mass 39.4 ± 7%). Weight, body mass-index and waist circumference decreased significantly (p < 0.01) with low fermentable oligo-di-mono-saccharides and polyols diet, but no significant effect on body composition was observed. All fibromyalgia symptoms, including somatic pain, declined significantly post-LFD (p < 0.01); as well for severity of fibromyalgia [Fibromyalgia survey questionnaire: M1 = 21.8; M2 = 16.9; M3 = 17.0 (p < 0.01)]. The intake of essential nutrients (fiber, calcium, magnesium and vitamin D) showed no significant difference. The significant reduction in FODMAP intake (M1 = 24.4 g; M2 = 2.6g; p < 0.01) reflected the "Diet adherence" (85%). "Satisfaction with improvement of symptoms" (76%), showed correlating with "diet adherence" (r = 0.65; p < 0.01). Conclusions: Results are highly encouraging, showing low fermentable oligo-di-mono-saccharides and polyols diets as a nutritionally balanced approach, contributing to weight loss and reducing the severity of FM fibromyalgia symptoms. Key words: FODMAP. Fibromyalgia. Irritable bowel syndrome. Pain. Diet. Short-chain. Carbohydrates.