Unraveling the Cardiac Effects Induced by Carvacrol in Spontaneously Hypertensive Rats: Involvement of Transient Receptor Potential Melastatin Subfamily 4 and 7 Channels.

ABSTRACT: Accumulating evidence indicates that transient receptor potential (TRP) channels are involved in the pathophysiological process in the heart, and monoterpenes, such as carvacrol, are able to modulate these channels activity. In this article, our purpose was to evaluate the direct cardiac effect of carvacrol on the contractility of cardiomyocytes and isolated right atria from spontaneously hypertensive and Wistar Kyoto rats. In this way, in vitro experiments were used to evaluate the ventricular cardiomyocytes contractility and the Ca2+ transient measuring, in addition to heart rhythm in the right atria. The role of TRPM channels in carvacrol-mediated cardiac activities was also investigated. The results demonstrated that carvacrol induced a significant reduction in ventricular cell contractility, without changes in transient Ca2+. In addition, carvacrol promoted a significant negative chronotropic response in spontaneously hypertensive and Wistar Kyoto rats' atria. Selective blockage of TRPM channels suggests the involvement of TRP melastatin subfamily 2 (TRPM2), TRPM4, and TRPM7 in the carvacrol-mediated cardiac effects. In silico studies were conducted to further investigate the putative role of TRPM4 in carvacrol-mediated cardiac action. FTMap underscores a conserved pocket in both TRPM4 and TRPM7, revealing a potential carvacrol binding site, and morphological similarity analysis demonstrated that carvacrol shares a more than 85% similarity to 9-phenanthrol. Taken together, these results suggest that carvacrol has direct cardiac actions, leading to reduced cellular contractility and inducing a negative chronotropic effect, which may be related to TRPM7 and TRPM4 modulation.

Benchmark Sets for Binding Hot Spot Identification in Fragment-Based Ligand Discovery.

Binding hot spots are regions of proteins that, due to their potentially high contribution to the binding free energy, have high propensity to bind small molecules. We present benchmark sets for testing computational methods for the identification of binding hot spots with emphasis on fragment-based ligand discovery. Each protein structure in the set binds a fragment, which is extended into larger ligands in other structures without substantial change in its binding mode. Structures of the same proteins without any bound ligand are also collected to form an unbound benchmark. We also discuss a set developed by Astex Pharmaceuticals for the validation of hot and warm spots for fragment binding. The set is based on the assumption that a fragment that occurs in diverse ligands in the same subpocket identifies a binding hot spot. Since this set includes only ligand-bound proteins, we added a set with unbound structures. All four sets were tested using FTMap, a computational analogue of fragment screening experiments to form a baseline for testing other prediction methods, and differences among the sets are discussed.

Cloning, expression, purification and spectrophotometric analysis of lanosterol 14-alpha demethylase from Leishmania braziliensis (LbCYP51).

Leishmaniasis, a neglected tropical disease, is a major cause of morbidity and mortality worldwide. Of the three main clinical forms, cutaneous leishmaniasis (CL) is the most common and 40 million people are at risk in the endemic areas. Currently, the available drugs to fight leishmaniasis have high toxicity and poor efficiency. Then, it is very important to search for effective and safe drugs that would target essential enzymes from the parasite, such as lanosterol 14-alpha demethylase (CYP51, EC 1.14.13.70) from Leishmania braziliensis. Because most drug design efforts have been directed for Leishmania non-braziliensis species, there is no structural or kinetic data regarding L. braziliensis CYP51. Herein, we present for the first time molecular biology efforts and purification protocol to obtain the enzyme LbCYP51. These results lay the ground for future investigation of drugs against this target.

Discovery of new inhibitors of Schistosoma mansoni PNP by pharmacophore-based virtual screening.

Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma mansoni , one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure-activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 µM. The most potent inhibitors 7, 10, and 17 with IC(50) of 2, 18, and 38 µM, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: kinetic and structural studies.

Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity.

Structure-based Druggability Assessment of Anti-virulence Targets from Pseudomonas aeruginosa.

Antimicrobial Resistance (AMR) represents a serious threat to health and the global economy. However, interest in antibacterial drug development has decreased substantially in recent decades. Meanwhile, anti-virulence drug development has emerged as an attractive alternative to fight AMR. Although several macromolecular targets have been explored for this goal, their druggability is a vital piece of information that has been overlooked. This review explores this subject by showing how structure- based freely available in silico tools, such as PockDrug and FTMap, might be useful for designing novel inhibitors of the pyocyanin biosynthesis pathway and improving the potency/selectivity of compounds that target the Pseudomonas aeruginosa quorum sensing mechanism. The information provided by hotspot analysis, along with binding site features, reveals novel druggable targets (PhzA and PhzS) that remain largely unexplored. However, it also highlights that in silico druggability prediction tools have several limitations that might be overcome in the near future. Meanwhile, anti-virulence drug targets should be assessed by complementary methods, such as the combined use of FTMap/PockDrug, once the consensus druggability classification reduces the risk of wasting resources on undruggable proteins.

Ligand-based design, synthesis and biochemical evaluation of potent and selective inhibitors of Schistosoma mansoni dihydroorotate dehydrogenase.

Schistosomiasis ranks second only to malaria as the most common parasitic disease worldwide. 700 million people are at risk and 240 million are already infected. Praziquantel is the anthelmintic of choice but decreasing efficacy has already been documented. In this work, we exploited the inhibition of Schistosoma mansoni dihydroorotate dehydrogenase (SmDHODH) as a strategy to develop new therapeutics to fight schistosomiasis. A series of quinones (atovaquone derivatives and precursors) was evaluated regarding potency and selectivity against both SmDHODH and human DHODH. The best compound identified is 17 (2-hydroxy-3-isopentylnaphthalene-1,4-dione) with IC50 = 23 ±â€¯4 nM and selectivity index of 30.83. Some of the new compounds are useful pharmacological tools and represent new lead structures for further optimization.

Structure-activity relationships for a class of selective inhibitors of the major cysteine protease from Trypanosoma cruzi.

Chagas' disease is a parasitic infection widely distributed throughout Latin America, with devastating consequences in terms of human morbidity and mortality. Cruzain, the major cysteine protease from Trypanosoma cruzi, is an attractive target for antitrypanosomal chemotherapy. In the present work, classical two-dimensional quantitative structure-activity relationships (2D QSAR) and hologram QSAR (HQSAR) studies were performed on a training set of 45 thiosemicarbazone and semicarbazone derivatives as inhibitors of T. cruzi cruzain. Significant statistical models (HQSAR, q(2) = 0.75 and r(2) = 0.96; classical QSAR, q(2) = 0.72 and r(2) = 0.83) were obtained, indicating their consistency for untested compounds. The models were then used to evaluate an external test set containing 10 compounds which were not included in the training set, and the predicted values were in good agreement with the experimental results (HQSAR, r(2)(pred) = 0.95; classical QSAR, r(2)(pred) = 0.91), indicating the existence of complementary between the two ligand-based drug design techniques.

Combined Strategies to Improve the Expression of Recombinant Sterol C24-Methyltransferase from Leishmania braziliensis in E. coli.

Among the neglected tropical diseases, leishmaniasis stands out for its worldwide distribution and diversity of symptoms. Cutaneous leishmaniasis (CL), for instance, is endemic in 18 countries, but the available drugs to fight it have high toxicity and low patient adherence. In order to overcome this, dilemma drugs that target enzymes which are absent in the human host, such as Leishmania braziliensis sterol C24-methyltransferase (SMT-C24, EC 2.1.1.41), are needed. However, medicinal chemistry efforts toward this goal have been hampered by the low yield of soluble recombinant SMT-C24 afforded by currently available expression systems. Herein, we show that a combination of molecular biology and chromatographic strategies may increase the yield of LbSMT-C24 in up to fivefold. These results lay the ground for future investigation of this enzyme as a drug target.

Molecular Cloning and Biochemical Characterization of Iron Superoxide Dismutase from Leishmania braziliensis.

Leishmaniasis is one of the most important neglected tropical diseases, with a broad spectrum of clinical manifestations. Among the clinical manifestations of the disease, cutaneous leishmaniasis, caused by species of Leishmania braziliensis, presents wide distribution in Brazil. In this work, we performed the cloning, expression, and purification of the enzyme superoxide dismutase of Leishmania braziliensis (LbSOD-B2) considered a promising target for the search of new compounds against leishmaniasis. In vitro assays based on pyrogallol oxidation showed that LbSOD-B2 is most active around pH 8 and hydrogen peroxide is a LbSOD-B2 inhibitor at low millimolar range (IC50 = 1 mM).

Synthesis and Biological Evaluation of Coumarins Derivatives as Potential Inhibitors of the Production of Pseudomonas aeruginosa Virulence Factor Pyocyanin.

Antimicrobial Resistance (AMR) is a serious problem for the humans since it threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi. One way around this problem is to act on the virulence factors, produced by bacteria, which increase their infection effectiveness. In view of these facts, new coumarin derivatives were synthesized and evaluated for their anti-virulence biological activity towards Pseudomonas aeruginosa. The results suggest that coumarin derivatives with a secondary carbon at C-3 position reduces P. aeruginosa growth whereas compounds with one additional substituent have a significant effect over pyocyanin production (10k EC50 7 ± 2 µM; 10l EC50 42 ± 13 µM). Moreover, 10k reduces P. aeruginosa motility and biofilm formation, what is compatible with a quorum sensing related mechanism of action.

Fragment-based and classical quantitative structure-activity relationships for a series of hydrazides as antituberculosis agents.

Worldwide, tuberculosis (TB) is the leading cause of death among curable infectious diseases. Multidrug-resistant Mycobacterium tuberculosis is an emerging problem of great importance to public health, and there is an urgent need for new anti-TB drugs. In the present work, classical 2D quantitative structure-activity relationships (QSAR) and hologram QSAR (HQSAR) studies were performed on a training set of 91 isoniazid derivatives. Significant statistical models (classical QSAR, q (2) = 0.68 and r (2) = 0.72; HQSAR, q (2) = 0.63 and r (2) = 0.86) were obtained, indicating their consistency for untested compounds. The models were then used to evaluate an external test set containing 24 compounds which were not included in the training set, and the predicted values were in good agreement with the experimental results (HQSAR, r(2)(pred) = 0.87; classical QSAR, r(2)(pred) = 0.75).

2D Quantitative structure-activity relationship studies on a series of cholesteryl ester transfer protein inhibitors.

Coronary heart disease (CHD) is one of the major causes of human death. The most successful therapeutic approach available is based on the reduction of low density-lipoprotein cholesterol (LDL-C). However, it is believed that the next paradigm in CHD treatment will rely on increased HDL-C levels. One of the most promising strategies for this goal is the inhibition of cholesteryl ester transfer protein (CETP). In the present work, robust classical 2D QSAR (r(2)=0.76, q(2)=0.72) and hologram QSAR (r(2)=0.88, q(2)=0.70) models were developed for a series of 85 CETP inhibitors (N-N-disubstituted trifluoro-3-amino-2-propanol derivatives). These models are complementary in nature and highlight important structural features for the design of novel CETP inhibitors with improved potency.

2D QSAR studies on thyroid hormone receptor ligands.

2D QSAR studies were carried out for a series of 55 ligands for the Thyroid receptors, TRalpha and TRbeta. Significant cross-validated correlation coefficients (q(2)=0.781 (TRalpha) and 0.693 (TRbeta)) were obtained. The models' predictive abilities were proved more valuable than the classical 2D-QSAR, and were further investigated by means of an external test set of 13 compounds. The predicted values are in good agreement with experimental values, suggesting that the models could be useful in the design of novel, more potent TR ligands. Contribution map analysis identified a number of positions that are promising for the development of receptor isoform specific ligands.

Two- and three-dimensional quantitative structure-activity relationships for a series of purine nucleoside phosphorylase inhibitors.

Comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis, and hologram quantitative structure-activity relationship (HQSAR) studies were conducted on a series of 52 training set inhibitors of calf spleen purine nucleoside phosphorylase (PNP). Significant cross-validated correlation coefficients (CoMFA, q(2)=0.68; CoMSIA, q(2)=0.66; and HQSAR, q(2)=0.70) were obtained, indicating the potential of the models for untested compounds. The models were then used to predict the inhibitory potency of 16 test set compounds that were not included in the training set, and the predicted values were in good agreement with the experimental results. The final QSAR models along with the information gathered from 3D contour and 2D contribution maps should be useful for the design of novel inhibitors of PNP having improved potency.

Evidence for the two phosphate binding sites of an analogue of the thioacyl intermediate for the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase-catalyzed reaction, from its crystal structure.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) into d-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD(+) and inorganic phosphate (P(i)). Within the active site, two anion-binding sites were ascribed to the binding of the C3 phosphate of GAP (P(s)) and to the binding of the attacking phosphate ion (P(i)). The role played by these two sites in the catalytic mechanism in connection with the functional role of coenzyme exchange (NADH-NAD(+) shuttle) has been investigated by several studies leading to the C3 phosphate flipping model proposed by Skarzynski et al. [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. This model has not yet received direct confirmation. To gain further insight into the role of both sites, we synthesized irreversible inhibitors which form with the essential cysteine residue a thioacyl enzyme analogue of the catalytic intermediate. Here we report the refined glycosomal Trypanosoma cruzi GAPDH in complex with a covalently bound GAP analogue at an improved resolution of 2.0-2.5 A. For this holo-thioacyl enzyme complex, a flip-flop movement is clearly characterized, the change from the P(i) to the P(s) binding site being correlated with the coenzyme exchange step: the weaker interaction of the intermediate when bound at the P(s) site with the cofactor allows its release and also the binding of the inorganic phosphate for the next catalytic step. This result gives strong experimental support for the generally accepted flip-flop model of the catalytic mechanism in GAPDH.

3D QSAR studies on binding affinities of coumarin natural products for glycosomal GAPDH of Trypanosoma cruzi.

Drug design strategies based on Comparative Molecular Field Analysis (CoMFA) have been used to predict the activity of new compounds. The major advantage of this approach is that it permits the analysis of a large number of quantitative descriptors and uses chemometric methods such as partial least squares (PLS) to correlate changes in bioactivity with changes in chemical structure. Because it is often difficult to rationalize all variables affecting the binding affinity of compounds using CoMFA solely, the program GRID was used to describe ligands in terms of their molecular interaction fields, MIFs. The program VolSurf that is able to compress the relevant information present in 3D maps into a few descriptors can treat these GRID fields. The binding affinities of a new set of compounds consisting of 13 coumarins, for one of which the three-dimensional ligand-enzyme bound structure is known, were studied. A final model based on the mentioned programs was independently validated by synthesizing and testing new coumarin derivatives. By relying on our knowledge of the real physical data (i.e., combining crystallographic and binding affinity results), it is also shown that ligand-based design agrees with structure-based design. The compound with the highest binding affinity was the coumarin chalepin, isolated from Rutaceae species, with an IC50 value of 55.5 microM towards the enzyme glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from glycosomes of the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. The proposed models from GRID MIFs have revealed the importance of lipophilic interactions in modulating the inhibition, but without excluding the dependence on stereo-electronic properties as found from CoMFA fields.

Crystal structure of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase complexed with an analogue of 1,3-bisphospho-d-glyceric acid.

We report here the first crystal structure of a stable isosteric analogue of 1,3-bisphospho-d-glyceric acid (1,3-BPGA) bound to the catalytic domain of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) in which the two phosphoryl moieties interact with Arg249. This complex possibly illustrates a step of the catalytic process by which Arg249 may induce compression of the product formed, allowing its expulsion from the active site. Structural modifications were introduced into this isosteric analogue and the respective inhibitory effects of the resulting diphosphorylated compounds on T. cruzi and Trypanosoma brucei gGAPDHs were investigated by enzymatic inhibition studies, fluorescence spectroscopy, site-directed mutagenesis, and molecular modelling. Despite the high homology between the two trypanomastid gGAPDHs (> 95%), we have identified specific interactions that could be used to design selective irreversible inhibitors against T. cruzi gGAPDH.

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors / Busca de inibidores da enzima glicossomal gliceraldeído 3-fosfato desidrogenase de Trypanosoma cruzi

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 μg/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA percent > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Nesse trabalho foi avaliada a atividade inibitória sobre a enzima glicossomal gliceraldeído-3-fosfato desidrogenase de T. cruzi (gGAPDH) de extratos vegetais oriundos de plantas das famílias Meliaceae e Rutaceae, na concentração de 100 μg/mL. Foram testados 46 extratos, dos quais 15 apresentaram atividade inibitória significativa ( por cento AI > 50). A maioria dos extratos de plantas da família Meliaceae (Cedrela fissilis, Cipadessa fruticosa e Trichilia ramalhoi) apresentou grande potencial em inibir a atividade enzimática. O fracionamento do extrato hexânico dos galhos de C. fruticosa permitiu o isolamento de três flavonóides: flavona, 7-metoxiflavona e 3',4',5',5,7-pentametoxiflavona. Os dois últimos foram ativos na inibição da atividade de gGAPDH. Desta forma, as três espécies de Meliaceae testadas podem ser consideradas promissoras na busca de compostos protótipos para o controle da doença de Chagas.