Identification of Active Compounds against Melanoma Growth by Virtual Screening for Non-Classical Human DHFR Inhibitors.

Antifolates such as methotrexate (MTX) have been largely known as anticancer agents because of their role in blocking nucleic acid synthesis and cell proliferation. Their mechanism of action lies in their ability to inhibit enzymes involved in the folic acid cycle, especially human dihydrofolate reductase (hDHFR). However, most of them have a classical structure that has proven ineffective against melanoma, and, therefore, inhibitors with a non-classical lipophilic structure are increasingly becoming an attractive alternative to circumvent this clinical resistance. In this study, we conducted a protocol combining virtual screening (VS) and cell-based assays to identify new potential non-classical hDHFR inhibitors. Among 173 hit compounds identified (average logP = 3.68; average MW = 378.34 Da), two-herein, called C1 and C2-exhibited activity against melanoma cell lines B16 and A375 by MTT and Trypan-Blue assays. C1 showed cell growth arrest (39% and 56%) and C2 showed potent cytotoxic activity (77% and 51%) in a dose-dependent manner. The effects of C2 on A375 cell viability were greater than MTX (98% vs 60%) at equivalent concentrations and times. Our results indicate that the integrated in silico/in vitro approach provided a benchmark to identify novel promising non-classical DHFR inhibitors showing activity against melanoma cells.

Computational Drug Repositioning for Chagas Disease Using Protein-Ligand Interaction Profiling.

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), affects nearly eight million people worldwide. There are currently only limited treatment options, which cause several side effects and have drug resistance. Thus, there is a great need for a novel, improved Chagas treatment. Bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) has emerged as a promising pharmacological target. Moreover, some human dihydrofolate reductase (HsDHFR) inhibitors such as trimetrexate also inhibit T. cruzi DHFR-TS (TcDHFR-TS). These compounds serve as a starting point and a reference in a screening campaign to search for new TcDHFR-TS inhibitors. In this paper, a novel virtual screening approach was developed that combines classical docking with protein-ligand interaction profiling to identify drug repositioning opportunities against T. cruzi infection. In this approach, some food and drug administration (FDA)-approved drugs that were predicted to bind with high affinity to TcDHFR-TS and whose predicted molecular interactions are conserved among known inhibitors were selected. Overall, ten putative TcDHFR-TS inhibitors were identified. These exhibited a similar interaction profile and a higher computed binding affinity, compared to trimetrexate. Nilotinib, glipizide, glyburide and gliquidone were tested on T. cruzi epimastigotes and showed growth inhibitory activity in the micromolar range. Therefore, these compounds could lead to the development of new treatment options for Chagas disease.

Targeting pteridine reductase 1 and dihydrofolate reductase: the old is a new trend for leishmaniasis drug discovery.

Leishmaniasis is one of the major neglected tropical diseases in the world and it is considered endemic in 88 countries. This disease is transmitted by a Leishmania spp. infected sandfly and it may lead to cutaneous or systemic manifestations. The preconized treatment has low efficacy and there are cases of resistance to some drugs. Therefore, the search for new efficient molecular targets that can lead to the preparation of new drugs must be pursued. This review aims to evaluate both Leishmania enzymes PTR1 and DHFR-TS as potential drug targets, highlight their inhibitors and to discuss critically the use of chemoinformatics to elucidate interactions and propose new molecules against these enzymes.

Crystal structures of the closed form of Mycobacterium tuberculosis dihydrofolate reductase in complex with dihydrofolate and antifolates.

Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M. tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, through Kd measurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highest Kd) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.

Structural and mechanistic insight from antiviral and antiparasitic enzyme drug targets for tropical infectious diseases.

With almost half of the world population living at risk, tropical infectious diseases cause millions of deaths every year in developing countries. Considering the lack of economic prospects for investment in this field, approaches aiming the rational design of compounds, such as structure-based drug discovery (SBDD), fragment screening, target-based drug discovery, and drug repurposing are of special interest. Herein, we focused in the advances on the field of SBDD targeting arboviruses such as dengue, yellow fever, zika and chikungunya enzymes of the RNA replication complex (RC) and enzymes involved in a variety of pathways essential to ensure parasitic survival in the host, for malaria, Chagas e leishmaniasis diseases. We also highlighted successful examples such as promising new inhibitors and molecules already in preclinical/clinical phase tests, major gaps in the field and perspectives for the future of drug design for tropical diseases.

Identification, Characterization and Molecular Modelling Studies of Schistosoma mansoni Dihydrofolate Reductase Inhibitors: From Assay Development to Hit Identification.

INTRODUCTION: Schistosoma mansoni is responsible for virtually all reported cases of schistosomiasis in Latin America and the emergence of praziquantel- and oxaminiquine-resistant strains makes it urgent to develop new schistosomicide agents. Dihydrofolate reductases (DHFR) from bacteria and protozoan parasites are considered validated macromolecular targets for this goal, but S. mansoni DHFR (SmDHFR) has been largely overlooked. To fill this gap in knowledge, the present work describes optimized conditions to carry out thermal shift assays with SmDHFR, as well as a balanced kinetic assay that supports 2,4-diaminopyrimidine derivatives as SmDHFR inhibitors. The most potent inhibitor (2a) shows a large shift of the melting temperature (ΔTm = + 8 ± 0,21 ºC) and a low micromolar IC50 value (12 ± 2,3 µM). Both thermal shift and classical kinectic assay suggest that 2a binds to the substrate binding site (competitive inhibition mechanism). This information guided docking and molecular dynamics studies that probed 2a interaction profile towards SmDHFR. CONCLUSION: In conclusion, this work not only provides standardized assay conditions to identify SmDHFR inhibitors, but also describes the binding profile of the first low micromolar inhibitor of this macromolecular target.

Folate biosynthesis pathway: mechanisms and insights into drug design for infectious diseases.

Folate pathway is a key target for the development of new drugs against infectious diseases since the discovery of sulfa drugs and trimethoprim. The knowledge about this pathway has increased in the last years and the catalytic mechanism and structures of all enzymes of the pathway are fairly understood. In addition, differences among enzymes from prokaryotes and eukaryotes could be used for the design of specific inhibitors. In this review, we show a panorama of progress that has been achieved within the folate pathway obtained in the last years. We explored the structure and mechanism of enzymes, several genetic features, strategies, and approaches used in the design of new inhibitors that have been used as targets in pathogen chemotherapy.

Analysis of Coxiela burnetti dihydrofolate reductase via in silico docking with inhibitors and molecular dynamics simulation.

Coxiella burnetii is a gram-negative bacterium able to infect several eukaryotic cells, mainly monocytes and macrophages. It is found widely in nature with ticks, birds, and mammals as major hosts. C. burnetii is also the biological warfare agent that causes Q fever, a disease that has no vaccine or proven chemotherapy available. Considering the current geopolitical context, this fact reinforces the need for discovering new treatments and molecular targets for drug design against C. burnetii. Among the main molecular targets against bacterial diseases reported, the enzyme dihydrofolate reductase (DHFR) has been investigated for several infectious diseases. In the present work, we applied molecular modeling techniques to evaluate the interactions of known DHFR inhibitors in the active sites of human and C. burnetii DHFR (HssDHFR and CbDHFR) in order to investigate their potential as selective inhibitors of CbDHFR. Results showed that most of the ligands studied compete for the binding site of the substrate more effectively than the reference drug trimethoprim. Also the most promising compounds were proposed as leads for the drug design of potential CbDHFR inhibitors.

Virtual screening, docking, and dynamics of potential new inhibitors of dihydrofolate reductase from Yersinia pestis.

In the present work, we propose to design drugs that target the enzyme dihydrofolate redutase (DHFR) as a means of a novel drug therapy against plague. Potential inhibitors of DHFR from Yersinia pestis (YpDHFR) were selected by virtual screening and subjected to docking, molecular dynamics (MD) simulations, and Poisson-Boltzmann surface area method, in order to evaluate their interactions in the active sites of YpDHFR and human DHFR (HssDHFR). The results suggested selectivity for three compounds that were further used to propose the structures of six new potential selective inhibitors for YpDHFR.

Molecular modeling toward selective inhibitors of dihydrofolate reductase from the biological warfare agent Bacillus anthracis.

In the present work, we applied docking and molecular dynamics techniques to study 11 compounds inside the enzymes dihydrofolate reductase (DHFR) from the biological warfare agent Bacillus anthracis (BaDHFR) and Homo sapiens sapiens (HssDHFR). Six of these compounds were selected for a study with the mutant BaF96IDHFR. Our results corroborated with experimental data and allowed the proposition of a new molecule with potential activity and better selectivity for BaDHFR.

Complementary data on four methods for sampling free-living ticks in the Brazilian Pantanal / Dados complementares de quatro métodos para amostragem de carrapatos em vida livre no Pantanal brasileiro

In this study, four methods for sampling free-living ticks that are used in ecological and human tick-bite risk studies were evaluated. Cloth dragging, carbon dioxide traps and visual searches and inspection of plant litter on the ground were used in field and forest areas within the Brazilian Pantanal. Among the three tick species collected, Amblyomma sculptum predominated, followed by Amblyomma parvum and Amblyomma ovale. Dragging, a cheap and simple technique, yielded the highest numbers of ticks, particularly nymphs. The visual search detected a high number of adult ticks and provided information on tick questing height. Even though laborious, plant litter examination showed that large numbers of ticks may use this stratum. Carbon dioxide (CO2) traps are expensive and difficult to handle, but they are highly efficient for adult ticks, especially A. parvum. These data indicate that one method alone is incapable of providing a representative sample of the tick fauna in a particular area and that multiple techniques should be used for tick population studies.

Neste estudo, foram avaliados quatro métodos de amostragem de carrapatos em vida livre, usados em estudos ecológicos e avaliação do risco de picadas em humanos. Arraste de flanela, armadilhas de gás carbônico (CO2), busca visual e inspeção de serrapilheira foram aplicados em áreas campestres e florestais no Pantanal brasileiro. Dentre três espécies coletadas, a predominância foi de Amblyomma sculptum, seguida por Amblyomma parvum e Amblyomma ovale. O arraste, técnica simples e de baixo custo, resultou em maior número de carrapatos, particularmente de ninfas. A busca visual detectou alto número de carrapatos adultos e forneceu informações sobre altura de espera por hospedeiros. Apesar de trabalhoso, o exame da serrapilheira demonstrou que grande número de carrapatos pode utilizar esse estrato. Armadilhas de CO2 têm custo elevado e são difíceis de manusear, entretanto, são altamente eficientes para carrapatos adultos, em especial para A. parvum. Esses dados indicam que somente um método é incapaz de fornecer amostra representativa da ixodofauna em uma área particular e que, para estudos populacionais, técnicas múltiplas devem ser usadas.

CoMFA/CoMSIA 3D-QSAR of pyrimidine inhibitors of Pneumocystis carinii dihydrofolate reductase.

Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.

Molecular modeling studies of Yersinia pestis dihydrofolate reductase.

Considering the risk represented by plague today as a potential biological warfare agent, we propose cytosolic Yersinia pestis dihydrofolate reductase (YpDHFR) as a new target to the design of selective plague chemotherapy. This enzyme has a low homology with the human enzyme and its crystallographic structure has been recently deposited in the Protein Data Bank (PDB). Comparisons of the docking energies and molecular dynamic behaviors of five known DHFR inhibitors inside a 3D model of YpDHFR (adapted from the crystallographic structure) and human DHFR (HssDHFR), revealed new potential interactions and suggested insights into the design of more potent HssDHFR inhibitors as well as selective inhibitors for YpDHFR.

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant.

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

Binding mode analysis of 2,4-diamino-5-methyl-5-deaza-6-substituted pteridines with Mycobacterium tuberculosis and human dihydrofolate reductases.

There are major differences between the structures of human dihydrofolate reductase (hDHFR) and Mycobacterium tuberculosis dihydrofolate reductase (mtDHFR). These differences may allow the design of more selective mtDHFR inhibitors. In this paper, we have used docking approaches to study the binding orientations and predict binding affinities of 2,4-diamino-5-methyl-5-deazapteridines derivatives in both hDHFR and mtDHFR. Our results of molecular docking combined with experimental data for inhibition of the human and mycobacterial dihydrofolate reductases suggest the presence of empty spaces around the 2,4-diaminodeazapteridine and N10-phenyl rings in the mtDHFR active site that are not found in the hDHFR-bound structures. Preparation of new analogs with substituents attached to C7 of the pteridine nucleus and positions 3 and 4 of the N10-phenyl group should increase the affinity and selectivity for mtDHFR.

Molecular modeling of wild-type and antifolate resistant mutant Plasmodium falciparum DHFR.

The development of drug resistance is reducing the efficiency of antifolates as antimalarials. This phenomenon has been linked to the occurrence of mutations in the parasite's dihydrofolate reductase (DHFR). In this way, the resistance to pyrimethamine and cycloguanil, two potent inhibitors of P. falciparum DHFR, is mainly related to mutations (single and crossed) at residues 16, 51, 59, 108 and 164 of the enzyme. In this work, we have refined a recently proposed homology-model of P. falciparum DHFR, and the resulting structure was used to obtain models for 14 mutant enzymes, employing molecular modeling. Ternary complexes of the mutant enzymes with these inhibitors have been superimposed to equivalent ternary complexes of the wild-type enzyme, allowing the proposition of hypotheses for the role of each mutation in drug resistance. Based on these results, possible reasons for antifolate resistance have been proposed.