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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548400

RESUMO

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.


Assuntos
Compostos de Boro/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Administração Oral , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Domínio Catalítico , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Plasmodium falciparum/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/química
2.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443484

RESUMO

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys11, Lys12,Lys13-(pBthTX-I)2K ((pBthTX-I)2K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)2K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC50 = 28-65 µM) and mostly low cytotoxic effect (CC50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (Mpro) and Papain-Like protease (PLpro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PLpro inhibition potencies (IC50s = 1.0-3.5 µM) and binding affinities (Kd = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against Mpro (IC50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PLpro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.


Assuntos
Venenos de Crotalídeos/química , Dimerização , Papaína/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , SARS-CoV-2/enzimologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Simulação de Acoplamento Molecular , Papaína/química , Papaína/metabolismo , Peptídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Conformação Proteica , SARS-CoV-2/efeitos dos fármacos
3.
J Med Chem ; 64(9): 6085-6136, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33876936

RESUMO

Dihydroorotate dehydrogenase (DHODH) has been clinically validated as a target for the development of new antimalarials. Experience with clinical candidate triazolopyrimidine DSM265 (1) suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa. We describe a structure-based computationally driven lead optimization program of a pyrrole-based series of DHODH inhibitors, leading to the discovery of two candidates for potential advancement to preclinical development. These compounds have improved physicochemical properties over prior series frontrunners and they show no time-dependent CYP inhibition, characteristic of earlier compounds. Frontrunners have potent antimalarial activity in vitro against blood and liver schizont stages and show good efficacy in Plasmodium falciparum SCID mouse models. They are equally active against P. falciparum and Plasmodium vivax field isolates and are selective for Plasmodium DHODHs versus mammalian enzymes.


Assuntos
Antimaláricos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirróis/farmacologia , Animais , Antimaláricos/química , Inibidores Enzimáticos/química , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Pirróis/química , Relação Estrutura-Atividade
4.
ACS Infect Dis ; 7(6): 1680-1689, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33929818

RESUMO

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner 1 and its active enantiomer 1-S exhibited low-double-digit nanomolar activity against resistant Plasmodium falciparum (Pf) laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of Pf and P. vivax. The slow killing profile and the relative high propensity to develop resistance in vitro (minimum inoculum resistance of 8 × 105 parasites at a selection pressure of 3 × IC50) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of Pf malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target Plasmodium PRS.


Assuntos
Aminoacil-tRNA Sintetases , Antimaláricos , Malária Falciparum , Malária , Animais , Antimaláricos/farmacologia , Humanos , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Camundongos , Plasmodium falciparum
5.
Nat Prod Res ; : 1-7, 2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33703954

RESUMO

Malaria is an infectious illness, affecting vulnerable populations in Third World countries. Inspired by natural products, indole alkaloids have been used as a nucleus to design new antimalarial drugs. So, eighteen oxindole derivatives, aza analogues were obtained with moderate to excellent yields. Also, the saturated derivatives of oxindole and aza derivatives via H2/Pd/C reduction were obtained in good yields, leading to racemic mixtures of each compound. Next, the inhibitory activity against P. falciparum of 18 compounds were tested, founding six compounds with IC50 < 20 µM. The most active of these compounds was 8c; however, their unsaturated derivative 7c was inactive. Then, a structure-activity relationship analysis was done, founding that focused LUMO lobe on the specific molecular zone is related to inhibitory activity against P. falciparum. Finally, we found a potential inhibition of lactate dehydrogenase by oxindole derivatives, using molecular docking virtual screening.

6.
Bioorg Chem ; 109: 104719, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636437

RESUMO

Although the widespread epidemic of Zika virus (ZIKV) and its neurological complications are well-known there are still no approved drugs available to treat this arboviral disease or vaccine to prevent the infection. Flavonoids from Pterogyne nitens have already demonstrated anti-flavivirus activity, although their target is unknown. In this study, we virtually screened an in-house database of 150 natural and semi-synthetic compounds against ZIKV NS2B-NS3 protease (NS2B-NS3p) using docking-based virtual screening, as part of the OpenZika project. As a result, we prioritized three flavonoids from P. nitens, quercetin, rutin and pedalitin, for experimental evaluation. We also used machine learning models, built with Assay Central® software, for predicting the activity and toxicity of these flavonoids. Biophysical and enzymatic assays generally agreed with the in silico predictions, confirming that the flavonoids inhibited ZIKV protease. The most promising hit, pedalitin, inhibited ZIKV NS2B-NS3p with an IC50 of 5 µM. In cell-based assays, pedalitin displayed significant activity at 250 and 500 µM, with slight toxicity in Vero cells. The results presented here demonstrate the potential of pedalitin as a candidate for hit-to-lead (H2L) optimization studies towards the discovery of antiviral drug candidates to treat ZIKV infections.


Assuntos
Antivirais/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Zika virus/metabolismo , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flavonas/farmacologia , Aprendizado de Máquina , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Quercetina/farmacologia , Rutina/farmacologia , Serina Endopeptidases , Células Vero
7.
Eur J Med Chem ; 209: 112941, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33158577

RESUMO

In this work, we designed and synthesized 35 new triazolopyrimidine, pyrazolopyrimidine and quinoline derivatives as P. falciparum inhibitors (3D7 strain). Thirty compounds exhibited anti-P. falciparum activity, with IC50 values ranging from 0.030 to 9.1 µM. The [1,2,4]triazolo[1,5-a]pyrimidine derivatives were more potent than the pyrazolo[1,5-a]pyrimidine and quinoline analogues. Compounds 20, 21, 23 and 24 were the most potent inhibitors, with IC50 values in the range of 0.030-0.086 µM and were equipotent to chloroquine. In addition, the compounds were selective, showing no cytotoxic activity against the human hepatoma cell line HepG2. All [1,2,4]triazolo[1,5-a]pyrimidine derivatives inhibited PfDHODH activity in the low micromolar to low nanomolar range (IC50 values of 0.08-1.3 µM) and did not show significant inhibition against the HsDHODH homologue (0-30% at 50 µM). Molecular docking studies indicated the binding mode of [1,2,4]triazolo[1,5-a]pyrimidine derivatives to PfDHODH, and the highest interaction affinities for the PfDHODH enzyme were in agreement with the in vitro experimental evaluation. Thus, the most active compounds against P. falciparum parasites 20 (R = CF3, R1 = F; IC50 = 0.086 µM), 21 (R = CF3; R1 = CH3; IC50 = 0.032 µM), 23, (R = CF3, R1 = CF3; IC50 = 0.030 µM) and 24 (R = CF3, 2-naphthyl; IC50 = 0.050 µM) and the most active inhibitor against PfDHODH 19 (R = CF3, R1 = Cl; IC50 = 0.08 µM - PfDHODH) stood out as new lead compounds for antimalarial drug discovery. Their potent in vitro activity against P. falciparum and the selective inhibition of the PfDHODH enzyme strongly suggest that this is the mechanism of action underlying this series of new [1,2,4]triazolo[1,5-a]pyrimidine derivatives.


Assuntos
Antimaláricos/síntese química , Inibidores Enzimáticos/química , Malária Falciparum/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirimidinas/síntese química , Quinolinas/síntese química , Triazóis/síntese química , Antimaláricos/química , Antimaláricos/farmacologia , Cloroquina/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Plasmodium falciparum/efeitos dos fármacos , Ligação Proteica , Pirimidinas/farmacologia , Quinolinas/farmacologia , Relação Estrutura-Atividade , Triazóis/farmacologia
8.
Drug Dev Res ; 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32045013

RESUMO

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium and transmitted by Anopheles spp. mosquitos. Due to the emerging resistance to currently available drugs, great efforts must be invested in discovering new molecular targets and drugs. N-myristoyltransferase (NMT) is an essential enzyme to parasites and has been validated as a chemically tractable target for the discovery of new drug candidates against malaria. In this work, 2D and 3D quantitative structure-activity relationship (QSAR) studies were conducted on a series of benzothiophene derivatives as P. falciparum NMT (PfNMT) and human NMT (HsNMT) inhibitors to shed light on the molecular requirements for inhibitor affinity and selectivity. A combination of Quantitative Structure-activity Relationship (QSAR) methods, including the hologram quantitative structure-activity relationship (HQSAR), comparative molecular field analysis (CoMFA), and comparative molecular similarity index analysis (CoMSIA) models, were used, and the impacts of the molecular alignment strategies (maximum common substructure and flexible ligand alignment) and atomic partial charge methods (Gasteiger-Hückel, MMFF94, AM1-BCC, CHELPG, and Mulliken) on the quality and reliability of the models were assessed. The best models exhibited internal consistency and could reasonably predict the inhibitory activity against both PfNMT (HQSAR: q2 /r2 /r2 pred = 0.83/0.98/0.81; CoMFA: q2 /r2 /r2 pred = 0.78/0.97/0.86; CoMSIA: q2 /r2 /r2 pred = 0.74/0.95/0.82) and HsNMT (HQSAR: q2 /r2 /r2 pred = 0.79/0.93/0.74; CoMFA: q2 /r2 /r2 pred = 0.82/0.98/0.60; CoMSIA: q2 /r2 /r2 pred = 0.62/0.95/0.56). The results enabled the identification of the polar interactions (electrostatic and hydrogen-bonding properties) as the major molecular features that affected the inhibitory activity and selectivity. These findings should be useful for the design of PfNMT inhibitors with high affinities and selectivities as antimalarial lead candidates.

9.
Bioorg Med Chem ; 28(2): 115252, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31864777

RESUMO

The mosquito Aedes aegypti is the vector of arboviruses such as Zika, Chikungunya, dengue and yellow fever. These infectious diseases have a major impact on public health. The unavailability of effective vaccines or drugs to prevent or treat most of these diseases makes vector control the main form of prevention. One strategy to promote mosquito population control is the use of synthetic insecticides to inhibit key enzymes in the metabolic pathway of these insects, particularly during larval stages. One of the main targets of the kynurenine detoxification pathway in mosquitoes is the enzyme 3-hydroxykynurenine transaminase (HKT), which catalyzes the conversion of 3-hydroxykynurenine (3-HK) into xanthurenic acid (XA). In this work, we report eleven newly synthesized oxadiazole derivatives and demonstrate that these compounds are potent noncompetitive inhibitors of HKT from Ae. aegypti. The present data provide direct evidence that HKT can be explored as a molecular target for the discovery of novel larvicides against Ae. aegypti. More importantly, it ensures that structural information derived from the HKT 3D-structure can be used to guide the development of more potent inhibitors.


Assuntos
Aedes/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Oxidiazóis/farmacologia , Transaminases/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Relação Estrutura-Atividade , Transaminases/metabolismo
10.
Nat Prod Res ; 34(23): 3423-3427, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30784314

RESUMO

Dermatophytosis is a dermic disease caused by fungi. The aim of this study was to search anti-dermatophyte bioactive compounds in Piper umbellatum leaves. Cytotoxicity evaluation was performed against MRC-5 and HepG2 as a selectivity parameter. Crude ethanol extract presented MIC value of 39.1 µg/mL against M. canis and no cytotoxicity to Hep G2 (human liver cancer) and MRC-5 (normal lung fibroblast). 4-nerolydilcatechol was isolated from P. umbellatum ethanolic extract. MIC values for 4-NC were 7.6µM to M. canisand 15.6µM to Trichophyton rubrum. 4-NC presented activity against M. canis14 times lower than to MRC-5 (non-tumoral human cell line), which suggest selective activity for this fungus. Molecular modeling suggests 4-NC could bind to CYP51, present in lanosterol synthesis, blocking fungi development. In conclusion, P. umbellatum crude ethanol extract and 4-NC demonstrated high and selective in vitro antifungal activity.[Formula: see text].


Assuntos
Antifúngicos/farmacologia , Arthrodermataceae/efeitos dos fármacos , Catecóis/farmacologia , Microsporum/efeitos dos fármacos , Piper/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Bioensaio , Domínio Catalítico , Catecóis/química , Catecóis/metabolismo , Dermatomicoses/microbiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Etanol/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Extratos Vegetais/química , Folhas de Planta/química , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/metabolismo
11.
ACS Med Chem Lett ; 10(1): 137-141, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30655961

RESUMO

Malaria is a major tropical disease where important needs are to mitigate symptoms and to prevent the establishment of infection. Cyclopeptides containing N-methyl amino acids with in vitro activity against erythrocytic forms as well as liver stage are presented. The synthesis, parasitological characterization, physicochemical properties, in vivo evaluation, and mice pharmacokinetics are described.

12.
Curr Med Chem ; 26(23): 4380-4402, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-28875841

RESUMO

Malaria remains a major health problem, especially because of the emergence of resistant P. falciparum strains to artemisinin derivatives. In this context, safe and affordable antimalarial drugs are desperately needed. New proteins have been investigated as molecular targets for research and development of innovative compounds with welldefined mechanism of action. In this review, we highlight genetically and clinically validated plasmodial proteins as drug targets for the next generation of therapeutics. The enzymes described herein are involved in hemoglobin hydrolysis, the invasion process, elongation factors for protein synthesis, pyrimidine biosynthesis, post-translational modifications such as prenylation, phosphorylation and histone acetylation, generation of ATP in mitochondrial metabolism and aminoacylation of RNAs. Significant advances on proteomics, genetics, structural biology, computational and biophysical methods provided invaluable molecular and structural information about these drug targets. Based on this, several strategies and models have been applied to identify and improve lead compounds. This review presents the recent progresses in the discovery of antimalarial drug candidates, highlighting the approaches, challenges, and perspectives to deliver affordable, safe and low single-dose medicines to treat malaria.


Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/química , Humanos
13.
Mol Biotechnol ; 60(8): 595-600, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29951736

RESUMO

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).


Assuntos
Leishmania braziliensis/genética , Superóxido Dismutase/genética , Brasil , Clonagem Molecular/métodos , Humanos , Peróxido de Hidrogênio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia
14.
J Nat Prod ; 81(1): 188-202, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29297684

RESUMO

The isolation and identification of a series of new pseudoceratidine (1) derivatives from the sponge Tedania brasiliensis enabled the evaluation of their antiparasitic activity against Plasmodium falciparum, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) infantum, and Trypanosoma cruzi, the causative agents of malaria, cutaneous leishmaniasis, visceral leishmaniasis, and Chagas disease, respectively. The new 3-debromopseudoceratidine (4), 20-debromopseudoceratidine (5), 4-bromopseudoceratidine (6), 19-bromopseudoceratidine (7), and 4,19-dibromopseudoceratidine (8) are reported. New tedamides A-D (9-12), with an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxamide moiety, are also described. Compounds 4 and 5, 6 and 7, 9 and 10, and 11 and 12 have been isolated as pairs of inseparable structural isomers differing in their sites of bromination or oxidation. Tedamides 9+10 and 11+12 were obtained as optically active pairs, indicating an enzymatic formation rather than an artifactual origin. N12-Acetylpseudoceratidine (2) and N12-formylpseudoceratidine (3) were obtained by derivatization of pseudoceratidine (1). The antiparasitic activity of pseudoceratidine (1) led us to synthesize 23 derivatives (16, 17, 20, 21, 23, 25, 27-29, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51) with variations in the polyamine chain and aromatic moiety in sufficient amounts for biological evaluation in antiparasitic assays. The measured antimalarial activity of pseudoceratidine (1) and derivatives 4, 5, 16, 23, 25, 31, and 50 provided an initial SAR evaluation of these compounds as potential leads for antiparasitics against Leishmania amastigotes and against P. falciparum. The results obtained indicate that pseudoceratidine represents a promising scaffold for the development of new antimalarial drugs.


Assuntos
Alcaloides/química , Alcaloides/farmacologia , Antiparasitários/química , Antiparasitários/farmacologia , Poríferos/química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Relação Estrutura-Atividade , Trypanosoma cruzi/efeitos dos fármacos
15.
PLoS One ; 12(10): e0186869, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29088253

RESUMO

Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC50 < 20 µM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 µM. Two compounds exhibited IC50 values below 5 µM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects.


Assuntos
Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Simulação de Acoplamento Molecular/métodos , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Domínio Catalítico , Catepsina K/química , Catepsina K/metabolismo , Células Cultivadas , Colagenases/química , Colagenases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Estrutura Molecular , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligação Proteica , Domínios Proteicos
16.
Planta Med ; 83(11): 912-920, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28264205

RESUMO

This is a comparative study on the intraspecific chemical variability of Aristolochia cordigera species, collected in two different regions of Brazil, Biome Cerrado (semiarid) and Biome Amazônia (coastal). The use of GC-MS and statistical methods led to the identification of 56 compounds. A higher percentage of palmitone and germacrene-D in the hexanes extracts of the leaves of plants from these respective biomes was observed. Phytochemical studies on the extracts led to the isolation and identification of 19 known compounds, including lignans, neolignans, aristolochic acids, indole-ß-carboline, and indole alkaloids. In addition, two new indole alkaloids, 3,4-dihydro-hyrtiosulawesine and 6-O-(ß-glucopyranosyl)hyrtiosulawesine, were isolated and a new neolignan, cis-eupomatenoid-7, was obtained in a mixture with its known isomer eupomatenoid-7. Their structures were determined by spectroscopic methods, mainly by 1D- and 2D-NMR. The occurrence of indole alkaloids is being described for the first time in the Aristolochiaceae family. Moreover, the in vitro susceptibility of intracellular amastigote and promastigote forms of Leishmania amazonensis to the alkaloids and eupomatenoid-7 were evaluated. This neolignan exhibited low activity against promastigotes (IC50 = 46 µM), while the alkaloids did not show inhibitory activity. The new alkaloid 6-O-(ß-glucopyranosyl)hyrtiosulawesine exhibited activity in the low micromolar range against Plasmodium falciparum, with an IC50 value of 5 µM and a selectivity index higher than 50.


Assuntos
Antiprotozoários/farmacologia , Aristolochia/química , Citotoxinas/farmacologia , Alcaloides Indólicos/farmacologia , Lignanas/farmacologia , Extratos Vegetais/farmacologia , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Brasil , Linhagem Celular Tumoral , Citotoxinas/química , Citotoxinas/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Alcaloides Indólicos/química , Alcaloides Indólicos/isolamento & purificação , Leishmania/efeitos dos fármacos , Lignanas/química , Lignanas/isolamento & purificação , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Plasmodium falciparum/efeitos dos fármacos
17.
Nat Commun ; 8: 14764, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28345596

RESUMO

The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution.


Assuntos
RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Zika virus/enzimologia , Antivirais/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas , Conformação Proteica , Recombinação Genética , Zika virus/efeitos dos fármacos , Zika virus/genética
18.
Sci Rep ; 6: 36858, 2016 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-27874020

RESUMO

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to ß-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.


Assuntos
Transferases Intramoleculares/metabolismo , Maytenus/enzimologia , Proteínas de Plantas/metabolismo , Triterpenos/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Transferases Intramoleculares/química , Transferases Intramoleculares/classificação , Transferases Intramoleculares/genética , Leucina/química , Leucina/metabolismo , Maytenus/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Alinhamento de Sequência , Triterpenos/análise , Triterpenos/química
19.
Sci Rep ; 6: 22093, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26915471

RESUMO

In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.


Assuntos
Biliverdina/metabolismo , Eritrócitos/parasitologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fosfopiruvato Hidratase/antagonistas & inibidores , Plasmodium falciparum/metabolismo , Sequência de Aminoácidos , Biliverdina/farmacologia , Eritrócitos/metabolismo , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Malária Falciparum/metabolismo , Modelos Moleculares , Proteínas de Protozoários/antagonistas & inibidores , Alinhamento de Sequência
20.
J Comput Biol ; 23(1): 27-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26540331

RESUMO

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.


Assuntos
Clonagem Molecular/métodos , Primers do DNA/genética , Mutagênese Sítio-Dirigida/métodos , Software , Algoritmos , Sequência de Bases
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