RESUMO
BACKGROUND: This study investigates the potential of eleven 1H-1,2,3-triazol-1,4-naphthoquinone conjugates as virulence factor inhibitors (like Pyocyanin) and their affinity for PhzM, a crucial enzyme for Pyocyanin biosynthesis in Pseudomonas aeruginosa infections. METHODS: A straightforward synthetic pathway enabled the production of these compounds, which were characterized and structurally confirmed through spectroscopic analyses. Evaluation of their impact on PhzM thermal stability identified promising candidates for PhzM binders. RESULTS: Concentration-response behavior elucidated their binding affinity, revealing them as the first reported micromolar affinity ligands for PhzM. Structure-activity relationship analysis emphasized the role of specific molecular moieties in binding affinity modulation, paving the way for future advanced inhibitors' development. CONCLUSION: These findings highlight the potential of naphthoquinone-triazole derivatives as leads for novel therapeutics against P. aeruginosa infections.
RESUMO
Tonantzitlolone B (TZL-B) is a diterpene isolated from the roots of Stillingia loranthacea. Its antinociceptive effects were investigated in male Swiss mice using the following models of pain: formalin test, inflammation induced by Complete Freund's Adjuvant (CFA), tail flick test, and cold plate test. The influence of TZL-B on the opioid system was assessed in vivo, using opioid antagonists; in silico, investigating the chemical similarity among TZL-B and opioid agonists; and ex vivo, measuring preproenkephalin (PENK) gene expression in the spinal cord by RT-qPCR. TZL-B (10-1000 µg/kg) promoted antinociception in the four experimental models without impairing mice's motor function. TZL-B did not alter paw edema during CFA-induced inflammation. The antinociceptive effects of TZL-B in the tail flick and cold plate tests were diminished by the opioid antagonists naloxone (5 mg/kg), NOR-BNI (0.5 mg/kg), naltrindole (3 mg/kg), and CTOP (1 mg/kg), indicating the involvement of κ-, δ-, and µ-opioid receptors. TZL-B showed no significant chemical similarity to opioid agonists, but the treatment with TZL-B (1000 µg/kg) increased PENK gene expression in the spinal cord of mice. These data suggest that TZL-B promotes antinociception by enhancing the transcription of PENK, hence modulating the endogenous opioid system.
Assuntos
Analgésicos Opioides , Diterpenos , Camundongos , Masculino , Animais , Analgésicos Opioides/farmacologia , Antagonistas de Entorpecentes/farmacologia , Diterpenos/farmacologia , Receptores Opioides mu , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Receptores Opioides kappaRESUMO
BACKGROUND: Dihydroorotate dehydrogenase (DHODH) has long been recognized as an important drug target for proliferative and parasitic diseases, including compounds that exhibit trypanocidal action and broad-spectrum antiviral activity. Despite numerous and successful efforts in structural and functional characterization of DHODHs, as well as in the development of inhibitors, DHODH hot spots remain largely unmapped and underexplored. OBJECTIVE: This review describes the tools that are currently available for the identification and characterization of hot spots in protein structures and how freely available webservers can be exploited to predict DHODH hot spots. Moreover, it provides for the first time a review of the antiviral properties of DHODH inhibitors. METHODS: X-ray structures from human (HsDHODH) and Trypanosoma cruzi DHODH (TcDHODH) had their hot spots predicted by both FTMap and Fragment Hotspot Maps web servers. RESULTS: FTMap showed that hot spot occupancy in HsDHODH is correlated with the ligand efficiency (LE) of its known inhibitors, and Fragment Hotspot Maps pointed out the contribution of selected moieties to the overall LE. The conformational flexibility of the active site loop in TcDHODH was found to have a major impact on the druggability of the orotate binding site. In addition, both FTMap and Fragment Hotspot Maps servers predict a novel pocket in TcDHODH dimer interface (S6 site). CONCLUSION: This review reports how hot spots can be exploited during hit-to-lead steps, docking studies or even to improve inhibitor binding profile and by doing so using DHODH as a model, points to new drug development opportunities.
Assuntos
Di-Hidro-Orotato Desidrogenase/antagonistas & inibidores , Di-Hidro-Orotato Desidrogenase/química , Desenvolvimento de Medicamentos/tendências , Antivirais , Di-Hidro-Orotato Desidrogenase/metabolismo , Humanos , Trypanosoma cruzi/enzimologiaRESUMO
The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechanism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results provided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydroquinazoline ring, found in the previous hit (A- Kd = 18 µM and LE = 0.20), is not required for PaPzhS inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach, pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-dione) with 10-fold improvement in affinity (Kd= 1.68 µM) and more than 100% increase in LE (0.45), which follows the same inhibition mode as the original hit compound (competitive to NADH).Executive summaryPhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa.Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS with one-digit micromolar affinity.Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS.The molecular simplification optimised the ligand efficiency and affinity of the lead compound.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Humanos , Ligantes , Tiazolidinedionas/síntese químicaRESUMO
Assessment of target druggability guided by search and characterization of hot spots is a pivotal step in early stages of drug-discovery. The raw output of FTMap provides the data to perform this task, but it relies on manual intervention to properly combine different sets of consensus sites, therefore allowing identification of hot spots and evaluation of strength, shape and distance among them. Thus, the user's previous experience on the target and the software has a direct impact on how data generated by FTMap server can be explored. DRUGpy plugin was developed to overcome this limitation. By automatically assembling and scoring all possible combinations of consensus sites, DRUGpy plugin provides FTMap users a straight-forward method to identify and characterize hot spots in protein targets. DRUGpy is available in all operating systems that support PyMOL software. DRUGpy promptly identifies and characterizes pockets that are predicted by FTMap to bind druglike molecules with high-affinity (druggable sites) or low-affinity (borderline sites) and reveals how protein conformational flexibility impacts on the target's druggability. The use of DRUGpy on the analysis of trypanothione reductases (TR), a validated drug target against trypanosomatids, showcases the usefulness of the plugin, and led to the identification of a druggable pocket in the conserved dimer interface present in this class of proteins, opening new perspectives to the design of selective inhibitors.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , NADH NADPH Oxirredutases/antagonistas & inibidores , Software , Sítios de Ligação , Inibidores Enzimáticos/química , Humanos , Ligantes , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
The proper pharmacological control of pain is a continuous challenge for patients and health care providers. Even the most widely used medications for pain treatment are still ineffective or unsafe for some patients, especially for those who suffer from chronic pain. Substances containing the chromone scaffold have shown a variety of biological activities, including analgesic effects. This work presents for the first time the centrally mediated antinociceptive activity of 5-O-methylcneorumchromone K (5-CK). Cold plate and tail flick tests in mice showed that the 5-CK-induced antinociception was dose-dependent, longer-lasting, and more efficacious than that induced by morphine. The 5-CK-induced antinociception was not reversed by the opioid antagonist naloxone. Topological descriptors (fingerprints) were employed to narrow the antagonist selection to further investigate 5-CK's mechanism of action. Next, based on the results of fingerprints analysis, functional antagonist assays were conducted on nociceptive tests. The effect of 5-CK was completely reversed in both cold plate and tail-flick tests by GABAA receptor antagonist bicuculline, but not by atropine or glibenclamide. Molecular docking studies suggest that 5-CK binds to the orthosteric binding site, with a similar binding profile to that observed for bicuculline and GABA. These results evidence that 5-CK has a centrally mediated antinociceptive effect, probably involving the activation of GABAergic pathways.
Assuntos
Analgésicos/farmacologia , Cromonas/farmacologia , Antagonistas GABAérgicos/farmacologia , Analgésicos/química , Animais , Sítios de Ligação , Cromonas/química , Antagonistas GABAérgicos/química , Camundongos , Simulação de Acoplamento Molecular , Nociceptividade , Ligação Proteica , Receptores de GABA/química , Receptores de GABA/metabolismoRESUMO
BACKGROUND: Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the terminal step of prostaglandin E2 (PGE2) production, which plays an important role in the regulation of febrile response. In our previous work, ligand-based pharmacophore models, built with mPGES-1 inhibitors, were employed to identify a novel series of compounds that reduce the febrile response in rats. OBJECTIVES: The study aimed to evaluate the mechanism of action of the most active compound (1). METHODS: For in vivo assays, rats were pretreated with the antipyretic compounds 1-8, 30 min before LPS injection. For in vitro assays, RAW 264.7 macrophage cells were incubated with the antipyretic compounds 1-8 for 1 hour before LPS stimulus. After 16 h, quantitative real-time PCR was carried out. Additionally, the PGE2 concentration in the hypothalamus was quantified by ELISA and the inhibitory effect of N-cyclopentyl-N'-[3-(3-cyclopropyl-1H-1,2,4-triazol- 5-yl)phenyl]ethanediamide (1) over human COX-2 enzymatic activity was determined with a COX Colorimetric Inhibitor Screening Assay Kit. RESULTS: Compound 1 and CAY10526 showed comparable efficacy to reduce the febrile response when injected i.v. (compound 1: 63.10%, CAY10526: 70.20%). Moreover, compound 1 significantly reduced the mPGES-1 mRNA levels, in RAW264.7 cells, under inflammatory conditions. A chemically-similar compound (8-) also significantly reduced the mRNA levels of the gene target. On the other hand, compounds 6 and 7, which are also somewhat similar to compound 1, did not significantly impact mPGES-1 mRNA levels. CONCLUSIONS: PGE2 concentration reduction in the hypothalamus, due to compound 1 central injection, is related to decreased mPGES-1 mRNA levels but not to COX-2 inhibition (IC50> 50 µM). Therefore, compound 1 is a promising lead for innovative antipyretic drug development.
Assuntos
Antipiréticos , Macrófagos , Prostaglandina-E Sintases , RNA Mensageiro , Animais , Antipiréticos/farmacologia , Ciclo-Oxigenase 2/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Prostaglandina-E Sintases/antagonistas & inibidores , Prostaglandina-E Sintases/genética , Células RAW 264.7 , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/economia , RatosRESUMO
Coumarins exhibit a wide variety of biological effects, including activities in the cardiovascular system and the aim of this study was to evaluate the vascular therapeutic potential of 7-Hydroxicoumarin (7-HC). The vascular effects induced by 7-HC (0.001 µM-300 µM), were investigated by in vitro approaches using isometric tension measurements in rat superior mesenteric arteries and by in silico assays using Ligand-based analysis. Our results suggest that the vasorelaxant effect of 7-HC seems to rely on potassium channels, notably through large conductance Ca2+-activated K+ (BKCa) channels activation. In fact, 7-HC (300 µM) significantly reduced CaCl2-induced contraction as well as the reduction of intracellular calcium mobilization. However, the relaxation induced by 7-HC was independent of store-operated calcium entry (SOCE). Moreover, in silico analysis suggests that potassium channels have a common binding pocket, where 7-HC may bind and hint that its binding profile is more similar to quinine's than verapamil's. These results are compatible with the inhibition of Ca2+ release from intracellular stores, which is prompted by phenylephrine and caffeine. Taken together, these results demonstrate a therapeutic potential of 7-HC on the cardiovascular system, making it a promising lead compound for the development of drugs useful in the treatment of cardiovascular diseases.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Artérias Mesentéricas/efeitos dos fármacos , Umbeliferonas/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Sinalização do Cálcio/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Masculino , Artérias Mesentéricas/fisiologia , Técnicas de Cultura de Órgãos , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Vasodilatação/fisiologiaRESUMO
Aim: Although bacterial resistance is a growing concern worldwide, the development of antibacterial drugs has been steadily decreasing. One alternative to fight this issue relies on reducing the bacteria virulence without killing it. PhzS plays a pivotal role in pyocyanin production in Pseudomonas aeruginosa. Results: A total of 31 thiazolidinedione derivatives were evaluated as putative PhzS inhibitors, using thermo shift assays. Compounds that significantly shifted PhzS's Tm had their mode of inhibition (cofactor competitor) and affinity calculated by thermo shift assays as well. The most promising compound (E)-5-(4-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methoxy)benzylidene)thiazolidine-2,4-dione had their affinity confirmed by microscale thermophoresis (Kd = 18 µM). Cellular assays suggest this compound reduces pyocyanin production in vitro, but does not affect P. aeruginosa viability. Conclusion: The first inhibitor of PhzS is described.
Assuntos
Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/metabolismo , Piocianina/biossíntese , Relação Estrutura-AtividadeRESUMO
Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC50 = 23.45 ± 4.54 µM), whereas 2a is the most potent against L. braziliensis (EC50 = 44.16 ± 5.77 µM). This result suggests that lipophilic substituents on either-meta and/or-para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE50 = 49.22 ± 7.71 µM) presented the highest selectivity index.
Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Animais , Antiprotozoários/química , Humanos , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Oxirredutases/antagonistas & inibidores , Testes de Sensibilidade Parasitária , Tiazolidinedionas/químicaRESUMO
Although bacterial resistance is a worldwide growing concern, the development of bacteriostatic and bactericidal drugs has been decreasing in the last decade. Compounds that modulate the microorganism virulence, without killing it, have been considered promising alternatives to combat bacterial infections. However, most signaling pathways that regulate virulence are complex and not completely understood. The rich chemical diversity of natural products offers a good starting point to identify key compounds that shed some light on this matter. Therefore, we investigated the role of Marcetia latifolia ethanolic extract, as well as its major constituent, calycopterin (5,4'-dihydroxy-3,6,7,8-tetramethoxylflavone), in the regulation of virulence-related phenotypes of Pseudomonas aeruginosa. Our results show that calycopterin inhibits pyocyanin production (EC50 = 32 µM), reduces motility and increases biofilm formation in a dose-dependent manner. Such biological profile suggests that calycopterin modulates targets that may act upstream the quorum sensing regulators and points to its utility as a chemical probe to further investigate P. aeruginosa transition from planktonic to sessile lifestyle.
Assuntos
Antibacterianos/farmacologia , Flavonas/farmacologia , Locomoção/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Biofilmes/efeitos dos fármacos , Melastomataceae/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Piocianina/biossíntese , Percepção de Quorum/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).
Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania infantum/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração Inibidora 50 , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismoRESUMO
Leishmaniasis is considered as one of the major neglected tropical diseases due to its magnitude and wide geographic distribution. Leishmania braziliensis, responsible for cutaneous leishmaniasis, is the most prevalent species in Brazil. Superoxide dismutase (SOD) belongs to the antioxidant pathway of the parasites and human host. Despite the differences between SOD of Leishmania braziliensis and human make this enzyme a promising target for drug development efforts. No medicinal chemistry effort has been made to identify LbSOD inhibitors. Herein, we show that thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) can be employed as primary and secondary screens to achieve this goal. Moreover, we show that thiazole derivatives bind to LbSOD with micromolar affinity.
Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania braziliensis/enzimologia , Superóxido Dismutase/antagonistas & inibidores , Tiazóis/farmacologia , Brasil , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Superóxido Dismutase/metabolismo , Tiazóis/síntese química , Tiazóis/químicaRESUMO
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.
Assuntos
Antagonistas do Ácido Fólico/análise , Antagonistas do Ácido Fólico/química , Modelos Moleculares , Pirimidinas/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Previous studies have shown that cowpea protein positively interferes with cholesterol metabolism. In this study, we evaluated the ability of the fraction containing peptides of <3â¯kDa, as well as that of the Gln-Asp-Phe (QDF) peptide, derived from cowpea ß-vignin protein, to inhibit HMG-CoA reductase activity. We established isolation and chromatography procedures to effectively obtain the protein with a purity above 95%. In silico predictions were performed to identify peptide sequences capable of interacting with HMG-CoA reductase. In vitro experiments showed that the fraction containing peptides of <3â¯kDa displayed inhibition of HMG-CoA reductase activity. The tripeptide QDF inhibits HMG-CoA reductase (IC50â¯=â¯12.8⯵M) in a dose-dependent manner. Furthermore, in silico studies revealed the binding profile of the QDF peptide and hinted at the molecular interactions that are responsible for its activity. Therefore, this study shows, for the first time, a peptide from cowpea ß-vignin protein that inhibits HMG-CoA reductase and the chemical modifications that should be investigated to evaluate its binding profile.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Peptídeos/metabolismo , Vigna/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Humanos , Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Simulação de Acoplamento Molecular , Peptídeos/análise , Peptídeos/químicaRESUMO
Adenosine receptors are considered as potential targets for drug development against several diseases. The discovery of subtype 2B adenosine receptors role in erythrocyte sickling process proved its importance to neglected diseases such as sickle cell anemia, which affects approximately 29.000 people around the world, but whose treatment is restricted to just one FDA approved drug (hydroxyurea). In order to widen the therapeutic arsenal available to treat sickle cell anemia patients, it is imperative to identify new lead compounds that modify the sickling course and not just its symptoms. In order to accomplish this goal, ligand-based pharmacophore models that differentiate true ligands from decoys and enlighten the structure-activity relationship of known RA2B antagonists were employed screen the lead-like subset of the ZINC database. Following a chemical diversity analysis, 18 compounds were selected for biological evaluation. Among them, one molecule Z1139491704 (pEC50 = 7.77 ± 0.17) has shown better anti-sickling activity than MRS1754 (pEC50 = 7.63 ± 0.12), a commercial RA2B antagonist. Moreover, these compounds exhibited no cytotoxic effect at low micromolar range on mammalian cells. In conclusion, the sound development of validated ligand-based pharmacophore models proved essential to identify novel chemical scaffolds that might be useful to develop anti-sickling drugs.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Anemia Falciforme/tratamento farmacológico , Receptor A2B de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Ligantes , Macrófagos/efeitos dos fármacos , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Despite the fact that Leishmania ssp are pteridine auxotrophs, Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) inhibitors are ineffective against Leishmania major. On the other hand Pteridine Reductase 1 (PTR1) inhibitors proved to be lethal to the parasite. Aiming at identifying hits that lie outside the chemical space of known PTR1 inhibitors, pharmacophore models that differentiate true-binders from decoys and explain the structure-activity relationships of known inhibitors were employed to virtually screen the lead-like subset of ZINC database. This approach leads to the identification of Z80393 (IC50 = 32.31 ± 1.18 µM), whose inhibition mechanism was investigated by Thermal Shift Assays. This experimental result supports a competitive mechanism and was crucial to establish the docking search space as well as select the best pose, which was then investigated by molecular dynamics studies that corroborate the hit putative binding profile towards LmPTR1. The information gathered from such studies shall be useful to design more potent non-nucleoside LmPTR1 inhibitors.
Assuntos
Leishmania major/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Descoberta de Drogas , Leishmania major/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Due to the absence of safety of the antipyretics to patients with cardiovascular dysfunction, new targets to treat inflammation have been pursued. mPGES-1 is a promising target because its inhibition would not cause the side-effects related to COX inhibition. To identify novel inhibitors of mPGES-1, we developed a ligand-based pharmacophore model that differentiates true inhibitors from decoys and enlightens the structure-activity relationships for known mPGES-1 inhibitors. The model (four hydrophobic centers, two hydrogen bond acceptor and two hydrogen bond donor points) was employed to select lead-like compounds from ZINC database for in vivo evaluation. Among the 18 compounds selected, five inhibited the fever induced by LPS. The most potent compound (5-(4-fluorophenyl)-3-({6-methylimidazo[1,2-a]pyridin-2-yl}methyl)-2,3dihydro-1,3,4-oxadiazol-2-one) is active peripherally (i.v.) or centrally (i.c.v.) (82.18% and 112% reduction, respectively) and reduces (69.13%) hypothalamic PGE2 production, without significant COX-1/2 inhibition. In conclusion, our in silico approach leads to the selection of a compound that presents the chemical features to inhibit mPGES-1 and reduces fever induced by LPS. Furthermore, the in vivo and in vitro results support the hypothesis that its mechanism of action does not depend on COX inhibition. Hence, it can be considered a promising lead compound for antipyretic development, once it would not have the side-effects of COX-1/2 inhibitors.
Assuntos
Antipiréticos/química , Antipiréticos/uso terapêutico , Febre/tratamento farmacológico , Oxidiazóis/química , Oxidiazóis/uso terapêutico , Prostaglandina-E Sintases/antagonistas & inibidores , Animais , Antipiréticos/farmacologia , Dinoprostona/metabolismo , Descoberta de Drogas , Febre/metabolismo , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Ligantes , Masculino , Oxidiazóis/farmacologia , Prostaglandina-E Sintases/metabolismo , Relação Quantitativa Estrutura-Atividade , Ratos WistarRESUMO
Leishmania major, as other protozoan parasites, plague human kind since pre-historic times but it remains a worldwide ailment for which the therapeutic arsenal remains scarce. Although L. major is pteridine- and purine-auxotroph, well-established folate biosynthesis inhibitors, such as methotrexate, have poor effect over the parasite survival. The lack of efficiency is related to an alternative biochemical pathway in which pteridine reductase 1 (PTR1) plays a major role. For this reason, this enzyme has been considered a promising target for anti-leishmanial drug development and several inhibitors that share the substrate scaffold have been reported. In order to design a novel class of PTR1 inhibitors, we employed the thiazolidinone ring as a bioisosteric replacement for pteridine/purine ring. Among seven novel thiazolidine-2,4-dione derivatives reported herein, 2d was identified as the most promising lead by thermal shift assays (ΔTm = 11 °C, p = 0,01). Kinetic assays reveal that 2d has IC50 = 44.67 ± 1.74 µM and shows a noncompetitive behavior. This information guided docking studies and molecular dynamics simulations (50 000 ps) that supports 2d putative binding profile (H-bonding to Ser-111 and Leu-66) and shall be useful to design more potent inhibitors.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Leishmania major/enzimologia , Oxirredutases/antagonistas & inibidores , Tiazolidinedionas/química , Tiazolidinedionas/farmacologia , Modelos Moleculares , Oxirredutases/química , Conformação ProteicaRESUMO
The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.