RESUMO
The human voltage-gated proton channel, hHV1, is highly expressed in various cell types including macrophages, B lymphocytes, microglia, sperm cells and also in various cancer cells. Overexpression of HV1 has been shown to promote tumor formation by highly metastatic cancer cells, and has been associated with neuroinflammatory diseases, immune response disorders and infertility, suggesting a potential use of hHV1 inhibitors in numerous therapeutic areas. To identify compounds targeting this channel, we performed a structure-based virtual screening on an open structure of the human HV1 channel. Twenty selected virtual screening hits were tested on Chinese hamster ovary (CHO) cells transiently expressing hHV1, with compound 13 showing strong block of the proton current with an IC50 value of 8.5 µM. Biological evaluation of twenty-three additional analogs of 13 led to the discovery of six other compounds that blocked the proton current by more than 50% at 50 µM concentration. This allowed for an investigation of structure-activity relationships. The antiproliferative activity of the selected promising hHV1 inhibitors was investigated in the cell lines MDA-MB-231 and THP-1, where compound 13 inhibited growth with an IC50 value of 9.0 and 8.1 µM, respectively. The identification of a new structural class of HV1 inhibitors contributes to our understanding of the structural requirements for inhibition of this ion channel and opens up the possibility of investigating the role of HV1 inhibitors in various pathological conditions and in cancer therapy.
Assuntos
Cricetulus , Canais Iônicos , Humanos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Células CHO , Animais , Relação Estrutura-Atividade , Avaliação Pré-Clínica de Medicamentos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Interface Usuário-Computador , Simulação de Acoplamento MolecularRESUMO
N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.
Assuntos
DNA Girase , Escherichia coli , Inibidores da Topoisomerase II , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/síntese química , DNA Girase/metabolismo , DNA Girase/química , Sítios de Ligação , Escherichia coli/enzimologia , Escherichia coli/efeitos dos fármacos , Relação Estrutura-Atividade , Benzotiazóis/química , Benzotiazóis/farmacologia , Benzotiazóis/síntese química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Estrutura Molecular , Teoria Quântica , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Modelos MolecularesRESUMO
Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins. ULD1 and ULD2 are excellently potent against a broad range of gram-positive bacteria. Notably, the efficacy of these compounds was tested against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. Antibiotics with clinical relevance against staphylococcal infections fail to inhibit a significant fraction of these isolates, whereas both ULD1 and ULD2 inhibit all of them (minimum inhibitory concentration [MIC] ≤1 µg/mL). Resistance mutations against these compounds are rare, have limited impact on compound susceptibility, and substantially reduce bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant bacterial infections.
Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Evolução Molecular Direcionada , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Testes de Sensibilidade Microbiana , Mutação/genética , Pele/efeitos dos fármacos , Pele/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Testes de ToxicidadeRESUMO
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Catepsina Z/metabolismo , Glioblastoma/metabolismo , Fosfopiruvato Hidratase/metabolismo , Microambiente Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Catepsina Z/antagonistas & inibidores , Catepsina Z/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Regulação para CimaRESUMO
Multidrug resistance (MDR) is the dominant cause of the failure of cancer chemotherapy. The design of antitumor drugs that are able to evade MDR is rapidly evolving, showing that this area of biomedical research attracts great interest in the scientific community. The current review explores promising recent approaches that have been developed with the aim of circumventing or overcoming MDR. Encouraging results have been obtained in the investigation of the MDR-modulating properties of various classes of natural compounds and their analogues. Inhibition of P-gp or downregulation of its expression have proven to be the main mechanisms by which MDR can be surmounted. The use of hybrid molecules that are able to simultaneously interact with two or more cancer cell targets is currently being explored as a means to circumvent drug resistance. This strategy is based on the design of hybrid compounds that are obtained either by merging the structural features of separate drugs, or by conjugating two drugs or pharmacophores via cleavable/non-cleavable linkers. The approach is highly promising due to the pharmacokinetic and pharmacodynamic advantages that can be achieved over the independent administration of the two individual components. However, it should be stressed that the task of obtaining successful multivalent drugs is a very challenging one. The conjugation of anticancer agents with nitric oxide (NO) donors has recently been developed, creating a particular class of hybrid that can combat tumor drug resistance. Appropriate NO donors have been shown to reverse drug resistance via nitration of ABC transporters and by interfering with a number of metabolic enzymes and signaling pathways. In fact, hybrid compounds that are produced by covalently attaching NO-donors and antitumor drugs have been shown to elicit a synergistic cytotoxic effect in a variety of drug resistant cancer cell lines. Another strategy to circumvent MDR is based on nanocarrier-mediated transport and the controlled release of chemotherapeutic drugs and P-gp inhibitors. Their pharmacokinetics are governed by the nanoparticle or polymer carrier and make use of the enhanced permeation and retention (EPR) effect, which can increase selective delivery to cancer cells. These systems are usually internalized by cancer cells via endocytosis and accumulate in endosomes and lysosomes, thus preventing rapid efflux. Other modalities to combat MDR are described in this review, including the pharmaco-modulation of acridine, which is a well-known scaffold in the development of bioactive compounds, the use of natural compounds as means to reverse MDR, and the conjugation of anticancer drugs with carriers that target specific tumor-cell components. Finally, the outstanding potential of in silico structure-based methods as a means to evaluate the ability of antitumor drugs to interact with drug transporters is also highlighted in this review. Structure-based design methods, which utilize 3D structural data of proteins and their complexes with ligands, are the most effective of the in silico methods available, as they provide a prediction regarding the interaction between transport proteins and their substrates and inhibitors. The recently resolved X-ray structure of human P-gp can help predict the interaction sites of designed compounds, providing insight into their binding mode and directing possible rational modifications to prevent them from becoming P-gp drug substrates. In summary, although major efforts were invested in the search for new tools to combat drug resistant tumors, they all require further implementation and methodological development. Further investigation and progress in the abovementioned strategies will provide significant advances in the rational combat against cancer MDR.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias/tratamento farmacológico , Tecnologia Farmacêutica/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acridinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Glicoconjugados/química , Humanos , Nanopartículas , Óxido Nítrico/metabolismo , Preparações de Plantas/farmacologia , Preparações de Plantas/uso terapêutico , Polímeros/químicaRESUMO
Quorum sensing (QS), a bacterial communication strategy, has been recognized as one of the control mechanisms of virulence in bacteria. Thus, targeting QS offers an interesting opportunity to impair bacterial pathogenicity and develop antivirulence agents. Aiming to enhance the discovery of QS inhibitors, we developed a bioreporter Escherichia coli JW5505 pET-Plsrlux and set up a cell-based assay for identifying inhibitors of autoinducer-2 (AI-2)-mediated QS. A comparative study on the performance of target- versus cell-based assays was performed, and 91 compounds selected with the potential to target the ATP binding pocket of LsrK, a key enzyme in AI-2 processing, were tested in an LsrK inhibition assay, providing 36 hits. The same set of compounds was tested by the AI-2-mediated QS interference assay, resulting in 24 active compounds. Among those, six were also found to be active against LsrK, whereas 18 might target other components of the pathway. Thus, this AI-2-mediated QS interference cell-based assay is an effective tool for complementing target-based assays, yet also stands as an independent assay for primary screening.
Assuntos
Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/metabolismo , Homosserina/análogos & derivados , Lactonas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Percepção de Quorum , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Homosserina/metabolismo , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor - siderophore mimic conjugates.
Assuntos
Desenho de Fármacos , Mimetismo Molecular , Sideróforos/farmacologia , Tiazóis/química , Inibidores da Topoisomerase II/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Human DNA topoisomerase II is one of the major targets in anticancer therapy, however ATP-competitive inhibitors of this target have not yet reached their full potential. ATPase domain of human DNA topoisomerase II belongs to the GHKL ATPase superfamily and shares a very high 3D structural similarity with other superfamily members, including bacterial topoisomerases. In this work we report the discovery of a new chemotype of ATP-competitive inhibitors of human DNA topoisomerase IIα that were discovered through screening of in-house library of ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV. Systematic screening of this library provided us with 20 hit compounds. 1,2,4-Substituted N-phenylpyrrolamides were selected for a further exploration which resulted in 13 new analogues, including 52 with potent activity in relaxation assay (IC50 = 3.2 µM) and ATPase assay (IC50 = 0.43 µM). Cytotoxic activity of all hits was determined in MCF-7 cancer cell line and the most potent compounds, 16 and 20, showed an IC50 value of 8.7 and 8.2 µM, respectively.
Assuntos
Adenosina Trifosfatases/metabolismo , Antineoplásicos/uso terapêutico , DNA Topoisomerases Tipo II/química , Inibidores da Topoisomerase II/uso terapêutico , Antineoplásicos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Inibidores da Topoisomerase II/farmacologiaRESUMO
The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes. Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Vemurafenib/farmacologiaRESUMO
Discovery of novel DNA gyrase B inhibitors remains an attractive field in the search for new antibacterial drugs to overcome the known bacterial resistance mechanisms. In the present study, we designed and synthesized novel ethylurea derivatives of 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine, 2-(2-aminothiazol-4-yl)acetic acid, and benzo[1,2-d]thiazole-2,6-diamine and evaluated their Escherichia coli DNA gyrase inhibition. The most potent DNA gyrase inhibitors in the prepared library of compounds were benzo[1,2-d]thiazoles 32-34, 36, and 37 with IC50 values in the low micromolar range. The most promising inhibitors identified were evaluated against selected Gram-positive and Gram-negative bacterial strains. Compound 33 showed a MIC of 50 µM against an E. coli efflux pump-defective strain, which suggests that efflux decreases the on-target concentrations of these compounds.
Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Tiazóis/farmacologia , Inibidores da Topoisomerase II/farmacologia , Ureia/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química , Ureia/análogos & derivados , Ureia/químicaRESUMO
Development of novel DNA gyrase B inhibitors is an important field of antibacterial drug discovery whose aim is to introduce a more effective representative of this mechanistic class into the clinic. In the present study, two new series of Escherichia coli DNA gyrase inhibitors bearing the 4,5-dibromopyrrolamide moiety have been designed and synthesized. 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives inhibited E. coli DNA gyrase in the submicromolar to low micromolar range (IC50 values between 0.891 and 10.4µM). Their "ring-opened" analogues, based on the 2-(2-aminothiazol-4-yl)acetic acid scaffold, displayed weaker DNA gyrase inhibition with IC50 values between 15.9 and 169µM. Molecular docking experiments were conducted to study the binding modes of inhibitors.
Assuntos
Acetanilidas/farmacologia , Antibacterianos/farmacologia , Benzotiazóis/farmacologia , Pirróis/farmacologia , Inibidores da Topoisomerase II/farmacologia , Acetanilidas/síntese química , Antibacterianos/síntese química , Benzotiazóis/síntese química , Desenho de Fármacos , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Pirróis/síntese química , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese químicaRESUMO
Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC50 = 20 µM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.
Assuntos
Alcaloides/farmacologia , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Indóis/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pirróis/farmacologia , Alcaloides/síntese química , Alcaloides/química , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirróis/síntese química , Pirróis/química , Relação Estrutura-AtividadeRESUMO
Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC90 (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound's potential as an antimicrobial lead, was found to be 2.9 for compound 6h.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Pirróis/farmacologia , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antifúngicos/química , Antifúngicos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Poríferos/química , Pirróis/química , Pirróis/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacosRESUMO
Clathrodin is a marine alkaloid and believed to be a modulator of voltage-gated sodium (Na(V)) channels. Since there is an urgent need for small molecule Na(V) channel ligands as novel therapeutics, clathrodin could represent an interesting lead compound. Therefore, clathrodin was reinvestigated for its potency and Na(V) channel subtype selectivity. Clathrodin and its synthetic analogues were subjected to screening on a broad range of Na(V) channel isoforms, both in voltage clamp and patch clamp conditions. Even though clathrodin was not found to exert any activity, some analogues were capable of modulating the Na(V) channels, hereby validating the pyrrole-2-aminoimidazole alkaloid structure as a core structure for future small molecule-based Na(V) channel modulators.
Assuntos
Pirróis/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Desenho de Fármacos , Feminino , Técnicas de Patch-Clamp , Pirróis/química , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/metabolismo , Xenopus laevisRESUMO
Despite existing experimental and computational tools to assess the risk, the non-specific chemical modification of protein thiol groups remains a significant source of false-positive hits, particularly in academic drug discovery. Herein, we describe the application of a simple NMR method in a systematic study on the reactivity of 5-benzylidenebarbiturates, 5-benzylidenerhodanines, and their related oxo-heterocycles, which have been associated with numerous biological activities and have recently gained a reputation as unselective promiscuous binders. Using this method, we confirmed the reactivity of 5-benzylidenebarbiturates, which are known to easily form Michael adducts with nucleophiles. In contrast, 5-benzylidene five-membered oxo-heterocycles revealed almost insignificant reactivity. We can conclude that the distinct binding profile of the most controversial compounds, 5-benzylidenerhodanines, is not necessarily related to their unspecific Michael acceptor reactivity.
RESUMO
DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests - parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.
Assuntos
Absorção Gastrointestinal , Relação Quantitativa Estrutura-Atividade , Inibidores da Topoisomerase II , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacocinética , Micelas , Modelos Lineares , Membranas Artificiais , DNA Girase/metabolismo , DNA Girase/química , Humanos , DNA Topoisomerase IV/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/químicaRESUMO
Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.
Assuntos
Membrana Celular , Ciona intestinalis , Fluorometria , Técnicas de Patch-Clamp , Fenilalanina , Animais , Membrana Celular/metabolismo , Membrana Celular/química , Fluorometria/métodos , Ciona intestinalis/metabolismo , Ciona intestinalis/química , Ciona intestinalis/genética , Fenilalanina/química , Fenilalanina/análogos & derivados , Oócitos/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Xenopus laevis , Canais Iônicos/metabolismo , Canais Iônicos/química , Corantes Fluorescentes/química , HumanosRESUMO
The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.
Assuntos
Antineoplásicos , Apoptose , Proteínas de Choque Térmico HSP90 , Triazóis , Peixe-Zebra , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Triazóis/farmacologia , Triazóis/química , Triazóis/síntese química , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Relação Estrutura-Atividade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células MCF-7 , Proliferação de Células/efeitos dos fármacosRESUMO
New 2-pyrrolamidobenzothiazole-based inhibitors of mycobacterial DNA gyrase were discovered. Among these, compounds 49 and 51, show excellent antibacterial activity against Mycobacterium tuberculosis and Mycobacterium abscessus with a notable preference for mycobacteria. Both compounds can penetrate infected macrophages and reduce intracellular M. tuberculosis load. Compound 51 is a potent inhibitor of DNA gyrase (M. tuberculosis DNA gyrase IC50 = 4.1 nM, Escherichia coli DNA gyrase IC50 of <10 nM), selective for bacterial topoisomerases. It displays low MIC90 values (M. tuberculosis: 0.63 µM; M. abscessus: 2.5 µM), showing specificity for mycobacteria, and no apparent toxicity. Compound 49 not only displays potent antimycobacterial activity (MIC90 values of 2.5 µM for M. tuberculosis and 0.63 µM for M. abscessus) and selectivity for mycobacteria but also exhibits favorable solubility (kinetic solubility = 55 µM) and plasma protein binding (with a fraction unbound of 2.9 % for human and 4.7 % for mouse). These findings underscore the potential of fine-tuning molecular properties to develop DNA gyrase B inhibitors that specifically target the mycobacterial chemical space, mitigating the risk of resistance development in non-target pathogens and minimizing harm to the microbiome.
Assuntos
Antibacterianos , DNA Girase , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Inibidores da Topoisomerase II , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/síntese química , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Estrutura Molecular , Camundongos , Animais , Relação Dose-Resposta a Droga , Antituberculosos/farmacologia , Antituberculosos/química , Antituberculosos/síntese química , Desenvolvimento de Medicamentos , Mycobacterium/efeitos dos fármacosRESUMO
Podosomes, specialized actin-rich structures in macrophages (Mfs), degrade the extra-cellular matrix (ECM) and are involved in cell migration. On two-dimensional (2D) surfaces Mfs form spot-like podosomes at the ventral cell surface that develop into protrusive structures in a three-dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U-937. Monocyte-to-Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time-lapse live-cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.