RESUMO
Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 =0.29â µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.
Assuntos
Lisina , Piruvato Quinase , Humanos , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Espectrometria de Massas em Tandem , Fígado , Domínio Catalítico , Regulação AlostéricaRESUMO
The combination of cytotoxic copper-thiosemicarbazone complexes with phenoxazines results in an up to 50-fold enhancement in the cytotoxic potential of the thiosemicarbazone against the MCF-7 human breast adenocarcinoma cell line over the effect attributable to drug additivity-allowing minimization of the more toxic copper-thiosemicarbazone component of the therapy. The combination of a benzophenoxazine with all classes of copper complex examined in this study proved more effective than combinations of the copper complexes with related isoelectronic azines. The combination approach results in rapid elevation of intracellular reactive oxygen levels followed by apoptotic cell death. Normal fibroblasts representative of non-cancerous cells (MRC-5) did not display a similar elevation of reactive oxygen levels when exposed to similar drug levels. The minimization of the copper-thiosemicarbazone component of the therapy results in an enhanced safety profile against normal fibroblasts.
Assuntos
Antineoplásicos/farmacologia , Cobre/farmacologia , Citotoxinas/farmacologia , Compostos Organometálicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tiossemicarbazonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cobre/química , Citotoxinas/síntese química , Citotoxinas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Oxazinas/química , Oxazinas/farmacologia , Oxirredução , Relação Estrutura-Atividade , Tiossemicarbazonas/químicaRESUMO
A series of bis-thiosemicarbazone agents with coordinating groups capable of multiple metal coordination modes has been generated and evaluated for potential cytotoxic effects against melanoma (MelRm) and breast adenocarcinoma (MCF-7) cell lines. The bis-thiosemicarbazones in this study generally demonstrated superior cytotoxic activity against MelRm than MCF-7 in the absence of metal ion supplementation, but in most cases could not be considered superior to the reference thiosemicarbazone Dp44mT. The key structural features for the cytotoxic activity were the central metal binding atom on the aromatic core, the thiocarbonyl residue and the nature of substitution on the N4-terminus in terms of size and lipophilicity. The cytotoxicity of bis-thiosemicarbazone ligands improved significantly with Cu(II) supplementation, particularly against MCF-7 cells. The mechanism of cytotoxicity of bis-thiosemicarbazones was proposed to be dependent on the combined effect of metal mobilisation and ROS generation which is so called a "double-punch effect".
Assuntos
Complexos de Coordenação/farmacologia , Cobre/farmacologia , Fibroblastos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tiossemicarbazonas/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Cobre/química , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Tiossemicarbazonas/químicaRESUMO
Zinc is the second most abundant transition metal in the human body, between 3 and 10% of human genes encoding for zinc binding proteins. We have investigated the interplay of reactive oxygen species and zinc homeostasis on the cytotoxicity of the thiosemicarbazone chelators against the MCF-7 cell line. The cytotoxicity of thiosemicarbazone chelators against MCF-7 can be improved through supplementation of ionic zinc provided the zinc ion is at a level exceeding the thiosemicarbazone concentration. Elimination of the entire cell population can be accomplished with this regime, unlike the plateau of cytotoxicity observed on thiosemicarbazone monotherapy. The cytotoxic effects of copper complexes of the thiosemicarbazone are not enhanced by zinc supplementation, displacement of copper from the complex being disfavoured. Treatment of MCF-7 with uncomplexed thiosemicarbazone initiates post G1 blockade alongside the induction of apoptosis, cell death being abrogated through subsequent supplementation with zinc ion after drug removal. This would implicate a metal depletion mechanism in the cytotoxic effect of the un-coordinated thiosemicarbazone. The metal complexes of the species, however, fail to initiate similar G1 blockade and apparently exert their cytotoxic effect through generation of reactive oxygen species, suggesting that multiple mechanisms of cytotoxicity can be associated with the thiosemicarbazones dependant on the level of metal ion association.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Cobre/metabolismo , Tiossemicarbazonas/administração & dosagem , Zinco/metabolismo , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cobre/administração & dosagem , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Tiossemicarbazonas/efeitos adversos , Zinco/administração & dosagemRESUMO
The cytotoxic activity of thiosemicarbazones (TSC) and thiocarbohydrazones was investigated against the MelRm melanoma cell line. In general, the melanoma line was susceptible to metal coordinating agents, the most useful of which incorporated the dipyridyl ketone hydrazone sub-structure. The impact of copper supplementation on the cytotoxic activity towards the melanoma line (MelRm) of metal coordinating agents when acting as ionophores is less predictable than the general improvement that has been seen in other cancer cells such as breast adenocarcinoma (MCF-7). The bimetallic nature of thiocarbohydrazone complexes with resultant loss of lipophilicity is a limiting factor in usage against MelRm. The cytotoxic activity of TSC against MelRm when used as copper ionophores could be markedly improved through combination with a partner drug capable of disrupting cellular defences to oxidative stress. In the absence of copper supplementation, both TSC and thiocarbohydrazones could be used to initiate cell cycle arrest and this could be employed to improve cytotoxicity profiles of other metallodrugs such as cisplatin.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Quelantes/farmacologia , Complexos de Coordenação/farmacologia , Melanoma/tratamento farmacológico , Melanoma/patologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quelantes/síntese química , Quelantes/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Cobre/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Hidrazinas/farmacologia , Melanoma/metabolismo , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologiaRESUMO
Cancer cell resistance to chemotherapy is still a heavy burden that impairs the response of many cancer patients to conventional chemotherapy. Using drug combinations is one therapeutic approach to overcome the developing resistance to any one drug. Oxidative stress is now a generally regarded hallmark of cancer that can be one approach to selectively target cancer cells while sparing normal cells. With the aim of increasing oxidative stress in cancer cells to a lethal set point, we have generated and combined several series of redox active compounds that act at different points of the cellular oxidative cascade. The premise of such combinations is to deplete of endogenous antioxidant defence proteins (e.g., Glutathione) while concomitantly increasing the generation of ROS via metal redox recycling and Fenton chemistry which eventually leads to the disruption of cellular redox homeostasis and induction of cell death. Through this approach, we have identified highly synergistic combinations of two distinctive classes of compounds (Azines and Copper(II) complexes of 2-pyridyl ketone thiosemicarbazones) which are capable of eliminating cancer cells without concomitant increase in toxicity toward normal cells. In one of our most potent combinations, a combination index (CI) value of 0.056 was observed, representing a 17 fold enhancement in activity beyond additive effects. Such new combination regimen of redox active compounds can be one step closer to potentially safer low dose chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Flavinas/farmacologia , Espécies Reativas de Oxigênio/agonistas , Tiossemicarbazonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , Cobre/química , Combinação de Medicamentos , Sinergismo Farmacológico , Flavinas/síntese química , Flavinas/metabolismo , Glutationa/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/metabolismoRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 µM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.
Assuntos
Taninos Hidrolisáveis , Hepatopatia Gordurosa não Alcoólica , Piruvato Quinase , Humanos , Fígado , Sulfonas/farmacologiaRESUMO
The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.
RESUMO
Liver pyruvate kinase (PKL) has recently emerged as a new target for non-alcoholic fatty liver disease (NAFLD), and inhibitors of this enzyme could represent a new therapeutic option. However, this breakthrough is complicated by selectivity issues since pyruvate kinase exists in four different isoforms. In this work, we report that ellagic acid (EA) and its derivatives, present in numerous fruits and vegetables, can inhibit PKL potently and selectively. Several polyphenolic analogues of EA were synthesized and tested to identify the chemical features responsible for the desired activity. Molecular modelling studies suggested that this inhibition is related to the stabilization of the PKL inactive state. This unique inhibition mechanism could potentially herald the development of new therapeutics for NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Piruvato Quinase/metabolismo , Ácido Elágico/química , Fígado/metabolismoRESUMO
Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC(50) of 19.8 µM.
Assuntos
Caproatos/síntese química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Hidrazinas/síntese química , Modelos Moleculares , Transaminases/antagonistas & inibidores , Caproatos/química , Caproatos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hidrazinas/química , Hidrazinas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Esquizofrenia/tratamento farmacológicoRESUMO
In our continued exploration of disubstituted piperazine derivatives as sigma (σ) receptor ligands with central nervous system (CNS) activity, a series of N-(2-benzofuranylmethyl)-N'-(methoxyphenylalkyl)piperazines (16-21 and 26-31) were synthesized, anticipating that these ligands would better suit the structural requirements of the current σ(1) pharmacophore. Affinities of these ligands for σ(1) and σ(2) receptors were investigated by means of radioligand binding assays, with the identification of N-(2-benzofuranylmethyl)-N'-[3-(4-methoxyphenyl)propyl]piperazine (29, K(i)=3.1 nM, σ(2)/σ(1)=45) as a selective σ(1) ligand. The σ(1) affinities and subtype selectivities of piperazines 16-21 and 26-31 were generally comparable to the corresponding benzylic analogs. Additionally, the affinities of 16-21 and 26-31 for the 5-HT(2B) receptor were much lower than the relatively nonselective methoxybenzylic analogs 2-4, indicating that elongation of the alkyl tether generally improved selectivity for σ(1) receptors.
Assuntos
Desenho de Fármacos , Descoberta de Drogas , Piperazinas/química , Receptores sigma/agonistas , Animais , Benzofuranos/química , Benzofuranos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Químicos , Estrutura Molecular , Células PC12 , Piperazinas/metabolismo , Ligação Proteica , Ensaio Radioligante , Ratos , Receptores de Serotonina/metabolismo , Receptores sigma/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The emergence of resistance to the artemisinins which are the current mainstays for antimalarial chemotheraphy has created an environment where the development of new drugs acting in a mechanistally discrete manner is a priority. OBJECTIVE: The goal of this work was to synthesize ane evaluate bis-thiosemicarbazones as potential antimalarial agents. METHODS: Fifteen compounds were generated using two condensation protocols and evaluated in vitro against the NF54 (CQ sensitive) strain of Plasmodium falciparum. A preliminary assessment of the potential for human toxicity was conducted in vitro against the MRC5 human lung fibroblast line. RESULTS: The activity of the bis-thiosemicarbazones was highly dependent on the nature of the arene at the core of the structure. The inclusion of a non-coordinating benzene core resulted in inactive compounds, while the inclusion of a pyridyl core resulted in compounds of moderate or potent antimalarial activity (4 compounds showing IC50 < 250 nM). CONCLUSION: Bis-thiosemicarbazones containing a central pyridyl core display potent antimalarial activity in vitro. Sequestration and activation of ferric iron appears to play a significant role in this activity. Ongoing studies are aimed at further development of this series as potential antimalarials.
Assuntos
Antimaláricos/farmacologia , Quelantes de Ferro/farmacologia , Tiossemicarbazonas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/toxicidade , Linhagem Celular , Cobre/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ferro/química , Quelantes de Ferro/síntese química , Quelantes de Ferro/química , Quelantes de Ferro/toxicidade , Plasmodium falciparum/efeitos dos fármacos , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/química , Tiossemicarbazonas/toxicidadeRESUMO
In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 µM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.