RESUMO
Since the discovery of cisplatin in the 1960s, the search for metallo-drugs that are more efficient than platinum complexes with negligible side effects has attracted much interest. Among the other metals that have been examined for potential applications as anticancer agents is copper. The interest in copper was recently boosted by the discovery of cuproptosis, a recently evidenced form of cell death mediated by copper. However, copper is also known to induce the proliferation of cancer cells. In view of these contradictory results, there is a need to find the most suitable copper chelators, among which Schiff-based derivatives offer a wide range of possibilities. Gathering several metal complexes in a single, larger entity may provide enhanced properties. Among the nanometric objects suitable for such purpose are dendrimers, precisely engineered hyperbranched macromolecules, which are outstanding candidates for improving therapy and diagnosis. In this review article, we present an overview of the use of a particular Schiff base, namely pyridine-imine, linked to the surface of dendrimers, suitable for complexing copper, and the use of such dendrimer complexes in biology, in particular against cancers.
Assuntos
Cobre , Iminas , Piridinas , Animais , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Quelantes/química , Quelantes/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Dendrímeros/química , Iminas/química , Neoplasias/tratamento farmacológico , Piridinas/química , Bases de Schiff/químicaRESUMO
A series of 25 chiral anti-cancer lipidic alkynylcarbinols (LACs) were devised by introducing an (hetero)aromatic ring between the aliphatic chain and the dialkynylcarbinol warhead. The resulting phenyl-dialkynylcarbinols (PACs) exhibit enhanced stability, while retaining cytotoxicity against HCT116 and U2OS cell lines with IC50 down to 40 nM for resolved eutomers. A clickable probe was used to confirm the PAC prodrug behavior: upon enantiospecific bio-oxidation of the carbinol by the HSD17B11 short-chain dehydrogenase/reductase (SDR), the resulting ynones covalently modify cellular proteins, leading to endoplasmic reticulum stress, ubiquitin-proteasome system inhibition, and apoptosis. Insights into the design of LAC prodrugs specifically bioactivated by HSD17B11 vs its paralogue HSD17B13 were obtained. The HSD17B11/HSD17B13-dependent cytotoxicity of PACs was exploited to develop a cellular assay to identify specific inhibitors of these enzymes. A docking study was performed with the HSD17B11 AlphaFold model, providing a molecular basis of the SDR substrates mimicry by PACs. The safety profile of a representative PAC was established in mice.
Assuntos
Alcinos , Antineoplásicos , Camundongos , Animais , Alcinos/farmacologia , Alcinos/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Acetileno , Estrutura Molecular , Lipídeos/química , Linhagem Celular TumoralRESUMO
Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.
Assuntos
Antineoplásicos , Redutases-Desidrogenases de Cadeia Curta , Antineoplásicos/farmacologia , Estresse do Retículo Endoplasmático , Humanos , Lipídeos , Resposta a Proteínas não DobradasRESUMO
A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using H2O2 (1 equiv) led to ETH-SO as the only stable S-oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETHâSâO vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SO2H) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SO2H), which can then lose a SOxH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SO2H, and the carbodiimide compound can also react with NH2-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.
Assuntos
Antituberculosos/metabolismo , Etionamida/metabolismo , Feniltioureia/análogos & derivados , Pró-Fármacos/metabolismo , Tioacetazona/metabolismo , Antituberculosos/química , Etionamida/química , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Oxirredução , Feniltioureia/química , Feniltioureia/metabolismo , Pró-Fármacos/química , Tioacetazona/químicaRESUMO
Nitric oxide (NO) and nitroxyl (HNO) have gained broad attention due to their roles in several physiological and pathophysiological processes. Remarkably, these sibling species can exhibit opposing effects including the promotion of angiogenic activity by NO compared to HNO, which blocks neovascularization. While many NO donors have been developed over the years, interest in HNO has led to the recent emergence of new donors. However, in both cases there is an expressive lack of iron-based compounds. Herein, we explored the novel chemical reactivity and stability of the trans-[Fe(cyclam)(NO)Cl]Cl2 (cyclam = 1,4,8,11-tetraazacyclotetradecane) complex. Interestingly, the half-life (t1/2) for NO release was 1.8 min upon light irradiation, vs 5.4 h upon thermal activation at 37 °C. Importantly, spectroscopic evidence supported the generation of HNO rather than NO induced by glutathione. Moreover, we observed significant inhibition of NO donor- or hypoxia-induced HIF-1α (hypoxia-inducible factor 1α) accumulation in breast cancer cells, as well as reduced vascular tube formation by endothelial cells pretreated with the trans-[Fe(cyclam)(NO)Cl]Cl2 complex. Together, these studies provide the first example of an iron-nitrosyl complex with anti-angiogenic activity as well as the potential dual activity of this compound as a NO/HNO releasing agent, which warrants further pharmacological investigation.
Assuntos
Inibidores da Angiogênese/farmacologia , Complexos de Coordenação/farmacologia , Doadores de Óxido Nítrico/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/efeitos da radiação , Animais , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/efeitos da radiação , Glutationa/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ferro/química , Ferro/efeitos da radiação , Camundongos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/síntese química , Doadores de Óxido Nítrico/efeitos da radiação , Óxidos de Nitrogênio/metabolismo , Ratos , Temperatura , Raios Ultravioleta , Vasodilatadores/síntese química , Vasodilatadores/farmacologia , Vasodilatadores/efeitos da radiaçãoRESUMO
A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabolites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplified by combining two anti-mycobacterial drugs: pyrazinamide (first line) and delamanid (third line) whose active metabolites are pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO.)), respectively. Aiming to generate those active species, a hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na3[FeII(CN)5] moiety. The corresponding pentacyanoferrate(II) complex Na4[FeII(CN)5(PyzCONHO-)] was synthesized and characterized by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with H2O2 was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis (Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both H2O2- and N-coordinated pyrazine FeII species are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively growing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly lesser toxic effects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as a possible pharmacophore model for an alternative vasodilator drug.
Assuntos
Antituberculosos/síntese química , Complexos de Coordenação/síntese química , Compostos Ferrosos/síntese química , Ácidos Hidroxâmicos/química , Ferro/química , Mycobacterium tuberculosis/efeitos dos fármacos , Óxidos de Nitrogênio/química , Amidoidrolases/metabolismo , Antituberculosos/farmacologia , Complexos de Coordenação/farmacologia , Descoberta de Drogas , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/química , Ligantes , Óxidos de Nitrogênio/metabolismo , Oxirredução , Pirazinamida/análogos & derivados , Pirazinamida/química , VasodilataçãoRESUMO
Tyrosine kinase inhibitors (TKI) are targeted anticancer drugs that have been successfully developed over the past 2 decades. To date, many of them (around 70%) require warnings for liver injury and five of them, including pazopanib and sunitinib, have Black Box Warning (BBW) labels. Although TKI-induced hepatotoxicity is the first cause of drug failures in clinical trials, BBW labels, and market withdrawals, the underlying mechanisms remain unclear. However, the recent discovery of new reactive metabolites (RM) with aldehyde structures during pazopanib and sunitinib metabolism offers new perspectives for investigating their involvement in the toxicity of these two TKI. These hard electrophiles have a high reactivity potential toward proteins and are thought to be responsible for cytochrome P450 inactivation, drug-drug interactions (DDI), and liver toxicity. We report here, for the first time, the presence of these aldehyde RM in human plasma samples obtained during drug monitoring. Docking experiments in the CYP3A4 active site were performed and showed that pazopanib and sunitinib fitting in the catalytic site are in accordance with their regioselective oxidation to aldehydes. They also suggested that aldehyde RM may react with lysine and arginine residues. Based on these results, we studied the reactivity of the aldehyde RM toward lysine and arginine residues as potential targets on the protein framework to better understand how these RM could be involved in liver toxicity and drug-drug interactions. Adduct formation with different hepatic and plasma proteins was investigated by LC-MS/MS, and adducts between pazopanib or sunitinib aldehyde derivatives and lysine residues on both CYP3A4 and plasma proteins were indeed shown for the first time.
Assuntos
Aldeídos/metabolismo , Inibidores da Angiogênese/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/farmacocinética , Sulfonamidas/farmacocinética , Sunitinibe/farmacocinética , Aldeídos/sangue , Inibidores da Angiogênese/efeitos adversos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Humanos , Indazóis , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/efeitos adversos , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/metabolismo , Sulfonamidas/efeitos adversos , Sunitinibe/efeitos adversosRESUMO
Oxidation of two tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib, using a chemical catalytic system able to mimic the cytochrome P450 type oxidation, allowed us to prepare putative reactive/toxic metabolites of these anticancer drugs. Among these metabolites, aromatic aldehyde derivatives were unambiguously characterized. Such biomimetic oxidation of TKI-type drugs was essential to facilitate the identification of low amounts of aldehydes generated from these TKIs when incubated with human liver microsomes (HLM), which are classical models of human hepatic metabolism. These TKI derivative aldehydes quickly react in vitro with amines. A similar reaction is expected to occur in vivo and may be at the origin of the potentially severe hepatotoxicity of these TKIs.
Assuntos
Aldeídos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Metaloporfirinas/farmacologia , Microssomos Hepáticos/patologia , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Sulfonamidas/química , Sunitinibe/química , Aldeídos/efeitos adversos , Aminas/química , Aminas/metabolismo , Catálise , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Indazóis , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oxirredução , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/efeitos adversos , Sulfonamidas/efeitos adversos , Sunitinibe/efeitos adversosRESUMO
The enoyl-ACP reductase InhA from the mycobacterial fatty acid biosynthesis pathway has become a target of interest for the development of new anti-tubercular drugs. This protein has been identified as essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis, and as the main target of two pro-drugs: isoniazid, the frontline anti-tubercular drug, and ethionamide, a second-line medicine. Since most cases of resistance to isoniazid and ethionamide result from mutations in the mycobacterial activating enzyme (KatG for isoniazid and EthA for ethionamide), research of direct InhA inhibitors, avoiding the activation step, has emerged as a promising strategy for combating tuberculosis. Thereby, InhA is drawing much attention and its three-dimensional structure has been particularly studied. A better understanding of key sites of interactions responsible for InhA inhibition arises thus as an essential tool for the rational design of new potent inhibitors. In this paper, we propose an overview of the 80 available crystal structures of wild-type and mutant InhA, in its apo form, in complex with its cofactor, with an analogue of its natural ligands (C16 fatty acid and/or NADH) or with inhibitors. We will first discuss structural and mechanistic aspects in order to highlight key features of the protein before delivering thorough inventory of structures of InhA in the presence of synthetic ligands to underline the key interactions implicated in high affinity inhibition.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Produtos Biológicos/farmacologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ligantes , Modelos Moleculares , Estrutura Molecular , Oxirredutases/metabolismo , Relação Estrutura-AtividadeRESUMO
The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by H2O2 and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using H2O2 with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.
Assuntos
Antituberculosos/farmacologia , Complexos de Coordenação/farmacologia , Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Isoniazida/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Compostos Ferrosos/síntese química , Compostos Ferrosos/química , Isoniazida/síntese química , Isoniazida/química , Testes de Sensibilidade Microbiana , Modelos Químicos , Mycobacterium tuberculosis/efeitos dos fármacos , OxirreduçãoRESUMO
The place of prodrugs in the current antitubercular therapeutic arsenal is preponderant, since two of the four first-line antitubercular agents, isoniazid (INH) and pyrazinamide (PZA), need to be activated by Mycobacterium tuberculosis before exerting their activity. In addition, six other prodrugs can be found in the second- and third-line therapeutic regimens. The emergence of mycobacterial strains resistant to one or several antitubercular agents is one of the main issues of the antitubercular therapy. In the case of prodrugs, the resistance phenomenon is often related to a mutation in the gene encoding for the activation enzymes, resulting thus in a default of these enzymes that are no more able to activate prodrugs. Consequently, identification of the prodrugs targets and a better understanding of their modes of action and also of their activation mechanisms are of crucial importance. Related to their molecular mechanism of activation, these prodrugs may thus be classified in four categories: activation via oxidation (catalase-peroxidase (KatG) or flavin monooxygenase (EthA) enzymes), condensation (FolP1 and FolC), hydrolysis (by the amidase PncA) and reduction (by the nitroreductase DnD) mechanisms. For each prodrug, these mechanisms are described in details, as well as the mechanism of action of its active metabolite. Finally, the reported resistance related to these mechanisms of activation/action are also addressed in a molecular perspective.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Estrutura MolecularRESUMO
Ethionamide (ETH), a second-line anti-tubercular drug that is regaining a lot of interest due to the increasing cases of drug-resistant tuberculosis, is a pro-drug that requires an enzymatic activation step to become active and to exert its therapeutic effect. The enzyme responsible for ETH bioactivation in Mycobacterium tuberculosis is a monooxygenase (EthA) that uses flavin adenine dinucleotide (FAD) as a cofactor and is NADPH- and O2-dependant to exert its catalytic activity. In this work, we investigated the activation of ETH by various oxygen-donor oxidants and the first biomimetic ETH activation methods were developed (KHSO5, H2O2, and m-CPBA). These simple oxidative systems, in the presence of ETH and NAD+, allowed the production of short-lived radical species and the first non-enzymatic formation of active and non-active ETH metabolites. The intermediates and the final compounds of the activation pathway were well characterized. Based on these results, we postulated a consistent mechanism for ETH activation, not involving sulfinic acid as a precursor of the iminoyl radical, as proposed so far, but putting forward a novel reactivity for the S-oxide ethionamide intermediate. We proposed that ETH is first oxidized into S-oxide ethionamide, which then behaves as a "ketene-like" compound via a formal [2 + 2] cycloaddition reaction with peroxide to give a dioxetane intermediate. This unstable 4-membered intermediate in equilibrium with its open tautomeric form decomposes through different pathways, which would explain the formation of the iminoyl radical and also that of different metabolites observed for ETH oxidation, including the ETH-NAD active adduct. The elucidation of this unprecedented ETH activation mechanism was supported by the application of isotopic labelling experiments.
Assuntos
Antituberculosos/metabolismo , Etionamida/metabolismo , Mycobacterium tuberculosis/enzimologia , Oxirredutases/metabolismo , Pró-Fármacos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Biomimética , Etionamida/farmacologia , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Oxidantes/metabolismo , Pró-Fármacos/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.
Assuntos
Antituberculosos/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Ácidos Micólicos/metabolismo , Antituberculosos/síntese química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Isoindóis/síntese química , Isoindóis/química , Isoindóis/metabolismo , Isoindóis/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , TermodinâmicaRESUMO
A series of fluorene-based derivatives was synthesized and evaluated for inhibiting both InhA and Mycobacterium tuberculosis growth. These compounds were inspired by the previously reported Genz-10850 molecule, a good InhA inhibitor, but with a poor activity against M. tuberculosis growth. Structure-activity relationships were performed by introducing the following chemical modifications: 1) the piperazine ring; 2) the amide group; 3) the aryl moiety; and 4) the fluorene moiety. Among these new derivatives, one of them was more effective against both the InhA activity and mycobacterial growth, compared to the hit compound. Docking studies were also performed to rationalize activities of these derivatives. Furthermore, we showed for the first time that efflux pump inhibitors potentiated the efficacy of Genz-10850 (GEQ) derivatives against M. tuberculosis growth, demonstrating that these compounds could be substrates of some efflux pumps.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Piperazinas/farmacologia , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Oxirredutases/metabolismo , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-AtividadeRESUMO
InhA is an enoyl-ACP reductase of Mycobacterium tuberculosis implicated in the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. To date, this enzyme is considered as a promising target for the discovery of novel antitubercular drugs. In this work, we describe the first crystal structure of the apo form of the wild-type InhA at 1.80Å resolution as well as the crystal structure of InhA in complex with the synthetic metabolite of the antitubercular drug isoniazid refined to 1.40Å. This metabolite, synthesized in the absence of InhA, is able to displace and replace the cofactor NADH in the enzyme active site. This work provides a unique opportunity to enlighten the structural adaptation of apo-InhA to the binding of the NADH cofactor or of the isoniazid adduct. In addition, a differential scanning fluorimetry study of InhA, in the apo-form as well as in the presence of NAD(+), NADH and INH-NADH was performed showing that binding of the INH-NADH adduct had a strong stabilizing effect.
Assuntos
Proteínas de Bactérias/química , Isoniazida/química , Mycobacterium tuberculosis/enzimologia , Oxirredutases/química , Biomimética/métodos , Domínio Catalítico , NAD/química , Ligação Proteica/fisiologiaRESUMO
InhA, the NADH-dependent enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis (Mtb) is the proposed main target of the first-line antituberculosis drug isoniazid (INH). INH activity is dependent on activation by the catalase peroxidase KatG, a Mtb enzyme whose mutations are linked to clinical resistance to INH. Other inhibitors of InhA that do not require any preliminary activation are known. The design of such direct potent inhibitors represents a promising approach to circumvent this resistance mechanism. An ensemble-docking process with four known InhA X-ray crystal structures and employing the Autodock Vina software was performed. Five InhA inhibitors whose bioactive conformations are known were sequentially docked in the substrate cavity of each protein. The efficiency of the docking was assessed and validated by comparing the calculated conformations to the crystallographic structures. For a same inhibitor, the docking results differed from one InhA conformation to another; however, docking poses that matched correctly or were very close to the expected bioactive conformations could be identified. The expected conformations were not systematically well ranked by the Autodock Vina scoring function. A post-docking optimization was carried out on all the docked conformations with the AMMP force field implemented on the VEGAZZ software, followed by a single point calculation of the interaction energy, using the MOPAC PM6-DH2 semi-empirical quantum chemistry method. The conformations were subsequently submitted to a PM6-DH2 optimization in partially flexible cavities. The resulting interaction energies combined with the multiple receptor conformations approach allowed us to retrieve the bioactive conformation of each ligand.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Humanos , Isoniazida/farmacologia , Ligantes , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Ligação Proteica , Tuberculose/tratamento farmacológicoRESUMO
Five lipophilic analogues 1-5 of the active metabolite of the antitubercular drug isoniazid (INH), selected as inhibitors of Mycobacterium smegmatis and Mycobacterium tuberculosis growth, were evaluated for their activity against Corynebacterium glutamicum (lacking in InhA activity), Escherichia coli (to test mycobacteria selectivity), and Plasmodium falciparum (as possible parasite target). Compound 3 was the only one that did not inhibit C. glutamicum growth. The poor InhA inhibitors 1 and 2 were able to inhibit C. glutamicum and their anti(myco)bacterial mechanisms of action involve targets other than InhA. For the effective InhA inhibitors 4 and 5, also active against C. glutamicum and M. tuberculosis strains, more than one pathway should be envisaged to explain their actions. Pyridine-base ring analogues (1, 2, and 3) have no ability to inhibit the growth of E. coli even at a high concentration. Compound 3 thus exhibited a selective inhibitory action toward M. tuberculosis, while it was inactive on C. glutamicum and on E. coli growth. It presented an activity profile similar to that of INH suggesting InhA inhibition as one of the possible mechanisms of action. Finally, although a homologue of the reductase InhA exists in the FAS-II system of P. falciparum, 3 was unable to display antiplasmodial activity.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Corynebacterium glutamicum/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Isoniazida/metabolismo , Isoniazida/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antibacterianos/química , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Isoniazida/química , Testes de Sensibilidade Microbiana , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismoRESUMO
The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on the framework were performed. These compounds were designed as InhA inhibitors and as anti-Mycobacterium tuberculosis agents. Structure-activity relationships concerning the length and the location of the lipophilic chain around the azaisoindolinone framework, the suppression of the phenyl group, the bioisosteric substitution of ether link and alkylating of the tertiary hydroxyl and the hemiamidal nitrogen were also investigated, revealing insightful information and thereby enabling further diversification of the azaisoindolinone scaffold for new antitubercular agents.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Indóis/química , Indóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Antibacterianos/síntese química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/síntese química , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Oxirredutases/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Relação Estrutura-AtividadeRESUMO
Isoniazid-NAD truncated adducts embedding a lipophilic fragment were designed, synthesized and evaluated as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (InhA) of Mycobacterium tuberculosis and as antimycobacterial agents. These compounds, planned as bi-substrate inhibitors and inspired from the active metabolite of isoniazid, combine both the nicotinamide moiety of the cofactor NAD and a lipophilic hydrocarbon chain mimic of the InhA substrate. The lipophilic fragment was introduced using either Suzuki-Miyaura cross-coupling or a classical nucleophilic substitution reaction. Several compounds developed in this work were indeed able to inhibit the InhA activity and showed promising antimycobacterial activities. However a direct correlation between the expressed activity and the bi-substrate mode of action could not yet be unambiguously demonstrated.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Isoniazida/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , NAD/análogos & derivados , Oxirredutases/antagonistas & inibidores , Antituberculosos/síntese química , Proteínas de Bactérias/metabolismo , Humanos , Isoniazida/síntese química , Isoniazida/química , Isoniazida/farmacologia , Mycobacterium tuberculosis/enzimologia , NAD/síntese química , NAD/química , NAD/farmacologia , Oxirredutases/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/enzimologiaRESUMO
The front-line antituberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis enoyl ACP-reductase from Mycobacterium tuberculosis, via formation of covalent adducts with NAD (INH-NAD adducts). While ring tautomers were found the main species formed in solution, only the 4S chain INH-NAD tautomer was evidenced in the crystallized InhA:INH-NAD complex. In this study we attempted to explore the modes of interaction and energy binding of the different isomers placed in the active site of InhA with the help of various molecular modelling techniques. Ligand and enzyme models were generated with the help of the Vega ZZ program package. Resulting ligands were then docked into the InhA active site individually using computational automated docking package AUTODOCK 3.0.5. The more relevant docked conformations were then used to compute the interaction energy between the ligands and the InhA cavity. The AM1 Hamiltonian and the QM/MM ONIOM methodologies were used and the results compared. The various tautomers were found docked in almost the same place where INH-NAD was present as predicted by earlier X-ray crystallographic studies. However, some changes of ligand conformation and of the interactions ligand-protein were evidenced. The lower binding energy was observed for the 4S chain adduct that probably represents the effective active form of the INH-NAD adducts, as compared to the 4R epimer. The two 4S,7R and 4R,7S ring tautomers show intermediate and similar binding energies contrasting with their different experimental inhibitory potency on InhA. As a possible explanation based on calculated conformations, we formulated the hypothesis of an initial binding of the two ring tautomers to InhA followed by opening of only the ring hemiamidal 4S,7R tautomer (possibly catalyzed by Tyr158 phenolate basic group) to give the 4S chain INH-NAD tight-binding inhibitor. The predictions of ligand-protein interactions at the molecular level can be of primary importance in elucidating the mechanisms of action of isoniazid and InhA-related resistances, in identifying the effective mycobactericidal entities and, in further step, in the design of a new generation of antitubercular drugs.