RESUMO
Staphylococcus aureus, a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1-diphenylmethyl triazole-based analogues (7-13) designed to interact with the entire binding site of S. aureus biotin protein ligase (SaBPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both SaBPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13, with added substituents to better interact with the adenine binding site, are particularly potent, with Ki values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 µg/mL, respectively.
Assuntos
Antibacterianos , Proteínas de Bactérias , Carbono-Nitrogênio Liases , Staphylococcus aureus , Triazóis , Biotina , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Triazóis/química , Triazóis/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Carbono-Nitrogênio Liases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidoresRESUMO
Diaryldienone derivatives with accessible ß-carbons show strong anti-neoplastic properties, related to their ability to make covalent adducts with free thiols by Michael addition, and low toxicity in vivo. Accumulation of poly-ubiquitylated proteins, activation of the unfolded protein response (UPR) and induction of cell death are universal hallmarks of their activities. These compounds have been characterized as inhibitors of isopeptidases, a family of cysteine-proteases, which de-conjugate ubiquitin and ubiquitin-like proteins from their targets. However, it is unclear whether they can also react with additional proteins. In this work, we utilized the biotin-conjugated diaryldienone-derivative named 2c, as a bait to purify novel cellular targets of these small molecules. Proteomic analyses have unveiled that, in addition to isopeptidases, these inhibitors can form stable covalent adducts with different intracellular proteins, thus potentially impacting on multiple functions of the cells, from cytoskeletal organization to metabolism. These widespread activities can explain the ability of diaryldienone derivatives to efficiently trigger different cell death pathways.
Assuntos
Carbono-Nitrogênio Liases/antagonistas & inibidores , Cicloexanonas/metabolismo , Proteômica/métodos , HumanosRESUMO
Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.
Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/efeitos dos fármacos , Carbono-Nitrogênio Liases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Bortezomib/farmacologia , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacosRESUMO
Bis-arylidenecycloalkanones structurally related to the nonselective isopeptidase inhibitor G5 were synthesized and tested for cytotoxic activity against glioblastoma cells. Cytotoxicities correlate well with Hammett σ constants for substituted arylidene groups, confirming the proposed inhibition mechanism. A new inhibitor (2c) based on the 4-hydroxycyclohexanone scaffold, which favors apoptosis over necrosis, was selected for further development. 2c inhibited representative deubiquitinases with micromolar IC50, and its proapoptotic activity was studied on several cancer cell lines. Inhibitor 2c was conjugated to PEG via dicarbamate and diester linkers. While the dicarbamate was inactive, the diester (2cPE) behaves like a prodrug and is converted into the active species 2c by secreted esterase activities. Finally, 2cPE was also tested in vivo on A549 lung carcinoma xenografts generated in mice. Intravenous treatment with 2cPE led to a significant reduction in primary tumor growth, without appreciable toxicity to mice.
Assuntos
Antineoplásicos/farmacologia , Carbono-Nitrogênio Liases/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Cetonas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Carbono-Nitrogênio Liases/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Células HT29 , Humanos , Cetonas/síntese química , Cetonas/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/patologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 µM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 µM PLP restored the catalytic activity of MidD in the reaction containing 50 µM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.
Assuntos
Amônia/metabolismo , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Mimosina/metabolismo , Piridonas/metabolismo , Ácido Pirúvico/metabolismo , Rhizobium/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Carbono-Nitrogênio Liases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Hidroxilamina/metabolismo , Hidroxilamina/farmacologia , Dados de Sequência Molecular , Peso Molecular , Fosfato de Piridoxal/metabolismo , Rhizobium/genética , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Gene products that modify the apoptotic susceptibility of cancer cells may offer novel drug response markers or therapeutic targets. In this study, we probed the contribution of 53 different isopeptidases to apoptosis triggered by bortezomib and etoposide. USP18, a type I IFN-induced protein that deconjugates the ubiquitin-like modifier ISG15 from target proteins, was found to limit apoptotic susceptibility to IFN-alpha or bortezomib. Ablating USP18 in cells treated with IFN-alpha increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) production; upregulated expression of transcription factors IFN-regulatory factor (IRF)-1, IRF-7, and IRF-9; and promoted the extrinsic pathway of apoptosis. The proapoptotic effects of ablating USP18 were abrogated by FLIP overexpression or TRAIL silencing. However, in bortezomib-treated cells, weak spontaneous signaling from type I IFNs was implicated in the proapoptotic effect of USP18 ablation. Ectopic USP18 repressed apoptotic signaling by IFN-alpha, TRAIL, or bortezomib. Similar effects were produced by a catalytically inactive USP18 mutant, indicating that the antiapoptotic function of USP18 is independent of its catalytic activity. These findings suggest that USP18 may significantly limit operation of the extrinsic apoptotic pathway triggered by type I IFN and drugs.
Assuntos
Apoptose/efeitos dos fármacos , Endopeptidases/fisiologia , Interferon-alfa/farmacologia , Apoptose/fisiologia , Ácidos Borônicos/farmacologia , Bortezomib , Carbono-Nitrogênio Liases/antagonistas & inibidores , Carbono-Nitrogênio Liases/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Endopeptidases/biossíntese , Endopeptidases/genética , Etoposídeo/farmacologia , Inativação Gênica , Humanos , Interferon alfa-2 , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/genética , Transcrição Gênica , Ubiquitina TiolesteraseRESUMO
Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO). We have discovered that (i) both inhibitors induce apoptosis in a Bax and Bak-dependent manner, (ii) both inhibitors elicit autophagy in WT and DKO cells, and (iii) only G5 can kill apoptosis-resistant DKO cells by activating a necrotic response. The induction of necrosis was confirmed by different experimental approaches, including time lapse analysis, HMGB1 release, and electron microscopy studies. Neither treatment with antinecrotic agents, such as antioxidants, poly(ADP-ribose) polymerase and JNK inhibitors, necrostatin, and the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, nor overexpression of Bcl-2 and Bcl-xL prevented necrosis induced by G5. This necrotic death is characterized by the absence of protein oxidation and by the rapid cyclosporin A-independent dissipation of the mitochondrial membrane potential. Notably, a peculiar feature of the G5-induced necrosis is an early and dramatic reorganization of the actin cytoskeleton, coupled to an alteration of cell adhesion. The importance of cell adhesion impairment in the G5-induced necrotic death of DKO cells was confirmed by the antagonist effect of the extracellular matrix-adhesive components, collagen and fibronectin.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Carbono-Nitrogênio Liases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Piranos/farmacologia , Pirazinas/farmacologia , Compostos de Sulfidrila/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Apoptose/genética , Bortezomib , Carbono-Nitrogênio Liases/genética , Carbono-Nitrogênio Liases/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Transformada , Colágeno/genética , Colágeno/metabolismo , Ciclosporina/farmacologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Knockout , Necrose/induzido quimicamente , Necrose/genética , Necrose/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The Pictet-Spengler reaction, which yields either a beta-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a beta-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the beta-carboline product is proposed from these data.
Assuntos
Carbolinas/metabolismo , Carbono-Nitrogênio Liases/química , Sítios de Ligação , Carbolinas/química , Carbono-Nitrogênio Liases/antagonistas & inibidores , Catálise , Cristalização , Inibidores Enzimáticos/química , Cinética , EstereoisomerismoRESUMO
Deacetylipecoside synthase (DIS), the enzyme catalyzing the condensation of dopamine and secologanin to form the (R)-epimer of deacetylipecoside, has been purified 570-fold from the leaves of Alangium lamarckii and partially characterized. The isolated enzyme is a single polypeptide with Mr 30,000, and has a pH optimum at 7.5 and a temperature optimum at 45 degrees C. The apparent Km values for dopamine and secologanin are 0.7 and 0.9 mM, respectively. DIS exhibits high substrate specificity toward dopamine, whereas neither tyramine nor tryptamine are utilized. The enzyme activity is not inhibited by its substrate dopamine, but is inhibited by alangimakine and dehydroalangimakine with similar I50 values of 10 microM. DIS presumably provides (R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that also possess an (R)-configuration at the same chiral center.
Assuntos
Carbono-Nitrogênio Liases/isolamento & purificação , Magnoliopsida/enzimologia , Carbono-Nitrogênio Liases/antagonistas & inibidores , Carbono-Nitrogênio Liases/metabolismo , Catálise , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Concentração de Íons de Hidrogênio , Folhas de Planta/enzimologia , Especificidade por SubstratoRESUMO
A series of nonhydrolyzable ubiquitin dimer analogues has been synthesized and evaluated as inhibitors of ubiquitin-dependent processes. Dimer analogues were synthesized by cross-linking ubiquitin containing a terminal cysteine (G76C) to ubiquitin containing cysteine at position 11 ((76-11)Ub(2)), 29 ((76-29)Ub(2)), 48 ((76-48)Ub(2)), or 63 ((76-63)Ub(2)). A head-to-head dimer of cysteine G76C ((76-76)Ub(2)) served as a control. These analogues are mimics of the different chain linkages observed in natural polyubiquitin chains. All analogues showed weak inhibition toward the catalytic domain of UCH-L3 and a UBP pseudogene. In the absence of ubiquitin, isopeptidase T was inhibited only by the dimer linked through residue 29. In the presence of 0.5 microM ubiquitin, isopeptidase T was inhibited by several of the dimer analogues, with the (76-29)Ub(2) dimer exhibiting a K(i) of 1.8 nM. However, USP14, the human homologue of yeast Ubp6, was not inhibited at the concentrations tested. Some analogues of ubiquitin dimer also acted as selective inhibitors of conjugation and deconjugation of ubiquitin catalyzed by reticulocyte fraction II. (76-76)Ub(2) and (76-11)Ub(2) did not inhibit the conjugation of ubiquitin, while (76-29)Ub(2), (76-48)Ub(2), and (76-63)Ub(2) were potent inhibitors of conjugation. This specificity is consistent with the known ability of cells to form K29-, K48-, and K63-linked polyubiquitin chains. While (76-11)Ub(2), (76-29)Ub(2), and (76-63)Ub(2) inhibited release of ubiquitin from a pool of total conjugates, (76-48)Ub(2) and (76-76)Ub(2) showed no significant inhibition. Isopeptidase T was shown to specifically disassemble two conjugates (assumed to be di- and triubiquitin with masses of 26 and 17 kDa) formed in the reticulocyte lysate system. This activity was inhibited differentially by all dimer analogues. The inhibitor selectivity for deconjugation of the 26 and 17 kDa conjugates was similar to that observed for isopeptidase T. The observations suggest that these two conjugated proteins of the reticulocyte lysate are specific substrates for isopeptidase T in lysates.