RESUMO
Inhibition of steroid sulfatase (STS) decreases estrogen production and thus, suppresses tumor proliferation. Inspired by irosustat, the first STS inhibitor in clinical trials, we explored twenty-one tricyclic and tetra-heterocyclic coumarin-based derivatives. Their STS enzyme kinetic parameters, docking models, and cytotoxicity toward breast cancer and normal cells were evaluated. Tricyclic derivative 9e and tetracyclic derivative 10c were the most promising irreversible inhibitors developed in this study, with KI of 0.05 and 0.4 nM, and kinact/KI ratios of 28.6 and 19.1 nM-1min-1 on human placenta STS, respectively.
Assuntos
Neoplasias da Mama , Esteril-Sulfatase , Gravidez , Feminino , Humanos , Cinética , Relação Estrutura-Atividade , Ácidos Sulfônicos , Neoplasias da Mama/tratamento farmacológico , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêuticoRESUMO
Competitive proteome profiling is a powerful approach for the identification of targets of small molecules. This approach usually employs an inhibitor-derived probe or a cysteine-reactive probe such as an IA-alkyne in a comparison between inhibitor-treated and untreated samples, thus enabling distinction between genuine targets and nonspecific labeling. We have developed an active probe derived from an EGFR inhibitor, afatinib, and a cysteine reactive probe, an alkyne-containing α,ß-unsaturated amide, to compare their characterization of cellular targets. In both approaches, myosin heavy chain 9 (MYH9) was identified as an off-target. Subsequent functional validation experiments suggested that MYH9 might be involved in the function of afatinib.
Assuntos
Cisteína , Proteoma , Afatinib , AlcinosRESUMO
Steroid sulfatase inhibitors block the local production of estrogenic steroids and are attractive agents for the treatment of estrogen-dependent cancers. Inspiration of coumarin-based inhibitors, we synthesized thirty-two 5-oxa-1,2,3,4-tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates, focusing on the substitution derivatives on the adjacent phenyl ring and evaluated their abilities to block STS from human placenta and MCF-7 cells. SAR analysis revealed that the incorporation of chlorine at either meta and/or para position of the adjacent phenyl ring of the tricyclic skeleton enhanced STS inhibition. Di-substitutions at the adjacent phenyl ring were superior to mono and tri-substitutions. Further kinetic analysis of these compounds revealed that chloride-bearing compounds, such as 19m, 19v, and 19w, had KI of 0.02 to 0.11 nM and kinact/KI ratios of 8.8-17.5 nM-1min-1, a parameter indicated for the efficiency of irreversible inhibition. We also used the docking model to illustrate the difference in STS inhibitory potency of compounds. Finally, the safety and anti-cancer activity of selected compounds 19m, 19v, and 19w were also studied, showing the results of low cytotoxicity on NHDF cell line and being more potent than irosustat on ZR-75-1 cell, which was a hormone-dependent cancer cell line with high STS expression.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Placenta , Esteril-Sulfatase , Ácidos Sulfônicos , Feminino , Humanos , Gravidez , Inibidores Enzimáticos/farmacologia , Cinética , Esteril-Sulfatase/antagonistas & inibidores , Relação Estrutura-Atividade , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Placenta/enzimologia , Células MCF-7RESUMO
Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM-based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis-acting QM-based substrates could be used as activity-based probes to identify various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indolquinonas/química , Indolquinonas/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Hidrólise , Organofosfatos/metabolismoRESUMO
Neurological and neurodegenerative diseases are debilitating conditions, and frequently lack an effective treatment. Monoacylglycerol lipase (MAGL) is a key enzyme involved in the metabolism of 2-AG (2-arachidonoylglycerol), a neuroprotective endocannabinoid intimately linked to the generation of pro- and anti-inflammatory molecules. Consequently, synthesizing selective MAGL inhibitors has become a focus point in drug design and development. The purpose of this review was to summarize the diverse synthetic scaffolds of MAGL inhibitors concerning their potency, mechanisms of action and potential therapeutic applications, focusing on the results of studies published in the past five years. The main irreversible inhibitors identified were derivatives of hexafluoroisopropyl alcohol carbamates, glycol carbamates, azetidone triazole ureas and benzisothiazolinone, whereas the most promising reversible inhibitors were derivatives of salicylketoxime, piperidine, pyrrolidone and azetidinyl amides. We reviewed the results of in-depth chemical, mechanistic and computational studies on MAGL inhibitors, in addition to the results of in vitro findings concerning selectivity and potency of inhibitors, using the half maximal inhibitory concentration (IC50) as an indicator of their effect on MAGL. Further, for highlighting the potential usefulness of highly selective and effective inhibitors, we examined the preclinical in vivo reports regarding the promising therapeutic applications of MAGL pharmacological inhibition.
Assuntos
Monoacilglicerol Lipases , Doenças Neurodegenerativas , Animais , Inibidores Enzimáticos/farmacologia , HumanosRESUMO
Steroid sulfatase (STS) is a sulfatase enzyme that catalyzes the conversion of sulfated steroid precursors to free steroid. The inhibition of STS could abate estrogenic steroids that stimulate the proliferation and development of breast cancer, and therefore STS is a potential target for adjuvant endocrine therapy. In this study, a series of 3-benzylaminocoumarin-7-O-sulfamate derivatives targeting STS were designed and synthesized. Structure-relationship activities (SAR) analysis revealed that attachment of a benzylamino group at the 3-position of coumarin improved inhibitory activity. Compound 3j was found to have the highest inhibition activity against human placenta isolated STS (IC50 0.13 µM) and MCF-7 cell lines (IC50 1.35 µM). Kinetic studies found compound 3j to be an irreversible inhibitor of STS, with KI and kinact value of 86.9 nM and 158.7 min-1, respectively.
Assuntos
Cumarínicos/química , Cumarínicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Esteril-Sulfatase/antagonistas & inibidores , Aminação , Compostos de Benzil/síntese química , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Cumarínicos/síntese química , Inibidores Enzimáticos/síntese química , Feminino , Humanos , Células MCF-7 , Placenta/enzimologia , Gravidez , Esteril-Sulfatase/metabolismo , Relação Estrutura-Atividade , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologiaRESUMO
Glutathione S-transferase π (GSTP1-1 ) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP1-1 is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP1-1 both in vitro and in cellulo with a long duration of action.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa/análogos & derivados , Glutationa/farmacologia , Sulfonas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Glutationa/síntese química , Glutationa S-Transferase pi/química , Humanos , Simulação de Acoplamento Molecular , Sulfonas/síntese química , Tirosina/químicaRESUMO
The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is the best recognized and most widely implicated regulator of caspase-1 activation. It is a key regulator of innate immune response and is involved in many pathophysiological processes. Recent evidences for its inappropriate activation in autoinflammatory, autoimmune, as well as in neurodegenerative diseases attract a growing interest toward the development of small molecules NLRP3 inhibitors. Based on the knowledge of biochemical and structural aspects of NLRP3 activation, one successful strategy in the identification of NLRP3 inhibitors relies on the development of covalent irreversible inhibitors. Covalent inhibitors are reactive electrophilic molecules able to alkylate nucleophiles in the target protein. These inhibitors could ensure good efficacy and prolonged duration of action both in vitro and in vivo. In spite of these advantages, effects on other signalling pathways, prone to alkylation, may occur. In this review, we will illustrate the chemistry and the biological action of the most studied covalent NLRP3 inhibitors developed so far. A description of what we know about their mechanism of action will address the reader toward a critical understanding of NLRP3 inhibition by electrophilic compounds.
Assuntos
Descoberta de Drogas/métodos , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Humanos , Inflamassomos/química , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Conformação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Mantle cell lymphoma (MCL) is characterized by the translocation t(11;14) (q13;q32), resulting in the overexpression of cyclin-D1. The progression of MCL is an interaction of multitarget and multilink regulation. It has been proven that Bruton's tyrosine kinase (BTK) is commonly overexpressed in MCL, which makes it a focus of targeted therapy for MCL. Irreversible inhibitors usually have great potency, rapid onset of inhibition and long duration of drug action. Herein, structural modification via an open-loop strategy based on lead compound ibrutinib (IBN) was performed, leading to a series of pyrazole derivatives. Compounds 19c, 19'c, 21c and 21'c showed potent effect in MCL cells with IC50 values lower than 1⯵M, and a more than 3-28-fold increase in antiproliferative activity compared with IBN.
Assuntos
Antineoplásicos/química , Desenho de Fármacos , Pirazóis/química , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Meia-Vida , Humanos , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Microssomos Hepáticos/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.
Assuntos
Descoberta de Drogas , Reposicionamento de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , IMP Desidrogenase/antagonistas & inibidores , Animais , Azóis/química , Azóis/isolamento & purificação , Azóis/farmacologia , Cryptosporidium parvum/enzimologia , Dissulfiram/química , Dissulfiram/isolamento & purificação , Dissulfiram/farmacologia , Inibidores Enzimáticos/química , Humanos , IMP Desidrogenase/metabolismo , Isoindóis , Cinética , Camundongos , Camundongos SCID , Compostos Organosselênicos/química , Compostos Organosselênicos/isolamento & purificação , Compostos Organosselênicos/farmacologia , Estudo de Prova de Conceito , Propilenoglicóis/química , Propilenoglicóis/isolamento & purificação , Propilenoglicóis/farmacologia , Bibliotecas de Moléculas PequenasRESUMO
Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis typeâ I (MPSâ I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme® ). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1-3 adopt a half-chair conformation in solution (4 H3 and 3 H4 ), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1-3 may need to overcome an energy barrier in order to switch from the 4 H3 conformation to the transition state (2, 5 B) binding conformation before reacting and adopting a covalent 5 S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPSâ I patients.
Assuntos
Aziridinas/química , Cicloexanóis/química , Inibidores Enzimáticos/química , Iduronidase/antagonistas & inibidores , Iduronidase/análise , Cromatografia Líquida , Ensaios Enzimáticos , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Proteínas Recombinantes/análise , Coloração e Rotulagem , Espectrometria de Massas em TandemRESUMO
In the present study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most promising compound 25h inhibited HCC827 and H1975 cells growth with the IC50 values of 0.025⯵M and 0.49⯵M, respectively. Meanwhile, 25h displayed potent inhibitory activity against the EGFRL858R (IC50â¯=â¯1.7â¯nM) and EGFRL858R/T790M (IC50â¯=â¯23.3â¯nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound 25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse model at 50â¯mg/kg in vivo. These results indicated that the 2,4-disubstituted 6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR inhibitors and potent anticancer agents.
Assuntos
Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The structure-based design of M-525 as the first-in-class, highly potent, irreversible small-molecule inhibitor of the menin-MLL interaction is presented. M-525 targets cellular menin protein at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL-regulated gene expression in MLL leukemia cells. M-525 demonstrates high cellular specificity over non-MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co-crystal structure of M-525 in complex with menin firmly establish its mode of action. A single administration of M-525 effectively suppresses MLL-regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M-525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.
Assuntos
Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteína de Leucina Linfoide-Mieloide/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.
RESUMO
Covalent drugs have attracted increasing attention in recent years due to good inhibitory activity and selectivity. Targeting noncatalytic cysteines with irreversible inhibitors is a powerful approach for enhancing pharmacological potency and selectivity because cysteines can form covalent bonds with inhibitors through their nucleophilic thiol groups. However, most human kinases have multiple noncatalytic cysteines within the active site; to accurately predict which cysteine is most likely to form covalent bonds is of great importance but remains a challenge when designing irreversible inhibitors. In this work, FTMap was first applied to check its ability in predicting covalent binding site defined as the region where covalent bonds are formed between cysteines and irreversible inhibitors. Results show that it has excellent performance in detecting the hot spots within the binding pocket, and its hydrogen bond interaction frequency analysis could give us some interesting instructions for identification of covalent binding cysteines. Furthermore, we proposed a simple but useful covalent fragment probing approach and showed that it successfully predicted the covalent binding site of seven targets. By adopting a distance-based method, we observed that the closer the nucleophiles of covalent warheads are to the thiol group of a cysteine, the higher the possibility that a cysteine is prone to form a covalent bond. We believe that the combination of FTMap and our distance-based covalent fragment probing method can become a useful tool in detecting the covalent binding site of these targets.
Assuntos
Cisteína/química , Inibidores de Proteínas Quinases/química , Sítios de Ligação , Cisteína/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Inibidores de Proteínas Quinases/metabolismo , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
We identified and characterized ß-aminoketones as prodrugs for irreversible MetAP inhibitors that are selective for the MetAP-1 subtype. ß-Aminoketones with certain structural features form α,ß-unsaturated ketones under physiological conditions, which bind covalently and selectively to cysteines in the S1 pocket of MetAP-1. The binding mode was confirmed by X-ray crystallography and assays with the MetAPs from Escherichia coli, Staphylococcus aureus and both human isoforms. The initially identified tetralone derivatives showed complete selectivity for E. coli MetAP versus human MetAP-1 and MetAP-2. Rational design of indanone analogs yielded compounds with selectivity for the human type-1 versus the human type-2 MetAP.
Assuntos
Aminopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Cetonas/química , Pró-Fármacos/química , Aminopeptidases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Humanos , Cetonas/metabolismo , Metionil Aminopeptidases , Simulação de Acoplamento Molecular , Pró-Fármacos/metabolismo , Estrutura Terciária de Proteína , Staphylococcus aureus/enzimologia , Relação Estrutura-AtividadeRESUMO
Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the protein arginine deiminases (PADs 1-4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.
Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Hidrolases/antagonistas & inibidores , Estreptonigrina/química , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Hidrolases/metabolismo , Cinética , Camundongos , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Desiminases de Arginina em Proteínas , Quinolinas/química , Estreptonigrina/metabolismo , Estreptonigrina/farmacologia , Relação Estrutura-AtividadeRESUMO
TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.
Assuntos
Alquilantes , Domínio Catalítico , Proteínas de Ligação ao GTP , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Transglutaminases/química , Transglutaminases/metabolismo , Transglutaminases/antagonistas & inibidores , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Alquilantes/química , Alquilantes/farmacologia , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia , Conformação Proteica , Cinética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologiaRESUMO
In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1h) and late (8h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5-6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.
Assuntos
Receptores ErbB/antagonistas & inibidores , Quinazolinas , Compostos de Anilina/química , Compostos de Anilina/farmacocinética , Compostos de Anilina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Química Farmacêutica , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Fatores de TempoRESUMO
Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer's disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases.