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1.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500646

RESUMO

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, ß, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.


Assuntos
Etanolaminas/metabolismo , Fosfolipases A2/metabolismo , Isoformas de Proteínas/metabolismo , Aciltransferases/metabolismo , Amidas/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Endocanabinoides/metabolismo , Etanolamina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos/metabolismo , Ácidos Palmíticos/metabolismo , Fosfatidiletanolaminas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Alcamidas Poli-Insaturadas/metabolismo
2.
Eur J Med Chem ; 223: 113668, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198149

RESUMO

Based on successful antitubercular isoniazid scaffold we have designed its "mee-too" analogues by a combination of this drug linked with substituted anilines through pyruvic acid as a bridge. Lipophilicity important for passive diffusion through impenetrable mycobacterial cell wall was increased by halogen substitution on the aniline. We prepared twenty new 2-(2-isonicotinoylhydrazineylidene)propanamides that were assayed against susceptible Mycobacterium tuberculosis H37Rv, nontuberculous mycobacteria, and also multidrug-resistant tuberculous strains (MDR-TB). All the compounds showed excellent activity not only against Mtb. (minimum inhibitory concentrations, MIC, from ≤0.03 µM), but also against M. kansasii (MIC ≥2 µM). The most active molecules have CF3 and OCF3 substituent in the position 4 on the aniline ring. MIC against MDR-TB were from 8 µM. The most effective derivatives were used for the mechanism of action investigation. The treatment of Mtb. H37Ra with tested compounds led to decreased production of mycolic acids and the strains overproducing InhA were more resistant to them. These results confirm that studied compounds inhibit the enoyl-acyl carrier protein reductase (InhA) in mycobacteria. The compounds did not show any cytotoxic and cytostatic activity for HepG2 cells. The amides can be considered as a promising scaffold for antitubercular drug discovery having better antimicrobial properties than original isoniazid together with a significantly improved pharmaco-toxicological profile.


Assuntos
Amidas/química , Antituberculosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Oxirredutases/antagonistas & inibidores , Amidas/metabolismo , Amidas/farmacologia , Amidas/uso terapêutico , Compostos de Anilina/química , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Proteínas de Bactérias/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Oxirredutases/metabolismo , Ácido Pirúvico/química , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico
3.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069940

RESUMO

All nations which have undergone a nutrition transition have experienced increased frequency and falling latency of chronic degenerative diseases, which are largely driven by chronic inflammatory stress. Dietary supplementation is a valid strategy to reduce the risk and severity of such disorders. Palmitoylethanolamide (PEA) is an endocannabinoid-like lipid mediator with extensively documented anti-inflammatory, analgesic, antimicrobial, immunomodulatory and neuroprotective effects. It is well tolerated and devoid of side effects in animals and humans. PEA's actions on multiple molecular targets while modulating multiple inflammatory mediators provide therapeutic benefits in many applications, including immunity, brain health, allergy, pain modulation, joint health, sleep and recovery. PEA's poor oral bioavailability, a major obstacle in early research, has been overcome by advanced delivery systems now licensed as food supplements. This review summarizes the functionality of PEA, supporting its use as an important dietary supplement for lifestyle management.


Assuntos
Amidas/metabolismo , Amidas/farmacologia , Etanolaminas/metabolismo , Etanolaminas/farmacologia , Ácidos Palmíticos/metabolismo , Ácidos Palmíticos/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Suplementos Nutricionais , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Humanos , Inflamação/imunologia , Mediadores da Inflamação/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Dor/tratamento farmacológico
4.
Int J Biol Macromol ; 185: 251-263, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34161821

RESUMO

The habit of chewing arecanut leads to fibrosis in the oral tissues, which can lead to cancer. Despite high mortality, fibrosis has limited clinical success owing to organ-specific variations, genetic predispositions, and slow progression. Fibrosis is a progressive condition that is unresponsive to medications in the severe phase. To understand underlying macromolecular changes we studied the extracellular matrix's (ECM) key molecular modifications in the early and late phase of arecanut-induced fibrosis in skin. To study the fibrosis, we topically applied arecanut extract on the mice skin. We observed that the matrix changes observe early and late phases based on ECM characteristics including the matrix proteins and the glycans. A spike in the levels of proteoglycans and ß-sheet structures are noted in the early phase. A significant drop in the proteoglycans and strengthening of amide covalent interactions is observed in the late phase. Although, almost no physical changes are noticeable only in the early phase; the late phase observes thick collagen bundling and a 4-fold stiffening of the skin tissue. The study indicates that the temporal interplay of proteins and glycans determine the matrix's severity state while opening avenues to research directed towards the phase-specific clinical discovery.


Assuntos
Areca/química , Matriz Extracelular/metabolismo , Extratos Vegetais/efeitos adversos , Pele/patologia , Células 3T3 , Amidas/metabolismo , Animais , Cromatografia Líquida , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Espectrometria de Massas , Camundongos , Proteoglicanas/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo
5.
Eur J Med Chem ; 222: 113603, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34126456

RESUMO

We reported three distinct series of novel benzothiopyranones, derived from an active metabolite (M-1) of anti-TB agent 6b. These small molecules were evaluated for their biological activities against a range of Mycobacterium tuberculosis (M. tuberculosis) strains. Preliminary druggability evaluation demonstrated that M-1 showed good aqueous solubility and hepatocyte stability. Benzothiopyranones with acyl, sulfonyl and phosphoryl groups exhibited potent in vitro inhibitory activity against M. tuberculosis H37Rv and low cytotoxicity. In particular, compound 3d, containing a benzoate fragment, displayed marked metabolic stability and potent in vitro activity against drug-resistant tuberculosis clinical strains. Further druggability evaluation based on the identified compounds 3d, 4e and 5b is ongoing for the discovery of promising anti-TB agents.


Assuntos
Amidas/farmacologia , Antituberculosos/farmacologia , Benzopiranos/farmacologia , Ésteres/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Amidas/química , Amidas/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Benzopiranos/química , Benzopiranos/metabolismo , Relação Dose-Resposta a Droga , Ésteres/química , Ésteres/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
6.
J Ind Microbiol Biotechnol ; 48(3-4)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-33982054

RESUMO

The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure-activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity.


Assuntos
Genoma Bacteriano , Pirróis/metabolismo , Quinolinas/metabolismo , Streptomyces/metabolismo , Amidas/química , Amidas/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Humanos , Família Multigênica , Pirróis/química , Quinolinas/química , Streptomyces/química , Streptomyces/genética
7.
Angew Chem Int Ed Engl ; 60(26): 14319-14323, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33856715

RESUMO

Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.


Assuntos
Aminoácidos/metabolismo , Metiltransferases/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas , Amidas/química , Amidas/metabolismo , Aminoácidos/química , Metilação , Metiltransferases/química , Peptídeos/química , Conformação Proteica
8.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799405

RESUMO

Palmitoylethanolamide (PEA) is an N-acylethanolamide produced on-demand by the enzyme N-acylphosphatidylethanolamine-preferring phospholipase D (NAPE-PLD). Being a key member of the larger family of bioactive autacoid local injury antagonist amides (ALIAmides), PEA significantly improves the clinical and histopathological stigmata in models of ulcerative colitis (UC). Despite its safety profile, high PEA doses are required in vivo to exert its therapeutic activity; therefore, PEA has been tested only in animals or human biopsy samples, to date. To overcome these limitations, we developed an NAPE-PLD-expressing Lactobacillus paracasei F19 (pNAPE-LP), able to produce PEA under the boost of ultra-low palmitate supply, and investigated its therapeutic potential in a murine model of UC. The coadministration of pNAPE-LP and palmitate led to a time-dependent release of PEA, resulting in a significant amelioration of the clinical and histological damage score, with a significantly reduced neutrophil infiltration, lower expression and release of pro-inflammatory cytokines and oxidative stress markers, and a markedly improved epithelial barrier integrity. We concluded that pNAPE-LP with ultra-low palmitate supply stands as a new method to increase the in situ intestinal delivery of PEA and as a new therapeutic able of controlling intestinal inflammation in inflammatory bowel disease.


Assuntos
Amidas/metabolismo , Colite/tratamento farmacológico , Etanolaminas/metabolismo , Inflamação/tratamento farmacológico , Lactobacillus paracasei/genética , Ácidos Palmíticos/metabolismo , Amidas/farmacologia , Animais , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Etanolaminas/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Lactobacillus paracasei/metabolismo , Engenharia Metabólica , Camundongos , Infiltração de Neutrófilos/efeitos dos fármacos , Ácidos Palmíticos/farmacologia
9.
Sci Rep ; 11(1): 7307, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790352

RESUMO

Outcomes of various clinical studies for the coronavirus disease 2019 (COVID-19) treatment indicated that the drug acts via inhibition of multiple pathways (targets) is likely to be more successful and promising. Keeping this hypothesis intact, the present study describes for the first-time, Grazoprevir, an FDA approved anti-viral drug primarily approved for Hepatitis C Virus (HCV), mediated multiple pathway control via synergistic inhibition of viral entry targeting host cell Angiotensin-Converting Enzyme 2 (ACE-2)/transmembrane serine protease 2 (TMPRSS2) and viral replication targeting RNA-dependent RNA polymerase (RdRP). Molecular modeling followed by in-depth structural analysis clearly demonstrated that Grazoprevir interacts with the key residues of these targets. Futher, Molecular Dynamics (MD) simulations showed stability and burial of key residues after the complex formation. Finally, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis identified the governing force of drug-receptor interactions and stability. Thus, we believe that Grazoprevir could be an effective therapeutics for the treatment of the COVID-19 pandemic with a promise of unlikely drug resistance owing to multiple inhibitions of eukaryotic and viral proteins, thus warrants further clinical studies.


Assuntos
Amidas/metabolismo , Amidas/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Carbamatos/metabolismo , Carbamatos/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Ciclopropanos/metabolismo , Ciclopropanos/farmacologia , Quinoxalinas/metabolismo , Quinoxalinas/farmacologia , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Enzima de Conversão de Angiotensina 2/química , Antivirais/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/química , Reposicionamento de Medicamentos , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Internalização do Vírus/efeitos dos fármacos
10.
Molecules ; 26(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925418

RESUMO

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.


Assuntos
Amidas/química , Ácidos Graxos/química , Lipídeos/química , Amidas/síntese química , Amidas/metabolismo , Animais , Abelhas/química , Bombyx/química , Linhagem Celular , Drosophila melanogaster/química , Ácidos Graxos/síntese química , Ácidos Graxos/metabolismo , Lipídeos/genética , Camundongos , Ovinos , Tribolium/química
11.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921211

RESUMO

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.


Assuntos
Amidas/metabolismo , Corantes Fluorescentes/metabolismo , Proteômica , Epitopos/metabolismo , Células HeLa , Peroxidase do Rábano Silvestre , Humanos , Proteínas do Fator Nuclear 90/metabolismo , Tonsila Palatina/metabolismo , Inclusão em Parafina , Análise de Célula Única , Fixação de Tecidos
12.
Plant Physiol ; 185(2): 369-384, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33721896

RESUMO

Jasmonates (JAs) are plant hormones that regulate the biosynthesis of many secondary metabolites, such as hydroxycinnamic acid amides (HCAAs), through jasmonic acid (JA)-responsive transcription factors (TFs). HCAAs are renowned for their role in plant defense against pathogens. The multidrug and toxic compound extrusion transporter DETOXIFICATION18 (DTX18) has been shown to mediate the extracellular accumulation of HCAAs p-coumaroylagmatine (CouAgm) at the plant surface for defense response. However, little is known about the regulatory mechanism of DTX18 gene expression by TFs. Yeast one-hybrid screening using the DTX18 promoter as bait isolated the key positive regulator redox-responsive TF 1 (RRTF1), which is a member of the AP2/ethylene-response factor family of proteins. RRTF1 is a JA-responsive factor that is required for the transcription of the DTX18 gene, and it thus promotes CouAgm secretion at the plant surface. As a result, overexpression of RRTF1 caused increased resistance against the fungus Botrytis cinerea, whereas rrtf1 mutant plants were more susceptible. Using yeast two-hybrid screening, we identified the BTB/POZ-MATH (BPM) protein BPM1 as an interacting partner of RRTF1. The BPM family of proteins acts as substrate adaptors of CUL3-based E3 ubiquitin ligases, and we found that only BPM1 and BPM3 were able to interact with RRTF1. In addition, we demonstrated that RRTF1 was subjected to degradation through the 26S proteasome pathway and that JA stabilized RRTF1. Knockout of BPM1 and BPM3 in bpm1/3 double mutants enhanced RRTF1 accumulation and DTX18 gene expression, thus increasing resistance to the fungus B. cinerea. Our results provide a better understanding of the fine-tuned regulation of JA-induced TFs in HCAA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Botrytis/fisiologia , Ácidos Cumáricos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Amidas/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Proteínas de Membrana Transportadoras/genética , Mutação , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
13.
Nat Commun ; 12(1): 1696, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727542

RESUMO

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a ß-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.


Assuntos
Amidas/metabolismo , Antibacterianos/biossíntese , Amidas/química , Asparagina/metabolismo , Vias Biossintéticas , Hidroxilação , Modelos Biológicos , Peso Molecular , Myxococcus xanthus/metabolismo , Especificidade por Substrato
14.
Phys Chem Chem Phys ; 23(11): 6746-6757, 2021 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-33711090

RESUMO

COVID-19, the disease caused by the newly discovered coronavirus-SARS-CoV-2, has created a global health, social, and economic crisis. As of mid-January 2021, there are over 90 million confirmed cases and more than 2 million reported deaths due to COVID-19. Currently, there are very limited therapeutics for the treatment or prevention of COVID-19. For this reason, it is important to find drug targets that will lead to the development of safe and effective therapeutics against the disease. The main protease (Mpro) of the virus is an attractive target for the development of effective antiviral therapeutics because it is required for proteolytic cleavage of viral polyproteins. Furthermore, the Mpro has no human homologues, so drugs designed to bind to this target directly have less risk for off-target effects. Recently, several high-resolution crystallographic structures of the Mpro in complex with inhibitors have been determined-to guide drug development and to spur efforts in structure-based drug design. One of the primary objectives of modern structure-based drug design is the accurate prediction of receptor-ligand binding affinities for rational drug design and discovery. Here, we perform rigorous alchemical absolute binding free energy calculations and QM/MM calculations to give insight into the total binding energy of two recently crystallized inhibitors of SARS-CoV-2 Mpro, namely, N3 and α-ketoamide 13b. The total binding energy consists of both covalent and non-covalent binding components since both compounds are covalent inhibitors of the Mpro. Our results indicate that the covalent and non-covalent binding free energy contributions of both inhibitors to the Mpro target differ significantly. The N3 inhibitor has more favourable non-covalent interactions, particularly hydrogen bonding, in the binding site of the Mpro than the α-ketoamide inhibitor. Also, the Gibbs energy of reaction for the Mpro-N3 covalent adduct is greater than the Gibbs reaction energy for the Mpro-α-ketoamide covalent adduct. These differences in the covalent and non-covalent binding free energy contributions for both inhibitors could be a plausible explanation for their in vitro differences in antiviral activity. Our findings are consistent with the reversible and irreversible character of both inhibitors as reported by experiment and highlight the importance of both covalent and non-covalent binding free energy contributions to the absolute binding affinity of a covalent inhibitor towards its target. This information could prove useful in the rational design, discovery, and evaluation of potent SARS-CoV-2 Mpro inhibitors for targeted antiviral therapy.


Assuntos
Peptidomiméticos/química , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Desenho de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Simulação de Dinâmica Molecular , Peptidomiméticos/metabolismo , Inibidores de Proteases/metabolismo , Teoria Quântica , SARS-CoV-2/isolamento & purificação , Termodinâmica , Proteínas da Matriz Viral/metabolismo
15.
Eur J Med Chem ; 215: 113267, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33639344

RESUMO

Inhibitors of the proteasome have been extensively studied for their applications in the treatment of human diseases such as hematologic malignancies, autoimmune disorders, and viral infections. Many of the proteasome inhibitors reported in the literature target the non-primed site of proteasome's substrate binding pocket. In this study, we designed, synthesized and characterized a series of novel α-keto phenylamide derivatives aimed at both the primed and non-primed sites of the proteasome. In these derivatives, different substituted phenyl groups at the head group targeting the primed site were incorporated in order to investigate their structure-activity relationship and optimize the potency of α-keto phenylamides. In addition, the biological effects of modifications at the cap moiety, P1, P2 and P3 side chain positions were explored. Many derivatives displayed highly potent biological activities in proteasome inhibition and anticancer activity against a panel of six cancer cell lines, which were further rationalized by molecular modeling analyses. Furthermore, a representative α-ketoamide derivative was tested and found to be active in inhibiting the cellular infection of SARS-CoV-2 which causes the COVID-19 pandemic. These results demonstrate that this new class of α-ketoamide derivatives are potent anticancer agents and provide experimental evidence of the anti-SARS-CoV-2 effect by one of them, thus suggesting a possible new lead to develop antiviral therapeutics for COVID-19.


Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Cetonas/farmacologia , Inibidores de Proteassoma/farmacologia , SARS-CoV-2/efeitos dos fármacos , Amidas/síntese química , Amidas/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antivirais/síntese química , Antivirais/metabolismo , Sítios de Ligação , Calpaína/química , Calpaína/metabolismo , Linhagem Celular Tumoral , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cetonas/síntese química , Cetonas/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
16.
Int J Mol Sci ; 22(3)2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494185

RESUMO

Oleoylethanolamide and palmitoylethanolamide are members of the fatty acid ethanolamide family, also known as acylethanolamides. Their physiological effects, including glucose homeostasis, anti-inflammation, anti-anaphylactic, analgesia, and hypophagia, have been reported. They have affinity for different receptor proteins, including nuclear receptors such as PPARα, channels such as TRPV1, and membrane receptors such as GPR119 and GPR55. In the present review, the pathophysiological functions of fatty acid ethanolamides have been discussed from the perspective of receptor pharmacology and drug discovery.


Assuntos
Amidas/metabolismo , Endocanabinoides/metabolismo , Etanolaminas/metabolismo , Ácidos Oleicos/metabolismo , Ácidos Palmíticos/metabolismo , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Analgesia , Animais , Anti-Inflamatórios/farmacologia , Desenvolvimento de Medicamentos , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Mobilização Lipídica , Receptores Acoplados a Proteínas G/antagonistas & inibidores
17.
Angew Chem Int Ed Engl ; 60(31): 16824-16855, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-33453143

RESUMO

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late-stage modification often superior to conventional de novo synthesis. Enzymes have proven to be useful for direct introduction of functional groups into complex scaffolds, as well as for rapid diversification of compound libraries. Particularly important and highly topical are enzyme-catalysed oxyfunctionalisations, halogenations, methylations, reductions, and amide bond formations due to the high prevalence of these motifs in pharmaceuticals. This Review gives an overview of the strengths and limitations of enzymatic late-stage modifications using native and engineered enzymes in synthesis while focusing on important examples in drug development.


Assuntos
Amidas/metabolismo , Enzimas/metabolismo , Amidas/química , Biocatálise
18.
Biophys Chem ; 269: 106510, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33285430

RESUMO

The search for therapeutic drugs that can neutralize the effects of COVID-2019 (SARS-CoV-2) infection is the main focus of current research. The coronavirus main protease (Mpro) is an attractive target for anti-coronavirus drug design. Further, α-ketoamide is proved to be very effective as a reversible covalent-inhibitor against cysteine proteases. Herein, we report on the non-covalent to the covalent adduct formation mechanism of α-ketoamide-based inhibitor with the enzyme active site amino acids by QM/SQM model (QM = quantum mechanical, SQM = semi-empirical QM). To uncover the mechanism, we focused on two approaches: a concerted and a stepwise fashion. The concerted pathway proceeds via deprotonation of the thiol of cysteine (here, Cys145 SγH) and simultaneous reversible nucleophilic attack of sulfur onto the α-ketoamide warhead. In this work, we propose three plausible concerted pathways. On the contrary, in a traditional two-stage pathway, the first step is proton transfer from Cys145 SγH to His41 Nδ forming an ion pair, and consecutively, in the second step, the thiolate ion attacks the α-keto group to form a thiohemiketal. In this reaction, we find that the stability of the tetrahedral intermediate oxyanion/hydroxyl group plays an important role. Moreover, as the α-keto group has two faces Si or Re for the nucleophilic attack, we considered both possibilities of attack leading to S- and R-thiohemiketal. We computed the structural, electronic, and energetic parameters of all stationary points including transition states via ONIOM and pure DFT method. Additionally, to characterize covalent, weak noncovalent interaction (NCI) and hydrogen-bonds, we applied NCI-reduced density gradient (NCI-RDG) methods along with Bader's Quantum Theory of Atoms-in-Molecules (QTAIM) and natural bonding orbital (NBO) analysis.


Assuntos
Amidas/química , Coronavirus/enzimologia , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Proteínas Virais/antagonistas & inibidores , Amidas/metabolismo , Sítios de Ligação , Domínio Catalítico , Coronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Proteínas M de Coronavírus/antagonistas & inibidores , Proteínas M de Coronavírus/metabolismo , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Teoria Quântica , Termodinâmica , Proteínas Virais/metabolismo
19.
Protein Expr Purif ; 177: 105751, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931916

RESUMO

A novel amidase (TAM) was identified and cloned from the genome of Thauera sinica K11. The recombinant protein was purified to homogeneity by one-step affinity chromatography for up to 26.4-fold with a yield of 38.1%. Gel filtration chromatography and SDS-PAGE revealed that the enzyme was a tetramer with a subunit of approximately 37.5 kDa. The amidase exhibited the maximum acyl transfer activity at 45 °C and pH 7.0, and it was highly stable over a wide pH range of 6.0-11.0. Inhibition of enzyme activity was observed in the presence of metal ions, thiol reagents and organic solvents. TAM showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides. For linear aliphatic monoamides, the acyl transfer activity of TAM was decreased with the extension of the carbon chain length, and thus the highest activity of 228.2 U/mg was obtained when formamide was used as substrate. This distinct selectivity of amidase to linear aliphatic monoamides expanded the findings of signature amidases to substrate specificity.


Assuntos
Amidas/metabolismo , Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Subunidades Proteicas/metabolismo , Thauera/enzimologia , Amidoidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Bacteriano , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Multimerização Proteica , Subunidades Proteicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura , Thauera/classificação , Thauera/genética
20.
Int J Mol Sci ; 21(23)2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33260658

RESUMO

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas; products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated; in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA's ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.


Assuntos
Amidas/química , Amidas/metabolismo , Etanolaminas/química , Etanolaminas/metabolismo , Ácidos Graxos/química , Modelos Moleculares , Ácidos Palmíticos/química , Ácidos Palmíticos/metabolismo , Animais , Forma Celular , Sobrevivência Celular , Humanos , Hidrólise , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ligantes , Camundongos , Microglia/metabolismo , NF-kappa B/metabolismo , PPAR alfa/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato , Células THP-1 , Termodinâmica , Fator de Necrose Tumoral alfa/metabolismo
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