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1.
Molecules ; 26(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34443552

RESUMO

The aims of this study were to evaluate the antioxidant properties, to investigate the content of major secondary metabolites in Ginkgo biloba cell cultures, and to determine the change in the production of phenolic acids by adding phenylalanine to the culture medium. Three in vitro methods, which depend on different mechanisms, were used for assessing the antioxidant activity of the extract: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays. The extract showed moderate activity both in the DPPH and in the reducing power assays (IC50 = 1.966 ± 0.058 mg/mL; ASE/mL = 16.31 ± 1.20); instead, it was found to possess good chelating properties reaching approximately 70% activity at the highest tested dose. The total phenolic, total flavonoid, and condensed tannin content of G. biloba cell culture extract was spectrophotometrically determined. The phenolic acid content was investigated by RP-HPLC, and the major metabolites-protocatechuic and p-hydroxybenzoic acids-were isolated and investigated by 1H NMR. The results showed that phenylalanine added to G. biloba cell cultures at concentrations of 100, 150, and 200 mg/150 mL increased the production of phenolic acids. Cultures that were grown for 3 weeks and collected after 4 days of phenylalanine supplementation at high concentration showed maximal content of phenolic acids (73.76 mg/100 g DW).


Assuntos
Antioxidantes/metabolismo , Ginkgo biloba/efeitos dos fármacos , Ginkgo biloba/metabolismo , Hidroxibenzoatos/metabolismo , Fenilalanina/farmacologia , Técnicas de Cultura de Células , Relação Dose-Resposta a Droga , Ginkgo biloba/citologia
2.
Nucleic Acids Res ; 49(16): 9374-9388, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34390346

RESUMO

The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA/biossíntese , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética , Benzofenonas/farmacologia , DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Mutação/genética , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Ubiquitina/química , Ubiquitina/ultraestrutura
3.
Molecules ; 26(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206893

RESUMO

PF74 is a capsid-targeting inhibitor of HIV replication that effectively perturbs the highly sensitive viral uncoating process. A lack of information regarding the optical purity (enantiomeric excess) of the single stereogenic centre of PF74 has resulted in ambiguity as to the potency of different samples of this compound. Herein is described the synthesis of enantiomerically enriched (S)- and (R)-PF74 and further enrichment of the samples (≥98%) using chiral HPLC resolution. The biological activities of each enantiomer were then evaluated, which determined (S)-PF74 (IC50 1.5 µM) to be significantly more active than (R)-PF74 (IC50 19 µM). Computational docking studies were then conducted to rationalise this large discrepancy in activity, which indicated different binding conformations for each enantiomer. The binding energy of the conformation adopted by the more active (S)-PF74 (ΔG = -73.8 kcal/mol) was calculated to be more favourable than the conformation adopted by the less active (R)-enantiomer (ΔG = -55.8 kcal/mol) in agreement with experimental observations.


Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Indóis/farmacologia , Fenilalanina/análogos & derivados , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Capsídeo/química , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Indóis/síntese química , Indóis/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Fenilalanina/síntese química , Fenilalanina/química , Fenilalanina/farmacologia , Estereoisomerismo
4.
RNA ; 27(10): 1148-1154, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34230059

RESUMO

CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5'-3' exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies show that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-molecule inhibitors against this enzyme. CPSF73 nuclease activity has so far been demonstrated using a gel-based end-point assay, using radiolabeled or fluorescently labeled RNA substrates. By taking advantage of unique properties of the U7 machinery, we have developed a novel, real-time fluorescence assay for the nuclease activity of CPSF73. This assay is facile and high-throughput, and should also be helpful for the discovery of new CPSF73 inhibitors.


Assuntos
Bioensaio , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Histonas/metabolismo , Processamento de Terminações 3' de RNA , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Sistema Livre de Células , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluorescência , Corantes Fluorescentes/química , Histonas/química , Histonas/genética , Humanos , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Proteólise , Precursores de RNA/química , Precursores de RNA/genética , Rodaminas/química , Ribonucleoproteína Nuclear Pequena U7/química , Ribonucleoproteína Nuclear Pequena U7/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
5.
Antimicrob Agents Chemother ; 65(10): e0103921, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34228546

RESUMO

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection and an attractive therapeutic target for antivirals. Here, we report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is 1 of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between reverse transcription and proviral DNA integration, without any effect on RT activity in vitro, suggesting that this compound may affect HIV-1 core disassembly (uncoating) as well as a CA inhibitor, PF74. Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and noncovalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues. These results indicate that ACAi-028 is a new CA inhibitor by binding to the novel hydrophobic pocket in CA-NTD. This study demonstrates that a compound, ACAi-028, targeting the hydrophobic pocket should be a promising anti-HIV-1 inhibitor.


Assuntos
Fármacos Anti-HIV , HIV-1 , Fármacos Anti-HIV/farmacologia , Capsídeo , Proteínas do Capsídeo/genética , Humanos , Fenilalanina/farmacologia , Replicação Viral
6.
Am J Respir Cell Mol Biol ; 65(5): 544-554, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34181859

RESUMO

Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.


Assuntos
Asma/genética , Interações Hospedeiro-Patógeno/genética , Pulmão/virologia , Infecções por Picornaviridae/genética , Doença Pulmonar Obstrutiva Crônica/genética , Idoso , Antivirais/farmacologia , Asma/patologia , Brônquios/patologia , Brônquios/fisiologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Isoxazóis/farmacologia , Pulmão/fisiologia , Masculino , Pessoa de Meia-Idade , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Pirrolidinonas/farmacologia , Rhinovirus/patogenicidade , Valina/análogos & derivados , Valina/farmacologia
7.
Food Funct ; 12(9): 4092-4104, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33977979

RESUMO

Baicalin shows excellent protective effects against Mycoplasma gallisepticum (MG) induced inflammatory injury as discussed in our previous studies. However, the physiological effects of baicalin are notable in contrast to its low bioavailability, and the critical mechanism for the protective effects of baicalin against MG infection is still unclear. The main objective of this study was to investigate whether baicalin alleviates MG-induced lung inflammatory injury through regulating gut microbiota. Using an MG infection model, results showed that baicalin treatment significantly reduced MG colonization and ameliorated the abnormal pathological changes in the lung. Baicalin treatment also reduced the level of proinflammatory cytokines and suppressed proinflammatory protein expression. Notably, MG infection changed the gut microbiota composition, however, the abnormal gut microbiota composition was partially alleviated by baicalin treatment. Baicalin significantly enriched the commensal bacterium Bacteroides fragilis, and gavaged with Bacteroides fragilis alleviating MG infection-induced inflammatory injury in the lung. In addition, baicalin reversed peripheral accumulation of phenylalanine induced by MG infection. Importantly, increased phenylalanine induced excessive necroptosis through the modulation of gga-miR-190a-3p-Fas-associated protein with death domain (FADD) axis in HD11 macrophages. Together, our findings highlighted the role of gut microbiota and phenylalanine metabolism in MG infection and confirmed that baicalin could effectively inhibit MG-induced inflammatory injury in the lung by remodeling the gut microbiota and phenylalanine metabolism.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Galinhas , Flavonoides/uso terapêutico , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum , Fenilalanina/metabolismo , Animais , Bacteroides fragilis/crescimento & desenvolvimento , Citocinas/metabolismo , Disbiose/tratamento farmacológico , Disbiose/veterinária , Inflamação/veterinária , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/metabolismo , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/patologia , Mycoplasma gallisepticum/crescimento & desenvolvimento , Necroptose , Necrose , Fenilalanina/farmacologia , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia
8.
Viruses ; 13(3)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804121

RESUMO

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 µM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.


Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/metabolismo , HIV-1/efeitos dos fármacos , Indóis/metabolismo , Fenilalanina/análogos & derivados , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fármacos Anti-HIV/isolamento & purificação , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Desenho de Fármacos , Células HEK293 , Humanos , Indóis/farmacologia , Fígado/efeitos dos fármacos , Camundongos , Microssomos/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Fenilalanina/metabolismo , Fenilalanina/farmacologia , Replicação Viral/efeitos dos fármacos
9.
PLoS One ; 16(4): e0250126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857222

RESUMO

Amino acid metabolism plays an important role in controlling blood pressure by regulating the production of NO and ROS. The present study examined amino acid levels in the serum of Dahl SS rats and SS.13BN rats fed a low or high salt diet. We observed that 8 of 27 amino acids responded to a high salt diet in SS rats. Thus, we hypothesized that a defect in amino acids may contribute to the development of salt-induced hypertension. L-phenylalanine was used to treat SS rats with a low or high salt diet. The results demonstrated that L-phenylalanine supplementation significantly enhanced the serum nitrite levels and attenuated the high salt-induced hypertension in SS rats. Low levels of BH4 and nitrite and the impaired vascular response to acetylcholine were rescued by L-phenylalanine supplementation. Moreover, increased GTP cyclohydrolase (GCH1) mRNA, levels of BH4 and nitrite, and reduced superoxide production were observed in the kidneys of hypertensive SS rats with L-phenylalanine. The antihypertensive effects of L-phenylalanine might be mediated by enhancing BH4 biosynthesis and decreasing superoxide production from NO synthase, thereby protecting vascular and kidney function with reduced ROS and elevated NO levels. The present study demonstrated that L-phenylalanine supplementation restored vascular function, suggesting L-phenylalanine represented a potential target to attenuate high salt-sensitive hypertension through GCH1-BH4.


Assuntos
Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , GTP Cicloidrolase/metabolismo , Hipertensão/tratamento farmacológico , Óxido Nítrico Sintase/metabolismo , Fenilalanina/uso terapêutico , Sódio na Dieta , Animais , Anti-Hipertensivos/farmacologia , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Fenilalanina/farmacologia , Ratos , Ratos Endogâmicos Dahl , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo
10.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33787221

RESUMO

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Assuntos
Antivirais/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Fenilalanina/farmacologia , Piperazinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Compostos de Tosil/farmacologia , Animais , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Testes de Sensibilidade Microbiana , Domínios Proteicos , Proteólise , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos
11.
mBio ; 12(2)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785634

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 µM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency-Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Azul Evans/farmacologia , Humanos , Simulação de Acoplamento Molecular , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Ligação Proteica/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Sulfonas/farmacologia , Ressonância de Plasmônio de Superfície , Células Vero
12.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33641565

RESUMO

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Ocludina/genética , Ocludina/metabolismo , Oxirredução/efeitos dos fármacos , Fenilalanina/análogos & derivados , Cultura Primária de Células , Serina Endopeptidases/genética
13.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673444

RESUMO

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic ß-lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3'-phenyl-2'-dibenzylamino)prop-1'-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca2+ entry to larger or lesser extent. Potency follows the order 3R,4R,2'R > 3S,4S,2'R ≅ 3R,4R,2'S > 3S,4S,2'S, with the most potent diastereoisomer showing a half inhibitory concentration (IC50) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these ß-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.


Assuntos
Neurônios/metabolismo , Fenilalanina/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , beta-Lactamas/farmacologia , Animais , Células Cultivadas , Gânglios Espinais/metabolismo , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Fenilalanina/análogos & derivados , Fenilalanina/química , Ratos , Relação Estrutura-Atividade , beta-Lactamas/química
14.
Sci Rep ; 11(1): 874, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441650

RESUMO

Currently, there is no appropriate treatment option for patients with sorafenib-resistant hepatocellular carcinoma (HCC). Meanwhile, pronounced anticancer activities of newly-developed mitochondria-accumulating self-assembly peptides (Mito-FF) have been demonstrated. This study intended to determine the anticancer effects of Mito-FF against sorafenib-resistant Huh7 (Huh7-R) cells. Compared to sorafenib, Mito-FF led to the generation of relatively higher amounts of mitochondrial reactive oxygen species (ROS) as well as the greater reduction in the expression of antioxidant enzymes (P < 0.05). Mito-FF was found to significantly promote cell apoptosis while inhibiting cell proliferation of Huh7-R cells. Mito-FF also reduces the expression of antioxidant enzymes while significantly increasing mitochondrial ROS in Huh7-R cells. The pro-apoptotic effect of Mito-FFs for Huh7-R cells is possibly caused by their up-regulation of mitochondrial ROS, which is caused by the destruction of the mitochondria of HCC cells.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Compostos Organofosforados/uso terapêutico , Peptídeos/farmacologia , Fenilalanina/uso terapêutico , Pirenos/uso terapêutico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Mitocôndrias/metabolismo , Compostos Organofosforados/farmacologia , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Fenilalanina/farmacologia , Pirenos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sorafenibe/farmacologia
15.
ACS Appl Mater Interfaces ; 13(2): 2165-2178, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33400482

RESUMO

Oxidative damage to cells from metabolites at a wound site is one of the trickiest factors inhibiting tissue regeneration, especially with bulk damage. In addition, an excessive inflammatory reaction by the body at the wound site can make it even worse. How to scavenge the reactive oxygen species (ROS) produced from metabolism and inflammatory reactions has become a critical issue in tissue engineering. Here, we utilize the natural bioactive small molecules l-arginine and l-phenylalanine and the growth factor inositol to synthesize a branched poly(ester amide) (BPEA) to fabricate BPEA nanocapsules for vitamin E delivery at wound sites. BPEA nanocapsules loaded with vitamin E (BPEA@VE NCs) could protect cells from both extracellular and intracellular damage by scavenging ROS. Simultaneously, the inflammatory reaction could also be downregulated, benefiting from the introduction of l-arginine. Furthermore, the biodegradation products of BPEA are natural metabolites of the body, such as amino acids and growth factors, guaranteeing the biocompatibility of the BPEA@VE NCs. The protective ability of the BPEA@VE NCs was also investigated in vivo for accelerated wound healing. All the results indicate that the BPEA@VE NCs have promising potential for the modulation of the local microenvironment in tissue engineering for excellent antioxidative and anti-inflammatory properties.


Assuntos
Aminoácidos/administração & dosagem , Antioxidantes/administração & dosagem , Inositol/administração & dosagem , Nanocápsulas/química , Vitamina E/administração & dosagem , Cicatrização/efeitos dos fármacos , Aminoácidos/farmacologia , Aminoácidos/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Arginina/administração & dosagem , Arginina/farmacologia , Arginina/uso terapêutico , Células Endoteliais da Veia Umbilical Humana , Humanos , Inositol/farmacologia , Inositol/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Masculino , Camundongos , Células NIH 3T3 , Fenilalanina/administração & dosagem , Fenilalanina/farmacologia , Fenilalanina/uso terapêutico , Poliésteres/química , Células RAW 264.7 , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Engenharia Tecidual , Vitamina E/farmacologia , Vitamina E/uso terapêutico
16.
Bioprocess Biosyst Eng ; 44(1): 127-140, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32816075

RESUMO

The use of Raman models for glucose and phenylalanine concentrations to provide the signal for a control algorithm to continuously adjust the feed rate of two separate supplemental feeds during the fed-batch culture of a CHOK1SV GS-KO® cell line in a platform process was evaluated. Automated feed rate adjustment of the glucose feed using a Raman model for glucose concentration, maintained the glucose concentration within the desired target (average deviation ± 0.49 g/L). Automated feed rate adjustment of the nutrient feed using a Raman model for phenylalanine concentration, maintained phenylalanine concentrations within the target (average deviation ± 29.97 mg/L). The novel use of a Raman model for phenylalanine concentration, combined with a Raman model for glucose concentration, to maintain target glucose and phenylalanine concentrations through feed-rate adjustments, reduced the average cumulative glucose and nutrient feed additions (19% and 27% respectively) compared to manually adjusted cultures. Additionally, the proposed automation strategy led to lower osmolality during culture, maintained the nutrient environment more consistently, and achieved higher harvest product concentration (≈ 20% higher) compared to typical fed-batch process control for the cell line and platform process evaluated. Furthermore, the proposed feeding strategy yielded similar glycosylation and charge variant profiles compared to manually adjusted fed-batch process control. The ability to continuously adjust the feed rate addition of two separate feeds in this manner helps enable a shift away from the current daily offline sampling needed to control fed-batch mammalian cell culture during clinical and commercial manufacturing on platform processes.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Meios de Cultura/farmacologia , Glucose/farmacologia , Modelos Biológicos , Fenilalanina/farmacologia , Animais , Células CHO , Cricetulus , Retroalimentação
17.
Bioorg Chem ; 106: 104483, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33268007

RESUMO

Two series of 5-aryl-furan derivatives bearing a phenylalanine- or isoleucine-derived rhodanine moiety were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, 5g was found to have the best PTP1B inhibitory potency (IC50 = 2.66 ± 0.16 µM) and the best cell division cycle 25 homolog B (CDC25B) inhibitory potency (IC50 = 0.25 ± 0.02 µM). Enzymatic data together with molecular modeling results demonstrated that the introduction of a sec-butyl group at the 2-position of the carboxyl group remarkably improved the PTP1B inhibitory activity.


Assuntos
Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Isoleucina/farmacologia , Fenilalanina/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Rodanina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Furanos/síntese química , Furanos/química , Humanos , Isoleucina/química , Estrutura Molecular , Fenilalanina/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Rodanina/química , Relação Estrutura-Atividade
18.
Nutrition ; 82: 111042, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33246675

RESUMO

OBJECTIVES: Amino acids are not only the building blocks of proteins, but also can be metabolized to energy substances or function as signaling molecules. The aim of this study was to profile whether amino acid treatment (essential amino acids and alanine) affects the energy metabolism (glycolysis, mitochondrial respiration) of cultured hepatocytes. METHODS: AML12 hepatocytes were treated with 5 mM of each amino acid for 1 h and the energy metabolism was then measured by using an extracellular flux analyzer. RESULTS: The results showed that phenylalanine and lysine decreased the extracellular acidification rate (ECAR), an indirect indicator of glycolysis, whereas isoleucine and histidine increased the ECAR. Amino acids did not affect the oxygen consumption rate, an indirect indicator of mitochondrial respiration. The glycolysis stress test revealed that treatment of the hepatocytes with phenylalanine inhibited glycolysis when the concentration of the substrate for glycolysis was sufficient in cultured media. We also investigated the effect of metabolites derived from conversion of phenylalanine on glycolysis in hepatocytes and found that phenylpyruvate inhibited glycolysis, whereas tyrosine and phenylethylamine did not affect glycolysis. CONCLUSIONS: The findings from the present study complement basic knowledge of the effects of amino acid treatment on energy metabolism in cultured hepatocytes and indicate that phenylalanine and phenylpyruvate inhibit glycolysis.


Assuntos
Aminoácidos , Metabolismo Energético , Fenilalanina , Aminoácidos/metabolismo , Glicólise , Hepatócitos/metabolismo , Fenilalanina/farmacologia , Ácidos Fenilpirúvicos
19.
Dalton Trans ; 50(1): 157-169, 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33290472

RESUMO

Four new ligand precursors (H2L1-H2L4), derived from the Mannich condensation of two amino acids (l-Val and l-Phe) and two 3,5-disubstituted phenols (t-Bu or Me), and the corresponding oxidovanadium(iv) (1-4) and copper(ii) (6-7) complexes are synthesized. Two other related compounds (H2L5 and H2L6), containing an additional 2-methyl-pyridine arm, and the corresponding VIVO (5) and CuII (8-9) complexes were also obtained. All metal complexes are monomeric in the solid state, having a solvent molecule or a chloride ion in the coordination sphere. The in vitro cytotoxic activity of all compounds is evaluated against cancer cells from different origins. The IC50 values at 72 h are in the range of 6-15 µM for HeLa cells, 4-17 µM for A-549 cells and >25 µM for MDA-MB-231 cells, except for [VIVOL1(CH3OH)] (1) and [CuL6(H2O)] (9). With the exception of H2L6, overall, the metal complexes are more cytotoxic than the corresponding ligand precursors. Globally, the cellular viability data show that (i) the l-Phe derived compounds are more cytotoxic than the corresponding l-Val complexes; (ii) the presence of the bulkier t-Bu groups increases the cytotoxicity; (iii) the presence of a 2-methyl-pyridine arm increases considerably the cytotoxicity; and (iv) the CuII-complexes are more cytotoxic than the VIVO-compounds. Complexes [VIVOL3(CH3OH)] (3), [CuL3(H2O)] (7) and [CuL5(H2O)] (8) were further evaluated and their mechanism of action was determined to be apoptosis, evidenced by AnnexinV staining and the increase in caspase 3/7 activity. Compounds 3, 7 and 8 also exhibit DNA cleavage activity, involving the formation of reactive oxygen species and were able to induce genomic damage in cells as determined by COMET assay.


Assuntos
Antineoplásicos , Complexos de Coordenação , Cobre , Fenóis , Fenilalanina , Valina , Vanadatos , Antineoplásicos/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Clivagem do DNA , Dano ao DNA , Células HeLa , Humanos , Ligantes , Fenóis/química , Fenóis/farmacologia , Fenilalanina/química , Fenilalanina/farmacologia , Valina/química , Valina/farmacologia , Vanadatos/química , Vanadatos/farmacologia
20.
Toxicon ; 190: 41-49, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33316297

RESUMO

Carcinogenic effects of ochratoxin A (OTA) on liver, kidneys, intestine, lung and eyes of Wistar rats exposed to 10 ppm or 5 ppm OTA in the diet and additionally supplemented or not with phenylalanine (PHE) were examined during 24-months experimental period. OTA was seen to provoke strong degenerative changes and slight pericapillary oedema in most internal organs, e.g. kidneys, liver, intestine, spleen and brain. Six of total nine neoplasms were identified as malignant and three as benign. Five of total six malignant neoplasms and two of total three benign neoplasms were seen in male rats. The pathological finding in rats after two weeks feeding with OTA-contaminated feed was dominated by degenerative changes in various internal organs, which were weaker in the group additionally supplemented with PHE. The protective effect of PHE was evident with respect to OTA-induced decrease of serum glucose and serum protein, but this protection was not singnificant with respect to serum enzymes activity. The number of neoplasms in PHE-supplemented group exposed to 10 ppm OTA was similar to that in the group exposed to twice lower feed levels of OTA alone, suggesting about a possible protective effect of PHE. The rats would not be able to serve as experimental model for humans with regard to OTA-induced tumorigenesis, because the target organ of OTA-toxicity in humans and pigs is mainly the kidney as opposed to the significant damages and carcinogenic effects seen in various organs in rats exposed to OTA.


Assuntos
Carcinógenos/toxicidade , Ocratoxinas/toxicidade , Fenilalanina/farmacologia , Substâncias Protetoras/farmacologia , Animais , Carcinogênese , Dieta , Rim , Fígado , Masculino , Ratos , Ratos Wistar , Baço
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