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1.
Chem Biol Interact ; 350: 109700, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34648813

RESUMO

AIM: To investigate the enzymatic properties of cytochrome P450 3A4 (CYP3A4) variants and their ability to metabolize vandetanib (VNT) in vitro, and to study potential drug interactions in combination with VNT. METHOD: Recombinant CYP3A4 cell microsomes were prepared using a Bac-to-Bac baculovirus expression system. Enzymatic reactions were carried out, and the metabolites were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: The activities of 27 CYP3A4 variants were determined to assess the degree of VNT metabolism that occurred. Analysis indicated that there was enhanced intrinsic clearance (Vmax/Km, CLint) for eight variants (CYP3A4.2, 3, 9, 15, 16, 29, 32, and 33), while there was a significant decrease in CYP3A4.5, 7, 8, 10-14, 17-20, 23, 24, 28, 31, and 34. Compared with CYP3A4.1, no significant differences were found for CYP3A4.6 and 30. Furthermore, the relative clearances were compared between VNT and cabozantinib, which were all metabolized by CYP3A4 with the same indications. When combined with ketoconazole, which is a CYP inhibitor, obvious differences were observed in the potency of VNT between different variants, including CYP3A4.2, 15, and 18. CONCLUSION: This comprehensive assessment of CYP3A4 variants provides significant insights into the allele-specific metabolism of VNT and drug interactions in vitro. We hope that these comprehensive data will provide references and predictions for the clinical application of VNT.


Assuntos
Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Piperidinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Quinazolinas/metabolismo , Alelos , Biotransformação , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Interações Medicamentosas , Estudos de Associação Genética , Variação Genética , Humanos , Técnicas In Vitro , Cetoconazol/administração & dosagem , Cinética , Taxa de Depuração Metabólica , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/administração & dosagem , Quinazolinas/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Forensic Sci Int ; 327: 110989, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34509061

RESUMO

After their first emergence in 2009, Novel synthetic opioids (NSO) have become an emerging class of New Psychoactive Substances (NPS) on the market for these new drugs. So far, 67 NSO have been reported to the Early Warning system of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). It is presumed that NSO mainly target the four known opioid receptors, i.e. the µ-opioid (MOR), the δ-opioid (DOR), the κ-opioid (KOR) and nociceptin receptors and that their consumption can result in serious adverse effects such as massive respiratory depression or death. In the present study we investigated the in vivo and in vitro metabolism of brorphine, a NSO that was first identified on the NPS market in August 2019 in the United States, using both a pooled human liver microsome assay and real forensic case samples. For the detection of metabolites LC-HR-MS/MS was used and quantification of brorphine was performed using an LC-MS/MS method. Additionally, we pharmacologically characterized brorphine regarding its activation of the MOR and KOR via G protein recruitment using the [35S]-GTPγS assay. In forensic urine samples, 14 distinct metabolites were identified, whereas in blood only four metabolites could be found. The pooled human liver microsome assay generated six distinct in vitro phase I metabolites. The most prominent in vivo metabolite was formed by N-oxydation, whereas the main in vitro metabolite was formed by hydroxylation. The pharmacological characterization at the MOR and KOR revealed brorphine to be a potent MOR agonist and a weak, partial KOR agonist in the [35S]-GTPγS assay.


Assuntos
Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Imidazóis/metabolismo , Imidazóis/farmacologia , Piperidinas/metabolismo , Piperidinas/farmacologia , Receptores Opioides/efeitos dos fármacos , Detecção do Abuso de Substâncias/métodos , Analgésicos Opioides/sangue , Analgésicos Opioides/urina , Cromatografia Líquida , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Imidazóis/sangue , Imidazóis/urina , Microssomos Hepáticos/metabolismo , Piperidinas/sangue , Piperidinas/urina , Espectrometria de Massas em Tandem
3.
ACS Chem Biol ; 16(9): 1644-1653, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34397208

RESUMO

Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.


Assuntos
Acrilamidas/química , Adenina/análogos & derivados , Afatinib/química , Compostos de Anilina/química , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Lisina/química , Inibidores de Fosfoinositídeo-3 Quinase/química , Piperidinas/química , Acetilação , Acrilamidas/metabolismo , Adenina/química , Adenina/metabolismo , Afatinib/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/metabolismo , Catálise , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Espectrometria de Massas , Simulação de Acoplamento Molecular , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
4.
J Med Chem ; 64(16): 12228-12244, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34387987

RESUMO

Cholesterol 24-hydroxylase (CH24H, CYP46A1), a brain-specific cytochrome P450 (CYP) family enzyme, plays a role in the homeostasis of brain cholesterol by converting cholesterol to 24S-hydroxycholesterol (24HC). Despite a wide range of potential of CH24H as a drug target, no potent and selective inhibitors have been identified. Here, we report on the structure-based drug design (SBDD) of novel 4-arylpyridine derivatives based on the X-ray co-crystal structure of hit derivative 1b. Optimization of 4-arylpyridine derivatives led us to identify 3v ((4-benzyl-4-hydroxypiperidin-1-yl)(2,4'-bipyridin-3-yl)methanone, IC50 = 7.4 nM) as a highly potent, selective, and brain-penetrant CH24H inhibitor. Following oral administration to mice, 3v resulted in a dose-dependent reduction of 24HC levels in the brain (1, 3, and 10 mg/kg). Compound 3v (soticlestat, also known as TAK-935) is currently under clinical investigation for the treatment of Dravet syndrome and Lennox-Gastaut syndrome as a novel drug class for epilepsies.


Assuntos
Colesterol 24-Hidroxilase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Colesterol 24-Hidroxilase/metabolismo , Cristalografia por Raios X , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/metabolismo , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Relação Estrutura-Atividade
5.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209188

RESUMO

Coronavirus disease (COVID)-19 is the leading global health threat to date caused by a severe acute respiratory syndrome coronavirus (SARS-CoV-2). Recent clinical trials reported that the use of Bruton's tyrosine kinase (BTK) inhibitors to treat COVID-19 patients could reduce dyspnea and hypoxia, thromboinflammation, hypercoagulability and improve oxygenation. However, the mechanism of action remains unclear. Thus, this study employs structure-based virtual screening (SBVS) to repurpose BTK inhibitors acalabrutinib, dasatinib, evobrutinib, fostamatinib, ibrutinib, inositol 1,3,4,5-tetrakisphosphate, spebrutinib, XL418 and zanubrutinib against SARS-CoV-2. Molecular docking is conducted with BTK inhibitors against structural and nonstructural proteins of SARS-CoV-2 and host targets (ACE2, TMPRSS2 and BTK). Molecular mechanics-generalized Born surface area (MM/GBSA) calculations and molecular dynamics (MD) simulations are then carried out on the selected complexes with high binding energy. Ibrutinib and zanubrutinib are found to be the most potent of the drugs screened based on the results of computational studies. Results further show that ibrutinib and zanubrutinib could exploit different mechanisms at the viral entry and replication stage and could be repurposed as potential inhibitors of SARS-CoV-2 pathogenesis.


Assuntos
Adenina/análogos & derivados , Reposicionamento de Medicamentos , Simulação de Dinâmica Molecular , Piperidinas/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Adenina/química , Adenina/metabolismo , Adenina/uso terapêutico , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Humanos , Simulação de Acoplamento Molecular , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
6.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34129030

RESUMO

We describe a previously unreported macroscopic Arabidopsis organ, the cantil, named for its 'cantilever' function of holding the pedicel at a distance from the stem. Cantil development is strongest at the first nodes after the vegetative to reproductive inflorescence transition; cantil magnitude and frequency decrease acropetally. Cantils develop in wild-type Arabidopsis accessions (e.g. Col-0, Ws and Di-G) as a consequence of delayed flowering in short days; cantil formation is observed in long days when flowering is delayed by null mutation of the floral regulator FLOWERING LOCUS T. The receptor-like kinase ERECTA is a global positive regulator of cantil formation; therefore, cantils never form in the Arabidopsis strain Ler. ERECTA functions genetically upstream of heterotrimeric G proteins. Cantil expressivity is repressed by the specific heterotrimeric complex subunits GPA1, AGB1 and AGG3, which also play independent roles: GPA1 suppresses distal spurs at cantil termini, while AGB1 and AGG3 suppress ectopic epidermal rippling. These G protein mutant traits are recapitulated in long-day flowering gpa1-3 ft-10 plants, demonstrating that cantils, spurs and ectopic rippling occur as a function of delayed phase transition, rather than as a function of photoperiod per se.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Benzilatos/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Piperidinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Heterotriméricas de Ligação ao GTP/genética , Mutação com Perda de Função , Fenótipo , Fotoperíodo , Plantas Geneticamente Modificadas/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
7.
J Photochem Photobiol B ; 221: 112238, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34130091

RESUMO

Nicotinamide adenine dinucleotide (NAD+) is a crucial coenzyme in energy production. The imbalance of NAD+ synthesis has been found to trigger age-related diseases, such as metabolic disorders, cancer, and neurodegenerative diseases. Also, UV irradiation induces NAD+ depletion in the skin. In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway and essential for NAD+ homeostasis. However, but few studies have focused on the role of NAMPT in response to UV irradiation. Here, we show that NAMPT prevents NAD+ depletion in epidermal keratinocytes to protect against the mild-dose UVA and UVB (UVA/B)-induced proliferation defects. We showed that poly(ADP-ribose) polymerase (PARP) inhibitor rescued the NAD+ depletion in UVA/B-irradiated human keratinocytes, confirming that PAPR transiently exhausts cellular NAD+ to repair DNA damage. Notably, the treatment with a NAMPT inhibitor exacerbated the UVA/B-induced loss of energy production and cell viability. Moreover, the NAMPT inhibitor abrogated the sirtuin-1 (SIRT1)-mediated deacetylation of p53 and significantly inhibited the proliferation of UVA/B-irradiated cells, suggesting that the NAMPT-NAD+-SIRT1 axis regulates p53 functions upon UVA/B stress. The supplementation with NAD+ intermediates, nicotinamide mononucleotide and nicotinamide riboside, rescued the UVA/B-induced phenotypes in the absence of NAMPT activity. Therefore, NAD+ homeostasis is likely essential for the protection of keratinocytes from UV stress in mild doses. Since the skin is continuously exposed to UVA/B irradiation, understanding the protective role of NAMPT in UV stress will help prevent and treat skin photoaging.


Assuntos
NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Acrilamidas/química , Acrilamidas/metabolismo , Acrilamidas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia
8.
J Med Chem ; 64(12): 8246-8262, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34107215

RESUMO

Adenosine A1/A2A receptors (A1R/A2AR) represent targets in nondopaminergic treatment of motor disorders such as Parkinson's disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of histamine H3 receptor (H3R) antagonists in combination with the "caffeine-like effects" of A1R/A2AR antagonists, we designed A1R/A2AR/H3R MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl H3R pharmacophore was introduced with overlap into an adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding profiles with overall nanomolar H3R affinities (Ki < 55 nM). Compound 4 (ST-2001, Ki (A1R) = 11.5 nM, Ki (A2AR) = 7.25 nM) and 12 (ST-1992, Ki (A1R) = 11.2 nM, Ki (A2AR) = 4.01 nM) were evaluated in vivo. l-DOPA-induced dyskinesia was improved after administration of compound 4 (1 mg kg-1, i.p. rats). Compound 12 (2 mg kg-1, p.o. mice) increased wakefulness representing novel pharmacological tools for PD therapy.


Assuntos
Antagonistas do Receptor A1 de Adenosina/uso terapêutico , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Antagonistas dos Receptores Histamínicos H3/uso terapêutico , Doença de Parkinson Secundária/tratamento farmacológico , Antagonistas do Receptor A1 de Adenosina/síntese química , Antagonistas do Receptor A1 de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/metabolismo , Animais , Discinesias/tratamento farmacológico , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/metabolismo , Humanos , Levodopa/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Piperidinas/síntese química , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Pirrolidinas/uso terapêutico , Ratos Sprague-Dawley , Receptor A2A de Adenosina/metabolismo , Receptores Histamínicos H3/metabolismo , Vigília/efeitos dos fármacos
9.
Sci Rep ; 11(1): 11985, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099830

RESUMO

Recent studies suggested that ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, developed for the treatment of chronic lymphocytic leukemia, may prevent NLRP3 inflammasome activation in macrophages, IL-1ß secretion and subsequent development of inflammation and organ fibrosis. The role of NLRP3 has been underlined in the various causes of acute kidney injury (AKI), a pathology characterized by high morbimortality and risk of transition toward chronic kidney disease (CKD). We therefore hypothesized that the BTK-inhibitor ibrutinib could be a candidate drug for AKI treatment. Here, we observed in both an AKI model (glycerol-induced rhabdomyolysis) and a model of rapidly progressive kidney fibrosis (unilateral ureteral obstruction), that ibrutinib did not prevent inflammatory cell recruitment in the kidney and fibrosis. Moreover, ibrutinib pre-exposure led to high mortality rate owing to severer rhabdomyolysis and AKI. In vitro, ibrutinib potentiated or had no effect on the secretion of IL-1ß by monocytes exposed to uromodulin or myoglobin, two danger-associated molecule patterns proteins involved in the AKI to CKD transition. According to these results, ibrutinib should not be considered a candidate drug for patients developing AKI.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Adenina/análogos & derivados , Antineoplásicos/farmacologia , Fibrose/prevenção & controle , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piperidinas/farmacologia , Injúria Renal Aguda/complicações , Adenina/metabolismo , Adenina/farmacologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Coleta de Amostras Sanguíneas , Humanos , Rim , Leucócitos Mononucleares , Macrófagos , Masculino , Camundongos Endogâmicos C57BL , Mortalidade , Mioglobina/metabolismo , Preparações Farmacêuticas , Piperidinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Rabdomiólise/complicações
10.
Bioorg Chem ; 112: 104967, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33975232

RESUMO

Nowadays, over 200 countries face a wellbeing emergency because of epidemiological disease COVID-19 caused by the SARS-CoV-2 virus. It will cause a very high effect on the world's economy and the worldwide health sector. The present work is an investigation of the newly synthesized 4-benzyl-1-(2,4,6-trimethyl-benzyl)-piperidine (M1BZP) molecule's inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. M1BZP crystallizes in monoclinic type with P1211 space group. For the title compound M1BZP, spectroscopic characterization like 1H NMR, 13C NMR, FTIR, were carried out. The geometry of the compound had been optimized by the DFT method and its results were compared with the X-ray diffraction data. The calculated energies for the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) showed the stability and reactivity of the title compound. Intermolecular interactions in the crystal network were determined using Hirshfeld surface analyses. The molecular electrostatic potential (MEP) picture was drawn using the same level of theory to visualize the chemical reactivity and charge distribution on the molecule. Molecular docking study performed for the synthesized compound revealed an efficient interaction with the COVID-19 protease and resulted in good activities. We hope the present study would help workers in the field to develop potential vaccines and therapeutics against the novel coronavirus. Virtual ADME studies were carried out as well and a relationship between biological, electronic, and physicochemical qualifications of the target compound was determined. Toxicity prediction by computational technique for the title compound was also carried out.


Assuntos
Antivirais/metabolismo , Piperidinas/química , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Antivirais/síntese química , Antivirais/química , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Cristalografia por Raios X , Teoria da Densidade Funcional , Meia-Vida , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Piperidinas/síntese química , Piperidinas/metabolismo , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/metabolismo
11.
J Med Chem ; 64(9): 5886-5904, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33902288

RESUMO

The ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for the suppression of hyperactive Wnt/ß-catenin signaling that is vigorously involved in cancer initiation and development. Herein, we describe the medicinal chemistry optimization of a screening hit to yield novel small-molecule inhibitors of the ß-catenin/BCL9 interaction. The best compound 30 can disrupt the ß-catenin/BCL9 interaction with a Ki of 3.6 µM in AlphaScreen competitive inhibition assays. Cell-based experiments revealed that 30 selectively disrupted the ß-catenin/BCL9 PPI, while leaving the ß-catenin/E-cadherin PPI unaffected, dose-dependently suppressed Wnt signaling transactivation, downregulated oncogenic Wnt target gene expression, and on-target selectively inhibited the growth of cancer cells harboring aberrant Wnt signaling. This compound with a new chemotype can serve as a lead compound for further optimization of inhibitors for ß-catenin/BCL9 PPI.


Assuntos
Desenho de Fármacos , Piperidinas/química , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Sítios de Ligação , Caderinas/antagonistas & inibidores , Caderinas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Humanos , Simulação de Acoplamento Molecular , Piperidinas/metabolismo , Piperidinas/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores
12.
Molecules ; 26(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918638

RESUMO

Gastrointestinal side effects of donepezil, including dyspepsia, nausea, vomiting or diarrhea, occur in 20-30% of patients. The pathogenesis of these dysmotility associated disorders has not been fully clarified yet. Pharmacokinetic parameters of donepezil and its active metabolite 6-O-desmethyldonepezil were investigated in experimental pigs with and without small intestinal injury induced by dextran sodium sulfate (DSS). Morphological features of this injury were evaluated by a video capsule endoscopy. The effect of a single and repeated doses of donepezil on gastric myoelectric activity was assessed. Both DSS-induced small intestinal injury and prolonged small intestinal transit time caused higher plasma concentrations of donepezil in experimental pigs. This has an important implication for clinical practice in humans, with a need to reduce doses of the drug if an underlying gastrointestinal disease is present. Donepezil had an undesirable impact on porcine myoelectric activity. This effect was further aggravated by DSS-induced small intestinal injury. These findings can explain donepezil-associated dyspepsia in humans.


Assuntos
Donepezila/farmacocinética , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiopatologia , Indanos/metabolismo , Metaboloma , Complexo Mioelétrico Migratório , Piperidinas/metabolismo , Estômago/fisiopatologia , Animais , Endoscopia por Cápsula , Sulfato de Dextrana , Donepezila/química , Donepezila/farmacologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Complexo Mioelétrico Migratório/efeitos dos fármacos , Estômago/efeitos dos fármacos , Suínos
13.
J Med Chem ; 64(8): 5099-5122, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33787273

RESUMO

A known zwitterionic, heterocyclic P2Y14R antagonist 3a was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2Y14R affinity. Piperidine amide 11, 3-aminopropynyl 19, and 5-(hydroxymethyl)isoxazol-3-yl) 29 congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing (32; MRS4654; IC50, 15 nM) and less active phenylamide-containing (33) scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2Y14R antagonists for in vivo application.


Assuntos
Piperidinas/química , Antagonistas do Receptor Purinérgico P2/química , Receptores Purinérgicos P2/metabolismo , Animais , Sítios de Ligação , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neuralgia/tratamento farmacológico , Piperidinas/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Antagonistas do Receptor Purinérgico P2/metabolismo , Antagonistas do Receptor Purinérgico P2/uso terapêutico , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Solubilidade , Relação Estrutura-Atividade , Triazóis/química
15.
Elife ; 102021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33755016

RESUMO

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.


Assuntos
Receptores ErbB/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteômica/métodos , Catálise , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ocludina/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacologia , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Domínios de Homologia de src
16.
J Biol Chem ; 296: 100538, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33722610

RESUMO

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.


Assuntos
Calgranulina A/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Piperidinas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Pirimidinas/metabolismo , Regulação Alostérica , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
17.
Biomed Chromatogr ; 35(6): e5081, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33522621

RESUMO

Tofacitinib is an orally available Janus kinase inhibitor. The aim of this study was to investigate the metabolism of tofacitinib in mouse, rat, monkey, and human liver microsomes fortified with ß-nicotinamide adenine dinucleotide phosphate tetrasodium salt and uridine diphosphate glucuronic acid. The biotransformation was executed at a temperature of 37°C for 60 min, and the samples were analyzed by ultra-high performance liquid chromatography combined with high-resolution mass spectrometry (UHPLC-HRMS) operated in positive electrospray ionization mode. The structures of the metabolites were elucidated according to their retention times, accurate masses, and MS/MS spectra. Under the current conditions, a total of 13 metabolites, including 1 glucuronide conjugate, were detected and structurally proposed. Oxygenation of the pyrrolopyrimidine ring, oxygenation of piperidine ring, N-demethylation, oxygenation of piperidine ring side chain, and glucuronidation were the primary metabolic pathways of tofacitinib. Among the tested species, tofacitinib showed significant species difference. Compared with other species, rat showed similar metabolic profiles to those of humans. The present study provides some new information regarding the metabolism of tofacitinib in animals and humans, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.


Assuntos
Microssomos Hepáticos/metabolismo , Piperidinas , Inibidores de Proteínas Quinases , Pirimidinas , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Haplorrinos , Humanos , Metaboloma , Camundongos , Piperidinas/análise , Piperidinas/metabolismo , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/análise , Pirimidinas/metabolismo , Ratos , Espectrometria de Massas em Tandem
18.
Curr Opin Chem Biol ; 62: 24-33, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33549806

RESUMO

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.


Assuntos
Quimera/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Ubiquitina-Proteína Ligases/metabolismo , Acrilamida/química , Acrilamida/metabolismo , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Animais , Descoberta de Drogas , Humanos , Ligantes , Simulação de Acoplamento Molecular , Piperidinas/química , Piperidinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica , Proteólise , Transdução de Sinais , Relação Estrutura-Atividade , Ubiquitinação
19.
J Med Chem ; 64(4): 1844-1855, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33570950

RESUMO

The acetylcholinesterase (AChE) inhibitors remain key therapeutic drugs for the treatment of Alzheimer's disease (AD). However, the low-safety window limits their maximum therapeutic benefits. Here, a novel kinetics-driven drug design strategy was employed to discover new-generation AChE inhibitors that possess a longer drug-target residence time and exhibit a larger safety window. After detailed investigations, compound 12 was identified as a highly potent, highly selective, orally bioavailable, and brain preferentially distributed AChE inhibitor. Moreover, it significantly ameliorated cognitive impairments in different mouse models with a lower effective dose than donepezil. The X-ray structure of the cocrystal complex provided a precise binding mode between 12 and AChE. Besides, the data from the phase I trials demonstrated that 12 had good safety, tolerance, and pharmacokinetic profiles at all preset doses in healthy volunteers, providing a solid basis for its further investigation in phase II trials for the treatment of AD.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/uso terapêutico , Indanos/uso terapêutico , Nootrópicos/uso terapêutico , Piperidinas/uso terapêutico , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/tratamento farmacológico , Animais , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/metabolismo , Cristalografia por Raios X , Cães , Desenho de Fármacos , Feminino , Humanos , Indanos/síntese química , Indanos/metabolismo , Cinética , Masculino , Camundongos Endogâmicos ICR , Estrutura Molecular , Nootrópicos/síntese química , Nootrópicos/metabolismo , Piperidinas/síntese química , Piperidinas/metabolismo , Ligação Proteica , Ratos Sprague-Dawley , Escopolamina , Relação Estrutura-Atividade
20.
Int J Mol Sci ; 22(4)2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-33562121

RESUMO

Bilastine, a zwitterionic second-generation antihistamine containing a carboxyl group, has higher selectivity for H1 receptors than first-generation antihistamines. Ligand-receptor docking simulations have suggested that the electrostatic interaction between the carboxyl group of second-generation antihistamines and the amino group of Lys179ECL2 and Lys1915.39 of human H1 receptors might contribute to increased affinity of these antihistamines to H1 receptors. In this study, we evaluated the roles of Lys179ECL2 and Lys1915.39 in regulating the electrostatic and hydrophobic binding of bilastine to H1 receptors by thermodynamic analyses. The binding enthalpy and entropy of bilastine were estimated from the van 't Hoff equation using the dissociation constants. These constants were obtained from the displacement curves against the binding of [3H] mepyramine to membrane preparations of Chinese hamster ovary cells expressing wild-type human H1 receptors and their Lys179ECL2 or Lys1915.39 mutants to alanine at various temperatures. We found that the binding of bilastine to wild-type H1 receptors occurred by enthalpy-dependent binding forces and, more dominantly, entropy-dependent binding forces. The mutation of Lys179ECL2 and Lys1915.39 to alanine reduced the affinity of bilastine to H1 receptors by reducing enthalpy- and entropy-dependent binding forces, respectively. These results suggest that Lys179ECL2 and Lys1915.39 differentially contribute to the increased binding affinity to bilastine via electrostatic and hydrophobic binding forces.


Assuntos
Benzimidazóis/metabolismo , Antagonistas não Sedativos dos Receptores H1 da Histamina/metabolismo , Piperidinas/metabolismo , Receptores Histamínicos H1/metabolismo , Termodinâmica , Animais , Células CHO , Linhagem Celular , Cricetulus , Entropia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Ligação Proteica , Eletricidade Estática
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