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1.
J Am Chem Soc ; 146(1): 187-200, 2024 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-38118119

RESUMO

The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (Kd) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular Kd values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular Ki values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular Ki values matched known biochemical Ki values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.


Assuntos
Ésteres de Forbol , Proteína Quinase C , Humanos , Células HEK293 , Proteína Quinase C/química , Ligação Competitiva
2.
Chemistry ; 29(24): e202300315, 2023 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-36855249

RESUMO

Synthesis-oriented design led us to the discovery of a series of novel cyanine-borondifluoride curcuminoid hybrids called Nanchang Red (NCR) dyes that overcome the intrinsic low synthetic yields of symmetrical cyanine-difluoroboronate (BF2 )-hybridized NIR dyes. The hybridization endows NCR dyes with high molar extinction coefficients, efficient red-to-NIR emission, and enlarged Stokes shifts. Quantum chemical calculations revealed that the asymmetrical layout of the three key electron-withdrawing and electron-donating fragments results in a special pattern of partial charge separation and inconsistent degrees of charge delocalization on their π-conjugated backbones. While the nature of the hemicyanine fragment exerts significant influence on the excitation modes of NCR dyes, the borondifluoride hemicurcuminoid fragment is the major contributor to the enlarged Stokes shifts. Cell imaging experiments illustrated that a subtle change in the N-heterocycle of the hemicyanine fragment has a remarkable effect on the subcellular localization of NCR dyes. Unlike other previously reported cyanine-BF2 hybridized dyes, which mainly target mitochondria, the benzothiazole and indole-based NCR dyes accumulate in both the endoplasmic reticulum (ER) and lipid droplets of HeLa cells, whereas the benzoxazole and quinoline-based NCR dyes stain the ER specifically.


Assuntos
Corantes Fluorescentes , Quinolinas , Humanos , Células HeLa , Corantes Fluorescentes/química , Carbocianinas/química , Quinolinas/química
3.
Molecules ; 27(15)2022 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-35956964

RESUMO

The root of Achyranthes bidentata Blume (AB) is a well-known traditional Chinese medicine for treating osteoporosis. Plenty of studies focused on the pharmacological mechanism of the whole extract; however, the contribution of different components to the anti-osteoporosis effect remains unknown. The aim of this study is to explore the anti-osteoporosis mechanism of different components of crude and salt-processed AB under the guidance of network pharmacology, metabolomics, and microbiomics. First, network pharmacology analysis was applied to constructing the compound-target-disease network of AB to provide a holistic view. Second, the anti-osteoporosis effects of the four components were evaluated in female Wistar rats. The subjects were divided into a normal group, a model group, a 17α-estradiol (E2)-treated group, a polysaccharide-component-treated groups, and a polysaccharide-knockout-component-treated groups. All the serum, urine, and feces samples of the six groups were collected after 16 weeks of treatment. Biochemical and microcomputed tomography (µCT) parameters were also acquired. Coupled with orthogonal partial least-squares discrimination analysis, one dimensional nuclear magnetic resonance (NMR) was used to monitor serum metabolic alterations. A total of twenty-two biomarkers, including lipids, amino acids, polyunsaturated fatty acids, glucose, and so on were identified for the different components-treated groups. Through pathway analysis, it is indicated that glyoxylate and dicarboxylate metabolism, glycine, serine, and threonine metabolism, alanine, aspartate, and glutamate metabolism, d-glutamine, and d-glutamate metabolism were the major intervened pathways. Levels of these biomarkers shifted away from the model group and were restored to normal after treatment with the four components. In addition, 16S rDNA sequencing demonstrated that the abundance of Anaerofilum, Rothia, and Turicibacter bacteria was positively correlated with an anti-osteoporosis effect, whereas the abundance of Oscillospira was negatively correlated. The osteoprotective effect of the polysaccharide components of crude and salt-processed AB is related to the regulation of the abundance of these gut microbiota.


Assuntos
Achyranthes , Medicamentos de Ervas Chinesas , Osteoporose , Extratos Vegetais , Achyranthes/química , Animais , Biomarcadores , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Humanos , Medicina Tradicional Chinesa , Metabolômica/métodos , Osteoporose/metabolismo , Osteoporose/prevenção & controle , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Raízes de Plantas/química , Polissacarídeos/química , Ratos , Ratos Wistar , Cloreto de Sódio , Cloreto de Sódio na Dieta , Microtomografia por Raio-X
4.
Methods Enzymol ; 640: 1-35, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32560793

RESUMO

Peroxynitrite (PNT) is a highly reactive oxidant that plays a key role in the destruction of foreign pathogens by specific phagocytic immune cells such as macrophages. However, when its production is dysregulated, this oxidant can contribute to cardiovascular disease, neurological diseases, and cancer. To facilitate the detection of PNT in living cells, we designed and synthesized a fluorescent sensor termed PS3 that accumulates in membranes of the endoplasmic reticulum (ER). This subcellular targeting enhances the proximity of PS3 to the phagosome of macrophages where PNT is generated. When PS3-treated macrophages are stimulated with 10 µm opsonized tentagel microspheres, antibody-dependent cellular phagocytosis (ADCP) of these particles results in production of endogenous PNT, oxidative cleavage of the fluorescence-quenching phenolic side chain of PS3, and increased fluorescence that can be detected by confocal laser scanning microscopy, flow cytometry, and other assays. We describe methods for the synthesis of PS3 and evaluation of its photophysical properties, selectivity, and reactivity. We further report differential production of PNT during ADCP by the phagocytic cell lines RAW 264.7, J774A.1, and THP-1, as detected by confocal microscopy and changes in fluorescence intensity on 96-well plates. This approach may be useful for identification of modulators of PNT and related studies of ADCP.


Assuntos
Ácido Peroxinitroso , Fagocitose , Citometria de Fluxo , Macrófagos , Fagossomos
5.
Yao Xue Xue Bao ; 50(6): 738-45, 2015 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-26521446

RESUMO

Poly(ADP-ribose)polymerase-1 (PARP-1) plays a significant role in the DNA repair process by catalyzing the transfer of ADP-ribose from NAD+ to its receptors. It is a promising anticancer drug target and many PARP-1 inhibitors have been developed and used in the clinical trial. In this work, a series of 3-(2-oxo-2-substituted acetamido)benzamides have been synthesized and their inhibitory activities against PARP-1 were evaluated. Of all the tested compounds, six compounds displayed inhibitory activities with IC50 values ranging from 0.23 to 5.78 µmol.L-1 . The binding pose of compound 5a was predicted using molecular docking to facilitate further structural modification.


Assuntos
Benzamidas/química , Desenho de Fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Antineoplásicos , Reparo do DNA , Humanos , Simulação de Acoplamento Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases
6.
Eur J Med Chem ; 68: 222-32, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23981529

RESUMO

A series of novel oxazole-bridged analogs of combretastatin A-4 bearing a benzo[d]-imidazole as B ring were synthesized and evaluated for antiproliferative activities against five human cancer cell lines. Among all the synthesized compounds, the N-unsubstituted benzoimidazole analog 5 and the analogs 6b, 7a and 7b with a small hydrophobic group on nitrogen atom of benzoimidazole ring were identified as the most potent inhibitors of tumor cell growth with IC50 values at nanomolar levels (5, IC50=8.4 nM, HT29; 6b, 7a, 7b, IC50=9.6 nM, 3.8 nM, 3.0 nM, A549). In a murine H22 tumor xenograft model, compound 5 exhibited significant antitumor activity. The binding mode of compound 5 in the colchicine binding site of tubulin was probed.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzimidazóis/química , Bibenzilas/química , Bibenzilas/farmacologia , Oxazóis/química , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Estrutura Molecular , Ensaios Antitumorais Modelo de Xenoenxerto
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