RESUMO
Although many redox signaling molecules are present at low concentrations, typically ranging from micromolar to submicromolar levels, they often play essential roles in a wide range of biological pathways and disease mechanisms. However, accurately measuring low-abundant analytes has been a significant challenge due to the lack of sensitivity and quantitative capability of existing measurement methods. In this study, we introduced a novel chemically induced amplifiable system for quantifying low-abundance redox signaling molecules in living cells. We utilized H2O2 as a proof-of-concept analyte and developed a probe that quantifies cellular peroxide levels by combining the NanoBiT system with androgen receptor dimerization as a reporting mechanism. Our system demonstrated a highly sensitive response to cellular peroxide changes induced both endogenously and exogenously. Furthermore, the system can be adapted for the quantification of other signaling molecules if provided with suitable probing chemistry.
Assuntos
Peróxido de Hidrogênio , Receptores Androgênicos , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/química , Humanos , Receptores Androgênicos/metabolismo , OxirreduçãoRESUMO
In the scope of targeted protein degradation (TPD), proteolysis-targeting chimeras (PROTACs), leveraging the ubiquitin-proteasome system, have been extensively studied. However, they are limited to the degradation of soluble and membrane proteins, excluding the aggregated and extracellular proteins and dysfunctional organelles. As an alternative protein degradation pathway, lysosomes serve as a feasible tool for accessing these untouched proteins and/or organelles by proteosomes. Here, we focus on reviewing the emerging lysosome-mediated TPD, such as AUTAC, ATTEC, AUTOTAC, LYTAC, and MoDE-A. Intracellular targets, such as soluble and aggregated proteins and organelles, can be degraded via the autophagy-lysosome pathway. Extracellular targets, such as membrane proteins, and secreted extracellular proteins can be degraded via the endosome-lysosome pathway. In addition, we summarize the mechanism and regulation of autophagy, available methods and assays for monitoring the autophagy process, and the recently developed chemical probes for perturbing the autophagy pathways.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Lisossomos/metabolismoRESUMO
In an effort to identify methylene blue analogues having improved antioxidant activity, a series of new methylene violet analogues have been designed and synthesized. The analogues were prepared following a synthetic route that is more efficient than the previously reported methods, both in terms of yield and purity of the final products. The route involves the Smiles rearrangement as one of the crucial steps. Smiles rearrangement of suitably substituted diphenyl sulfide intermediates afforded the corresponding phenothiazine analogues in high yields, which were subsequently converted to the final products. The methylene violet analogues were evaluated for their ability to preserve mitochondrial function in Friedreich's ataxia (FRDA) lymphocytes. The analogues were shown to be efficient ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I. The analogues also preserved mitochondrial membrane potential and augmented ATP production. The analogues were found to be better antioxidants than the parent compounds methylene blue and methylene violet.
Assuntos
Mitocôndrias/efeitos dos fármacos , Fenotiazinas/farmacologia , Trifosfato de Adenosina/biossíntese , Células Cultivadas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenotiazinas/síntese química , Fenotiazinas/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
The synthesis and incorporation into position 66 of green fluorescent protein (GFP) by in vitro protein translation of novel oxazole and thiazole based dipeptidomimetics are described. The compounds may be regarded as GFP chromophore analogues, and are strongly fluorescent. An α-amido-ß-ketoester intermediate was obtained via bisacylation of a protected glycine. The intermediate underwent dehydrative cyclization to afford the 1,3-oxazole and was treated with Lawesson's reagent to furnish the 1,3-thiazole. When these fluorophores were introduced into position 66 of GFP in place of Tyr66, the resulting GFP analogues exhibited fluorescence emission several-fold greater than wild-type GFP; the emission was also shifted to shorter wavelength. It may be noted that compared to the typical fluorophores formed in the natural and modified fluorescent proteins, the oxazole and thiazole fluorophores are completely stable and do not require activation by posttranslational modification to exhibit fluorescence.
Assuntos
Corantes Fluorescentes/síntese química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ribossomos/metabolismo , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Estrutura Molecular , Peptidomiméticos/químicaRESUMO
Steroid receptor coactivator 3 (SRC-3) is a critical mediator of many intracellular signaling pathways that are crucial for cancer proliferation and metastasis. In this study, we performed structure-activity relationship exploration and drug-like optimization of the hit compound SI-2, guided by in vitro/in vivo metabolism studies and cytotoxicity assays. Our efforts led to the discovery of two lead compounds, SI-10 and SI-12. Both compounds exhibit potent cytotoxicity against a panel of human cancer cell lines and demonstrate acceptable pharmacokinetic properties. A biotinylated estrogen response element pull-down assay demonstrated that SI-12 could disrupt the recruitment of SRC-3 and p300 in the estrogen receptor complex. Importantly, SI-10 and SI-12 significantly inhibited tumor growth and metastasis in vivo without appreciable acute toxicity. These results demonstrate the potential of SI-10 and SI-12 as drug candidates for cancer therapy, given their potent SRC-3 inhibition and promising pharmacokinetic and toxicity profiles.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Coativador 3 de Receptor Nuclear/metabolismo , Linhagem Celular , Relação Estrutura-Atividade , Transdução de Sinais , Proliferação de Células , Linhagem Celular Tumoral , Antineoplásicos/farmacologiaRESUMO
AIM: This study aimsâ to report preclinical validation, and the first clinical treatment of total bone marrow irradiation (TMI) and total bone marrow and lymph nodal irradiation (TMLI) using Volumetric modulated arc therapy in Halcyon-E ring gantry linear accelerator. Preclinical validation includes simulation, planning, patient-specific QA, and dry run. MATERIAL AND METHOD: Four patients, two female and two male, with body weights of 116 kg, 52 kg, 64 kg, and 62 kg; with two with chronic myeloid leukemia, one each with acute lymphoblastic leukemia and acute myeloid leukemia (AML) were simulated and planned for TMI/TMLI. Patients were immobilized with a full-body vacuum bag. Head first supine (HFS) and Feet first supine (FFS) CT scans were acquired from head to knee and knee to toe. Planning target volume (PTV) was created with a uniform margin of 6 mm over the total bone marrow/bone marrow + lymph nodes. HFS and FFS PTVs were optimized independently using 6MV unflatten energy for 12 Gy in 6 fractions. Plans were merged to create the resultant dose distribution using a junction bias dose matching technique. The total number of isocenters was ≤ 10 per CT set, and two to four full arcs were used for each isocenter. A junction dose gradient technique was used for dose feathering between arcs between adjacent isocenters. RESULT: Only one female patient diagnosed as AML received the TMLI treatment, while the other three patients dropped out due to clinical complications and comorbidities that developed in the time between simulation and treatment. The result presented has been averaged over all four patients. For PTV, 95% dose was normalised to 95% volume, PTV_V107% receiving 3.3 ± 3.1%. Total lung mean and V12Gy were 1048.6 ± 107.1 cGy and 19.5 ± 12.1%. Maximum lens doses were 489.5 ± 35.5 cGy (left: L) and 497 ± 69.2 cGy (right: R). The mean cardiac and bilateral kidney doses were 921.75 ± 89.2 cGy, 917.9 ± 63.2 cGy (L), and 805.9 ± 9.7 cGy (R). Average Monitor Unit was 7738.25 ± 1056.6. The median number of isocenters was 17(HFS+FFS), average MU/Dose (cGy) ratio per isocenter was 2.28 ± 0.3. CONCLUSION: Halcyon-E ring gantry linear accelerator capable of planning and delivering TMI/TMLI.ââ.
Assuntos
Leucemia Mieloide Aguda , Radioterapia de Intensidade Modulada , Feminino , Humanos , Masculino , Medula Óssea/efeitos da radiação , Aceleradores de Partículas , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia de Intensidade Modulada/métodosRESUMO
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that is linked to transcriptional repression of the nuclear FXN gene encoding the essential mitochondrial protein frataxin (FXN). Compounds that increase frataxin levels may enable effective therapeutic intervention for blunting disease progression. Recently, we showed that lipophilic methylene violet (MV) and methylene blue (MB) analogues both conferred benefit to cultured FRDA cells in several regards, including ROS suppression, maintenance of mitochondrial membrane potential and increased ATP production. Some of the MB analogues were also shown to promote increased frataxin levels and mitochondrial biogenesis. Presently, we report that two of the MV analogues studied previously (1 and 2) also increased frataxin levels and mitochondrial biogenesis significantly. Because the substitution pattern in the two series of compounds was not the same, we also prepared new MV derivatives having the same substitution pattern as the original MB derivatives studied to enable a more direct comparison. Two of the new MV compounds, 4b and 6b, exhibited enhanced antioxidant capability, increased frataxin levels and mitochondrial biogenesis, and improved aconitase activity. These encouraging findings demonstrated that the MV analogues had better overall activity with less cytotoxicity.