RESUMO
Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously inâ situ. Herein, we report a multifunctional molecular probe (BnN3 -OPD-Alk) by a "three-in-one" molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN3 -OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both inâ vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.
Assuntos
Sondas Moleculares/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Neoplasias/diagnóstico por imagem , Proteínas Proto-Oncogênicas c-bcl-2/química , Fluorescência , Humanos , Sondas Moleculares/síntese química , Imagem ÓpticaRESUMO
Proteolysis targeting chimera (PROTAC) recruits an E3 ligase to a target protein to induce its ubiquitination and subsequent degradation. We reported success in the development of two PROTACs (C3 and C5) that potently and selectively induce the degradation of Mcl-1 and Bcl-2 (DC50 = 0.7 and 3.0 µM), respectively, by introducing the E3 ligase cereblon-binding ligand pomalidomide to Mcl-1/Bcl-2 dual inhibitors S1-6 and Nap-1 with micromolar-range affinity. C3-induced Mcl-1 ubiquitination translated into much more lethality in Mcl-1-dependent H23 cells than the most potent Mcl-1 occupancy-based inhibitor A-1210477 with nanomolar-range affinity. Moreover, structure-activity relationship analysis and molecular dynamic simulations discovered the structural basis for turning nonselective or promiscuous Bcl-2 family ligands into selective PROTACs. C3 and C5 exhibited reversible depletion in living cells, which provides a new potent toolkit for gain-of-function studies to probe the dynamic roles of Bcl-2 and Mcl-1 in apoptosis networks.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Talidomida/análogos & derivados , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-Atividade , Talidomida/síntese química , Talidomida/química , Talidomida/farmacologia , Células Tumorais CultivadasRESUMO
Based on Nap-1, a Mcl-1/Bcl-2 dual inhibitor reported by our group, we carried out a structure-guided molecular design and structure-activity relationship (SAR) analysis to study structural features contributing to Mcl-1 binding selectivity and affinity. A series of derivatives of Nap-1 with various pharmacophores were synthesized and among them a dual Mcl-1/Bcl-2 inhibitor A4 with enhanced affinities (IC50â¯=â¯0.15⯵M for Mcl-1, 0.43⯵M for Bcl-2) and a selective Mcl-1 inhibitor B9 with a 20-fold selectivity over Bcl-2 (IC50â¯=â¯0.51⯵M vs 9.46⯵M) were obtained by enzyme linked immunosorbent assay (ELISA). The SAR data and binding modes of A4 and B9 investigated by 2D-NMR derived docking study illustrated that p2 pockets exhibiting different geometry and binding features between Mcl-1 and Bcl-2 contribute to specific binding properties of Mcl-1. In addition, apoptosis-inducing potencies of A4 and B9 were consistent with their binding selectivity determined in vitro.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Células HeLa , Humanos , Células K562 , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-AtividadeRESUMO
We synthesized six 1-oxo-1H-phenalene-2,3-dicarbonitrile (OPD)-based probes with various leaving groups using an arylthioether linker and for the first time identified the probe O-NH2 capable of highly selective detection of glutathione over cysteine/homocysteine in vitro and in vivo based on an aromatic nuclear substitution reaction (SNAr) mechanism. The fluorescence of the probe O-NH2 was quenched because of the photoinduced electron transfer (PET) process, but switched on by a glutathione-triggered specific recognition reaction between the probe O-NH2 and glutathione. The recognition mechanism for glutathione was explored and verified by theoretical calculations and ESI-MS analysis. Using O-NH2 as the probe, the GSH fluorescence images were demonstrated in HeLa cells and the intracellular GSH levels in different imatinib-resistant K562 tumor cells were firstly determined. Further, O-NH2 was utilized to detect glutathione in D. magna and zebrafish embryos. The combined results indicate that O-NH2 can be applied as an effective tool for detecting glutathione in biological investigations.
Assuntos
Corantes Fluorescentes/química , Glutationa/análise , Nitrilas/química , Fenalenos/química , Animais , Linhagem Celular Tumoral , Daphnia , Desenho de Fármacos , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Modelos Químicos , Nitrilas/síntese química , Nitrilas/toxicidade , Fenalenos/síntese química , Fenalenos/toxicidade , Espectrometria de Fluorescência/métodos , Peixe-ZebraRESUMO
Selective inhibition of proteins of the Bcl-2 family by small-molecule inhibitors is a promising new approach in drug discovery. However, information about how these molecules interact with their cellular targets (on- and off-) is highly limited. We have designed and synthesized photoreactive and "clickable" affinity-based probes (AfBPs)-Nap-2 and Nap-5-by introducing photo-crosslinkers onto Nap-1, a fluorescent derivative of small-molecule Bcl-2 inhibitor S1-6. The resulting trifunctional probes Nap-2 and Nap-5 can enrich, visualize, and enable the identification of cellular on- and off-targets of Bcl-2 inhibitors both in vitro and in situ. Tubulin was validated as an off-target of Bcl-2 inhibitors (Nap-1 and S1-6) by large-scale cell-based proteome profiling and pull-down/western blotting (PD/WB) with Nap-2 and Nap-5. It was preliminarily illustrated to be a BH3-containing protein because some well-known Bcl-2 inhibitors can block the labeling of tubulin by Nap-2.