A. Chełkowski Institute of Physics, University of Silesia in Katowice, Chorzów, Poland.
Institute of Chemistry, University of Silesia in Katowice, Katowice, Poland.
Int J Nanomedicine. 2023 Mar 31;18:1709-1724. doi: 10.2147/IJN.S403058. eCollection 2023.
Thanks to recent advances in synthetic methodology, water-soluble fullerene nanomaterials that interfere with biomolecules, especially DNA/RNA and selected proteins, have been found with tremendous potential for applications in nanomedicine. Herein, we describe the synthesis and evaluation of a water-soluble glycine-derived [60]fullerene hexakisadduct (HDGF) with T symmetry, which is a first-in-class BTK protein inhibitor.
We synthesized and characterized glycine derived [60]fullerene using NMR, ESI-MS, and ATR-FT-IR. DLS and zeta potential were measured and high-resolution transmission electron microscopy (HRTEM) observations were performed. The chemical composition of the water-soluble fullerene nanomaterial was examined by X-ray photoelectron spectrometry. To observe aggregate formation, the cryo-TEM analysis was carried out. The docking studies and molecular dynamic simulations were performed to determine interactions between HDGF and BTK. The in vitro cytotoxicity was evaluated on RAJI and K562 blood cancer cell lines. Subsequently, we examined the induction of cell death by autophagy and apoptosis by determining the expression levels of crucial genes and caspases. We investigated the direct association of HDGF on inhibition of the BTK signalling pathway by examining changes in the calcium levels in RAJI cells after treatment. The inhibitory potential of HDGF against non-receptor tyrosine kinases was evaluated. Finally, we assessed the effects of HDGF and ibrutinib on the expression of the BTK protein and downstream signal transduction in RAJI cells following anti-IgM stimulation.
Computational studies revealed that the inhibitory activity of the obtained [60]fullerene derivative is multifaceted: it hampers the BTK active site, interacting directly with the catalytic residues, rendering it inaccessible to phosphorylation, and binds to residues that form the ATP binding pocket. The anticancer activity of produced carbon nanomaterial revealed that it inhibited the BTK protein and its downstream pathways, including PLC and Akt proteins, at the cellular level. The mechanistic studies suggested the formation of autophagosomes (increased gene expression of and ) and two caspases (caspase-3 and -9) were responsible for the activation and progression of apoptosis.
These data illustrate the potential of fullerene-based BTK protein inhibitors as nanotherapeutics for blood cancer and provide helpful information to support the future development of fullerene nanomaterials as a novel class of enzyme inhibitors.
由于合成方法的最新进展，已经发现具有干扰生物分子（特别是 DNA/RNA 和选定蛋白质）能力的水溶性富勒烯纳米材料在纳米医学中有巨大的应用潜力。本文描述了一种水溶性甘氨酸衍生的 [60]富勒烯六加成物（HDGF）的合成与评价，它是一种首创的 BTK 蛋白抑制剂。
我们使用 NMR、ESI-MS 和 ATR-FT-IR 合成并表征了甘氨酸衍生的 [60]富勒烯。通过 DLS 和 ζ 电位测量以及高分辨率透射电子显微镜（HRTEM）观察进行了评估。通过 X 射线光电子能谱检查了水溶性富勒烯纳米材料的化学组成。为了观察聚集形成，进行了冷冻透射电子显微镜（cryo-TEM）分析。进行了对接研究和分子动力学模拟，以确定 HDGF 和 BTK 之间的相互作用。在 RAJI 和 K562 血液癌细胞系上评估了体外细胞毒性。随后，通过测定关键基因和半胱天冬酶的表达水平，研究了自噬和细胞凋亡诱导的细胞死亡。通过检测 RAJI 细胞治疗后钙水平的变化，研究了 HDGF 对 BTK 信号通路抑制的直接关联。评估了 HDGF 对非受体酪氨酸激酶的抑制潜力。最后，评估了 HDGF 和 ibrutinib 对 RAJI 细胞中 BTK 蛋白及其下游信号转导表达的影响，以及在抗 IgM 刺激后。
计算研究表明，所获得的[60]富勒烯衍生物的抑制活性是多方面的：它阻碍 BTK 活性位点，直接与催化残基相互作用，使其无法进行磷酸化，并与形成 ATP 结合口袋的残基结合。所产生的碳纳米材料的抗癌活性表明，它在细胞水平上抑制了 BTK 蛋白及其下游途径，包括 PLC 和 Akt 蛋白。机制研究表明，自噬体的形成（和的基因表达增加）和两个半胱天冬酶（caspase-3 和 -9）负责凋亡的激活和进展。
这些数据说明了基于富勒烯的 BTK 蛋白抑制剂作为血液癌的纳米治疗剂的潜力，并提供了有助于支持富勒烯纳米材料作为新型酶抑制剂的未来发展的信息。
