Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells

Abstract

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

“…Next, we considered a complex of a 70-nucleotide DNA aptamer and the CYP24 protein, which has been demonstrated to be relevant for antiproliferative activity in cancer cells and was previously observed under HS-AFM ( Biyani et al, 2022 ). The 3D atomistic structure of the complex as predicted from molecular docking simulations and the computed molecular surface representation with charge coloring according to the electrostatic potential are shown in Figure 2B .…”

Vitamin D inhibits osteosarcoma by reprogramming nonsense-mediated RNA decay and SNAI2-mediated epithelial-to-mesenchymal transition

“…In general, increased 24-hydroxylation of 1,25(OH) 2 D results in the much less active vitamin D 3 metabolite, 1,24,25(OH) 2 D, potentially supporting tumorigenicity. Indeed, overexpression of CYP24A1 has been linked to a poor prognosis in some human cancers 121 , and recently novel small molecule DNA aptamers have been identified that target CYP24A1 with promising anticancer effects 122 . However, in our studies, 1,25(OH) 2 D did not induce CYP24A1 in MG63 cells, which may reflect an enhanced antitumor response via increased 1,25(OH) 2 D but not the 1,24,25(OH) 2 D metabolite.…”

Vitamin D inhibits osteosarcoma by reprogramming nonsense-mediated RNA decay and SNAI2-mediated epithelial-to-mesenchymal transition

“…In general, increased 24-hydroxylation of 1,25(OH) 2 D results in less active vitamin D 3 metabolite, 1,24,25 (OH) 2 D, potentially supporting tumorigenicity. Indeed, overexpression of CYP24A1 has been linked to a poor prognosis in some human cancers (126), and recently novel small molecule DNA aptamers have been identified that target CYP24A1 with promising anticancer effects (127). However, in our studies, 1,25 (OH) 2 D did not induce CYP24A1 in MG63 cells, which may reflect an enhanced antitumor response via increased 1,25(OH) 2 D but not the 1,24,25(OH) 2 D metabolite.…”