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

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.