Discovery of APOBEC Cytidine Deaminases Inhibitors Using a BspH1 Restriction Enzyme‐Based Biosensor

Abstract

Apolipoprotein B mRNA‐editing enzyme catalytic polypeptide like (APOBEC) DNA cytidine deaminases convert cytosine to uracil in pathogen‐related single‐stranded DNAs. Aberrant activation of APOBEC enzymes in tumour cells leads to hypermutations that drive heterogeneity and clonal evolution, which results in tumour progression and treatment adaptation. APOBEC3 family member APOBEC3B (A3B) is a promising drug target to combat drug resistance, but the discovery of potent lead compounds remained challenging. Here, we devised a BspH1 restriction enzyme‐based biosensor to measure APOBEC deaminase activity, with superior simplicity and without the need of counter assays. Using this method, we performed a proof‐of‐concept screening using series of flavonoids and dihydrochalcones, from which bona fide inhibitors of recombinant A3B were identified and further validated by isothermal titration calorimetry. Our results demonstrate the capability of the BspH1‐based biosensor as a method for HTS, and prospects of developing potent A3B inhibitors using flavonoid and dihydrochalcone backbones.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A

“…During our study, two articles have been published on drug discovery efforts to obtain small molecule inhibitors of A3. 63,64 None approaches the potency of our inhibitors. This highlights the interest and need for powerful A3 inhibitors.…”

“…As K m (and K i ) depends strongly on the surrounding environment (e.g., buffer composition, pH, and ionic strengthsee Table ), inhibition with even lower K i may be observed in the cellular environment, because K m and K d values in the low μM , and nM range have been reported. During our study, two articles have been published on drug discovery efforts to obtain small molecule inhibitors of A3. , None approaches the potency of our inhibitors. This highlights the interest and need for powerful A3 inhibitors.…”

Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer

“…The pivotal role of APOBEC3B was further confirmed using two APOBEC3B inhibitors, phloretin and icariside I. 89 While SYNCRIP-deficient cells demonstrated resistance to enzalutamide, they exhibited substantial responsiveness to both APOBEC3B inhibitors ( Figure 3I ). We then examined whether augmented levels of APOBEC proteins could bypass the restriction of endogenous SYNCRIP proteins and induce resistance in sensitive PCa cells.…”

Characterisation of APOBEC3B-Mediated RNA Editing in Breast Cancer Cells Reveals Regulatory Roles of NEAT1 and MALAT1 lncRNAs

“…We incorporated ve breast cancer cell lines with documented A3A and A3B expression pro les into this study [20], and gauged the activity of intrinsic A3B in these lines. A3B activity was measured by immunoprecipitating the A3B complex and measuring enzymatic function using a DNA deaminase biosensor incorporating BspHI restriction enzyme [39]. In the four A3B expressing cell lines, NEAT1 and MALAT1 depletion led to an enhanced enzymatic activity of A3B, underscoring that sustained suppression of their expression augments A3B activity (Fig.…”