DRONE: Direct Tracking of DNA Cytidine Deamination and Other DNA Modifying Activities

Abstract

Enzymes that catalyze DNA modifying activities including cytidine deamination and cytosine methylation play important biological roles and have been implicated pathologically in diseases such as cancer. Here, we report Direct Resolution of ONE dalton difference (DRONE), an UHPLC-based analytical method to track a single dalton change in the cytosine-to-uracil conversion catalyzed by the human apolipoprotein B m-RNA editing catalytic polypeptide-like 3 (APOBEC3) cytidine deaminases, implicated in cancer and antiviral defense. Additionally, we demonstrate broad applicability by tracking other important DNA modifications and assessing epigenetic enzyme inhibition. We have extended our methodology to obtain data on two distinct deamination events in the same oligonucleotide substrate designed from a putative APOBEC substrate, diversifying the utility of the described method. DRONE provides an important foundation for in-depth analysis of DNA-modifying enzymes and versatile detection of novel DNA modifications of interest.

The current toolbox for APOBEC drug discovery

“…However, LC-MS/MS is often used in drug development for the characterization of covalent inhibitors discovered through HTS [98,99]. A significant advance in this area was the development of DRONE (direct resolution of one Dalton difference), which is an ultra-high performance LC (UHPLC)-based A3 activity assay that monitors ssDNA C-to-U deamination with HPLC separation [100]. Because A3-mediated deamination results in only a single Dalton difference between substrate and product, sensitive analytical techniques are necessary to distinguish between the two.…”

“…Because A3-mediated deamination results in only a single Dalton difference between substrate and product, sensitive analytical techniques are necessary to distinguish between the two. In this assay, the authors leveraged a variety of LC parameters, including a hexafluoroisopropanol/triethylamine buffer system in the mobile phase to increase chromatographic resolution [100]. Limitations of this assay include the need for sample processing before analysis, which decreases its adaptability for HTS, and the assay does not report real-time deamination (Table 2).…”

Structure-Guided Identification of DNMT3B Inhibitors

“…The computed aqueous solubilities of 10 −3 to 10 −6 M and octanol/water log P values of 1−3 are all in the normal ranges for oral drugs. 24 To confirm the potency of compound 33h, we subsequently performed a Direct Resolution of One Dalton difference (DRONE) assay described recently by Sasaki et al 25 In this assay an ultrahigh-performance liquid chromatography (UHPLC)-based analytical method is used to directly monitor reaction kinetics of DNA modifying enzymes including cytidine deamination and cytosine methylation.…”

“…This assay was previously applied to inactivation of DNMT3B by nanaomycin A and gave an IC 50 of 730 nM, in good accord with the value of 1.5 μM found here in the fluorogenic assay. When the DRONE assay was performed for compound 33h , an IC 50 of 4.8 μM was obtained (Figure A), again about a factor of 2 lower than in the fluorogenic assay.…”

“…(A) Inhibition of methyltransferase activity by the addition of DNMT3B inhibitor 33h . (B) Docking pose from Glide XP for 33h .…”