Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily‐Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3

Abstract

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic "thermal tag" with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC =2.6 μm) and ALKBH3 (8; IC =3.7 μm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.

A fluorescent methylation-switchable probe for highly sensitive analysis of FTON6-methyladenosine demethylase activity in cells

“…To assess the performance of our assay, a set of structurally-diverse small molecules ( Fig. 5a ) consisting of known FTO inhibitors (24PDCA, 50 LipotF, 25 Rhein, 24 IOX3 50 ) and non-inhibitor isonicotinic acid 40 were individually incubated at various concentrations with FTO (50 nM) and m 6 A-probe (10 μM) in a 384-well plate, and their fluorescence spectra simultaneously recorded on a fluorescence microplate reader. The inhibition curves are shown in Fig.…”

Targeting the RNA demethylase FTO for cancer therapy

“…By combining DSF with multiprotein dynamic combinatorial chemistry, Das et al developed an assay that simultaneously measures the melting temperatures of ALKBH3, ALKBH5, and FTO in dynamic chemistry libraries, which enabled the identification of selective inhibitors for FTO (with IC 50 = 2.6 μM) and ALKBH3. 70 …”

Recent Advances of m6A Demethylases Inhibitors and Their Biological Functions in Human Diseases

“…Later on, Svensen and Jaffrey reported an approach to identify FTO inhibitors by using a fluorometric RNA substrate based on broccoli aptamer [ 104 ]. Das and co-workers designed a multi-protein dynamic combinatorial chemistry (DCC) system for screening FTO inhibitors [ 105 ]. More recently, Zhang et al developed a single quantum dot-based Förster resonance energy transfer (FRET) nanosensor for FTO inhibitor screening [ 106 ].…”

“…The molecule MO-I-500 displayed anticonvulsant activity in the 6 Hz mouse model at a nontoxic dose, increased the total m6A level of cellular RNA, and altered the production of relative microRNAs. Through using a multi-protein DCC strategy, compound 12 ( Figure 3 ) was identified as a FTO (IC 50 = 2.6 μM) selective inhibitor, in comparison with ALKBH5 (IC 50 = 201.3 μM) [ 105 ]. The structural model of FTO- 12 revealed that compound 12 coordinated with Fe 2+ in a bidentate manner, which was further stabilized by a combination of hydrogen-bonding and salt bridge interactions with Arg96, Arg319, Tyr295, and Ser318 of side chains from FTO.…”