Selectively Disrupting m⁶A-Dependent Protein–RNA Interactions with Fragments

Abstract

We report a crystallographic analysis of small-molecule ligands of the human YTHDC1 domain that recognizes N6methylated adenine (m 6 A) in RNA. The 30 binders are fragments (molecular weight < 300 g mol −1 ) that represent 10 different chemotypes identified by virtual screening. Despite the structural disorder of the binding site loop (residues 429−439), most of the 30 fragments emulate the two main interactions of the −NHCH 3 group of m 6 A. These interactions are the hydrogen bond to the backbone carbonyl of Ser378 and the van der Waals contacts with the tryptophan cage. Different chemical groups are involved in the conserved binding motifs. Some of the fragments show favorable ligand efficiency for YTHDC1 and selectivity against other m 6 A reader domains. The structural information is useful for the design of modulators of m 6 A recognition by YTHDC1.

Fast fragment- and compound-screening pipeline at the Swiss Light Source

“…Based on results from initial optimization we instead performed DMSO-free soaking, in which preselected fragments dissolved in DMSO were deposited onto a crystallization plate with the Echo device, air-dried to remove the solvent and redissolved in the crystallization buffer. Subsequently, 70 YTHDC1 domain crystals were manually transferred to crystallization drops for soaking and harvested (both steps were assisted by the Crystal Shifter), resulting in 30 fragment-bound structures (Bedi et al, 2020). The dedicated Redissolve tab was created in the FFCS GUI on demand for this particular project, enabling the tracking of a DMSO-free soaking experiment in the FFCS DB.…”

“…After soaking has been completed, the Echo experiment report is sent to the FFCS DB, the Soaking tab timer is stopped, and the soaking status displayed in the FFCS GUI is updated accordingly. The Redissolve tab is an optional step of the FFCS pipeline which was created on demand for a challenging project that required DMSO-free soaking (Bedi et al, 2020).…”

Atomistic and Thermodynamic Analysis of N6-Methyladenosine (m6A) Recognition by the Reader Domain of YTHDC1

“…All of these regulatory functions are achieved through its binding to m 6 A-modified RNA and therefore it is necessary to fully understand its molecular recognition mechanism. YTHDC1 is the first human reader protein whose structure was successfully determined and was recently validated as a target of small-molecule inhibitors . These pioneer studies make YTHDC1 an ideal model system for studying the binding mechanisms of reader proteins and their binders.…”

Targeting the RNA demethylase FTO for cancer therapy

“… 109 Bedi et al performed computational screening of fragments and found thirty m 6 A mimics with the potential to bind to reader proteins. 110 There are also few selective modulators for another m 6 A demethylase, ALKBH5. For example, Li et al identified the ALKBH5 inhibitor ALK-04 (the structure remains unreleased) through in silico screening, which significantly enhances the efficacy of anti-PD-1 therapy.…”

“…Most recently, STM2457, a potent and selective METT3 inhibitor has been developed and showed pharmacological effects on AML in mice model 109. Recently, Bedi et al performed computational screening of fragments and found thirty m6 A mimics with the potential to bind to reader proteins 110. There are also few selective modulators for another m6 A demethylase, ALKBH5.…”