As an emerging field of RNA epigenetics in molecular biology, m⁶A modification has been shown to affect transcription and expression of key pathogenic gene sets in various diseases. ALKBH5, an eraser protein of m⁶A modification, can affect disease progression by regulating m⁶A modification in RNA. However, regulating ALKBH5 activity through chemical small molecule intervention remains a challenge.

In a study published in Angewandte Chemie International Edition, a team led by Prof. LIU Hong and Prof. YANG Caiguang from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences identified compound 18l as a covalent inhibitor targeting non-catalytic Cys200 residue of ALKBH5, verified its Cys200 covalent mechanism, in vitro and in vivo activity, and most importantly, demonstrated intracellular targeting of ALKBH5.

Cys200 residue is a non-catalytic covalent site unique to ALKBH5, located near single-stranded RNA (ssRNA) substrate binding pocket of ALKBH5. Researchers envisioned that targeting this site for covalent modification could interfere with substrate binding to ALKBH5 and achieve selective activity inhibition. Through preliminary screening and structure-activity relationship studies, they obtained compound 18l (ALKBH5 IC₅₀ = 620 nM) with maleimide as a covalent warhead. 

A variety of in vitro experiments confirmed that 18l inhibited ALKBH5 enzymatic activity by covalently modifying Cys200, preventing substrate binding to ALKBH5. The 18l exhibited good selectivity for ALKBH5 over FTO and various Cys proteases. At the cellular level, compound 18l was able to inhibit AML cell proliferation, promote AML cell apoptosis, induce differentiation into normal cells, block cell cycle, and inhibit ALKBH5-mediated signaling pathways. The 18l could increase m⁶A level in RNA in NB4 cells, indicating that the compound could inhibit ALKBH5 in cells. 

To verify the correlation between 18l's enzyme-level ALKBH5 inhibitory activity and anti-AML cell phenotype, researchers conducted non-modification targeting verification and ABPP-based chemical proteomics studies. In the non-modification experiment, 18l could change protease resistance and thermal stability of ALKBH5, proving its intracellular targeting engagement. In ABPP proteomics experiment, a chemical probe based on 18l could bind to ALKBH5, confirming that 18l can bind to ALKBH5 in AML cells. 

Finally, an in vivo antitumor pharmacodynamic study was conducted in an AML cell mouse xenograft tumor model. Researchers found that at doses of 1 mg/kg and 2.5 mg/kg, 18l effectively inhibited tumor growth, achieving tumor inhibition rates of 66.3% and 76.8%, respectively, without observable adverse effects, demonstrating the efficacy and safety in vivo.

The findings of this study provide new ideas for precise chemical regulation in the field of RNA epigenetics and pave the way for developing novel lead compound targeting ALKBH5.