Structural basis for NONO specific modification by the α-chloroacetamide compound <i>(R)</i> -SKBG-1

SUMMARY Among the many proteins involved in cancer progression an increasing number of RNA Binding Proteins (RBPs) are central to the function of a cell and tightly associated to genetic diseases as well as cancer appearance and progression. In a recent study, small molecule inhibitors have been identified as targeting NONO, a RBP known to be involved in mRNA splicing, DNA repair and membraneless organelles stability. Here we report the molecular basis of NONO-targeting by the α-chloroacetamide (R)- SKBG-1. We explore the specific binding and enantiomer specificity of NONO towards (R) -SKBG-1 using mass spectrometry and structure determination. We have determined the crystal structure of (R )-SKBG-1-bound to NONO homodimer. This study sheds light on the conformational plasticity of (R) -SKBG-1 when covalently bound to NONO. Altogether these results give an experimental rationale for ligand modification and optimization in a future use as a drug against cancer. SIGNIFICANCE DBHS proteins form a family of three proteins encoded by three different and essential genes. They form obligate homodimers and heterodimers to fulfil their function. In the cell, they are involved in mRNA splicing, DNA repair and membraneless organelles formation. Recently, NONO has been identified as a target of small-molecule inhibitors in prostate cancer cells. Treatment with α-chloroacetamide modifies a specific cysteine residue only found in NONO and not in its paralogue proteins SFPQ and PSPC1. Here we provide the molecular basis of α-chloroacetamide covalent binding to NONO and we explore the enantiomer specificity of binding. We also demonstrate that α-chloroacetamide can target NONO in homodimers and heterodimers and that both binding sites are equivalently modified. Finally, we provide show that α-chloroacetamide binding to NONO is driven by the combination of covalent binding and conformational flexibility of the ligand. Altogether, we believe that this study provides useful information for ligand improvement aiming at targeting NONO in cancer cells. OUTLINE NONO residue C145 is targeted by (R)- SKBG-1 The two binding sites are equally modified in NONO homodimers NONO is specifically targeted and not SFPQ and PSPC1 (R)- SKBG-1 adopts multiple conformations in the absence of RNA

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