P025 Obefazimod and its active metabolites ABX-464-N-Glu act by stabilizing protein-protein interaction among key RNA biogenesis partners, CBC and ARS2

Abstract Background Obefazimod is an oral, once-daily, small molecule in phase 3 clinical trials for the treatment of patients with ulcerative colitis (UC). Obefazimod selectively enhances the expression of a single micro-RNA (miRNA), miR-124. miRNAs function by binding to specific mRNA targets and reducing their translation into their respective proteins. Enhanced expression of miR-124 results in a decrease in cytokines and immune cells, helping to reduce inflammation and potentially controlling the progression of chronic conditions like UC. Obefazimod and its main active metabolite, ABX464-N-Glu, bind the nuclear Cap Binding Complex (CBC) that directs cellular RNA biogenesis by recognizing co-transcriptionally the cap structure of RNA polymerase II transcripts. The CBC complex is made of Cbp20 and Cbp80, and interacts with a key partner involved in microRNA biogenesis, ARS2. Here, we explore the structural basis for targeting the CBC complex. Methods Microscale thermophoresis analyses were performed to determine the binding affinities between the two components of CBC, Cbp20 and Cbp80, in presence or absence of obefazimod or its metabolite ABX464-N-Glu, and their interaction with ARS2 also in presence or absence of the two molecules. Cryo-electron microscopy (cryo-EM) datasets of CBC-apo, CBC-obefazimod, CBC-ABX464-N-Glu, CBC-ARS2-Apo, CBC-ARS2-obefazimod, CBC-ARS2-ABX464-N-Glu were processed and after 2D classification and 3D refinement the different structures were solved. To understand and explore the conformational dynamics of the complex, 3D multibody refinement was performed on CBC-Apo, CBC-obefazimod and CBC:ABX464-N-Glu. Results The cryo-EM structures showed that obefazimod or ABX464-N-Glu bind the same location at the interface of CBC complex to act as orthosteric stabilizers of Cbp20 and CBp80. The conformational change induced by the binding of obefazimod or ABX464-N-Glu to the CBC complex strengthens the interaction between the two subunits of CBC, resulting in a more rigid structure of CBC complex. Thermophoresis revealed that the binding of obefazimod or ABX464-N-Glu improves the interaction of CBC with ARS2, a key partner of CBC to establish the fate of RNA in the cell. The cryo-EM structures of CBC-ARS2, obefazimod-CBC-ARS2 or ABX464-N-Glu-CBC-ARS2 show that ARS2 passes through the site where Obefazimod and ABX464-NGlu bind. We propose that the orthosteric effect of obefazimod or ABX464-N-Glu on CBC is maintained even when these molecules leave their binding site, allowing for better affinity of ARS2 for CBC. Conclusion Our results identify a unique interfacial stabilizing mode of action for anti-inflammatory drugs targeting the CBC complex enhancing its interaction with ARS2 to generate specific microRNA.