Ricerc@Sapienza

- LSD2 is a histone demethylase which employs a flavin-dependent reaction mechanism to process H3K4me1 and H3K4me2. LSD2 forms a complex with the cytokine-like nuclear factor NPAC and the two proteins are critical for gene transcription elongation. NPAC is a multidomain protein containing a 9-residue linker segment that binds and activates LSD2, increasing its binding affinity for nucleosomes. Using the Q Exactive UHMR, we are going to identify the minimal NPAC linker sequence capable of activating LSD2. To this end, we are monitoring LSD2-nucleosome complex formation in the presence of different peptides. These experiments pave the way for rational design of peptidomimetic modulators which will be initially assessed in vitro and then in cell. - RNA modifications influence its structure, stability and function and have been found in a diverse subset of RNA classes. The most common modification is methylation which is catalysed by a wide range of S-adenosyl-L-methionine (SAM)-dependent RNA methyltransferases. Among them, the heterodimer METTL3-METTL14 catalyses the transfer of one methyl moiety to adenosine exocyclic nitrogen N6, yielding 6-methyladenosine (m6A), which influences RNA stability, splicing, nuclear export and translation. The catalytic subunit METTL3 has been reported to support initiation and maintenance of AML and METTL14 was indicated to promote leukemogenesis in vitro and in vivo. Hence, the METTL3-METTL14 complex is an attractive target for the development of anti-cancer drugs. The reported crystal structure of METTL3-METTL14 accelerated the discovery of small molecule inhibitors, such as the recently described STM2457, a potent and selective inhibitor of METTL3. Based on STM2457-METTL3-METTL14 cocrystal we plan to develop novel inhibitors. We are using Native MS as a platform to quickly screen our in-house library in the context of the multiprotein complex formed by METTL3 and partners (i.e., WTAP and KIAA1429). Thanks to the wide m/z range of the Q Exactive UHMR we are able to study these complexes and the influence of small molecules and additional proteins on enzymatic activity and substrate recognition. Through the integration of native MS with other biophysical techniques (such as SPR), we aim to gain structure-activity relationship information that will inform the synthesis of novel compounds.