We solved the structure of a PBM peptide bound to its cognate site on PRMT5, which is distinct and distal from the catalytic domain. Specifically, we identified a conserved 7 residue peptide sequence, GQF(D/E)DA(D/E), present in all three known PRMT5 SAPs (pICln, RIOK1 and COPR5), that mediates substrate adaptor binding to PRMT5, which we termed the PRMT5 Binding Motif (PBM). We recently elucidated the molecular mechanism mediating the interaction between PRMT5 and its SAPs. This mechanism would be distinct from catalytic site inhibition and potentially provide synergy with, or a different therapeutic profile than, the previously mentioned approaches. Large-scale shRNA screens have also implicated PRMT5 SAPs, pICln and RIOK1, as a potential alternative route to develop MTAP-null selective therapeutics 10. The efficacy and safety profile of MAT2A inhibitors are currently being evaluated in clinical trials ( NCT03435250). However, SAM is a ubiquitous methyl donor, and a reduction of its levels has an effect on multiple methyltransferases, potentially posing a toxicity risk to this approach. Preclinical data support an MTAP-dependent and synergistic anti-tumor activity when used in combination with taxanes or gemcitabine 9. In addition, inhibitors of the upstream enzyme MAT2A, which catalyzes the synthesis of SAM, have been generated to exploit the unbalanced SAM/MTA ratio of MTAP-deleted cancers. An MTA-cooperative compound might potentially leverage this synthetic lethality, however such an inhibitor is not yet available 8. However, these molecules act with either a SAM cooperative 4 or SAM/MTA-competitive mode of action 5, 6, 7 and thus do not appear to take advantage of MTAP deletion to provide the desired therapeutic window. Several potent and selective inhibitors of the PRMT5 catalytic pocket have been developed. This observed synthetic lethal phenotype has led to the hypothesis that pharmacological PRMT5 inhibition could be a viable strategy with a suitable therapeutic window for clinical use against MTAP-deleted cancers 3, 1, 2. This partial inhibition of PRMT5 sensitizes MTAP-deleted cells to further loss of PRMT5 function by siRNA. However, MTA appears to have selective inhibitory activity on PRMT5 alone, likely due to key structural features within its SAM binding pocket 1, 2. PRMT5 belongs to the PRMT family, which consists of nine members, all of which use SAM as their methyl donor cofactor. PRMT5 has an obligate partner, WDR77, with which it forms a hetero-octamer, also known as the methylosome complex. While loss of MTAP has as yet unknown functional consequences for tumorigenesis, it leads to accumulation of its substrate methylthioadenosine (MTA), which acts as an endogenous competitive inhibitor of S-adenosylmethionine (SAM), the methyl donor used by the protein arginine N-methyltransferase, PRMT5, which is involved in the regulation of gene expression, mRNA splicing, protein translation, DNA damage response and immune functions. The MTAP gene is in close proximity to the tumor suppressor CDKN2A locus on chromosome 9, and is hence frequently codeleted. For example, 15-50% of pancreatic cancers, glioblastoma and mesotheliomas, cancers for which there is a critical need for new drugs, bear deletions of the methylthioadenosine phosphorylase gene, MTAP. Recent advances in cancer genomics and molecular biology have led to the identification of novel tumor selective therapeutic opportunities. BRD0639 is a first-in-class PBM-competitive small molecule that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts the PRMT5-RIOK1 complex, and reduces substrate methylation. Mode of action and structure determination studies revealed that these compounds form a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells.
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