The PRMT5–MEP50 methyltransferase complex plays a key role in various cancers and is regulated by different protein–protein interactions. Several proteins have been reported to act as adaptor proteins that recruit substrate proteins to the active site of PRMT5 for the methylation of arginine residues. To define the interaction between these adaptor proteins and PRMT5, we employed peptide truncation and mutation studies and prepared truncated protein constructs. We report the characterisation of the interface between the TIM barrel of PRMT5 and the adaptor proteins pICln, RioK1 and COPR5, and identify the consensus amino acid sequence GQF[D/E]DA[E/D] involved in binding. Protein crystallography revealed that the RioK1 derived peptide interacts with a novel PPI site.

The scaffolding protein RbAp48 is part of several epigenetic regulation complexes and is overexpressed in av ariety of cancers.I no rder to develop tool compounds for the study of RbAp48 function, we have developed peptide inhibitors targeting the protein-protein interaction interface between RbAp48 and the scaffold protein MTA1. Based on aM TA1-derived linear peptide with lowm icromolar affinity and informed by crystallographic analysis,abicyclic peptide was developed that inhibits the RbAp48/MTA1 interaction with av ery low nanomolar K D value of 8.56 nM, and which showed appreciable stability against cellular proteases.Design included exchange of ap olar amide cyclization strategy to hydrophobic aromatic linkers enabling mono-and bicyclization by means of cysteine alkylation, whichi mproved affinity by direct interaction of the linkers with ahydrophobic residue on RbAp48. Our results demonstrate that stepwise evolution of as tructure-based design is as uitable strategy for inhibitor development targeting PPIs.

The PRMT5-MEP50 methyltransferase is a major target for anticancer drug discovery, and modulators of its interactions with different regulatory proteins are in high demand because they modulate PRMT5 substrate selectivity. We describe a strategy for the development of a PRMT5/adaptor protein PPI inhibitor, which includes the design and synthesis of macrocyclic peptides based on the motif for the interaction of PRMT5 with its adaptor protein RioK1. After the initial exploration of different macrocycle sizes and cyclization linkages, analysis of a peptide library identified hot spots for the variation of the amino acid structure. The incorporation of nonproteinogenic amino acids into the macrocyclic peptide led to a potent cyclic PRMT5 binding peptide (K i = 66 nM), which selectively inhibits the interaction of PRMT5 with the adaptor proteins RioK1 and pICln (IC 50 = 654 nM) but not with the alternative adaptor protein MEP50. The inhibitor is a promising tool for further biological investigation of this intriguing protein interface.

Pseudo‐natural products (pseudo‐NPs) are de novo combinations of natural product (NP) fragments that define novel bioactive chemotypes. For their discovery, new design principles are being sought. Previously, pseudo‐NPs were synthesized by the combination of fragments originating from biosynthetically unrelated NPs to guarantee structural novelty and novel bioactivity. We report the combination of fragments from biosynthetically related NPs in novel arrangements to yield a novel chemotype with activity not shared by the guiding fragments. We describe the synthesis of the polyketide pseudo‐NP grismonone and identify it as a structurally novel and potent inhibitor of Hedgehog signaling. The insight that the de novo combination of fragments derived from biosynthetically related NPs may also yield new biologically relevant compound classes with unexpected bioactivity may be considered a chemical extension or diversion of existing biosynthetic pathways and greatly expands the opportunities for exploration of biologically relevant chemical space by means of the pseudo‐NP principle.