Discovery and Structural Basis of the Selectivity of Potent Cyclic Peptide Inhibitors of MAGE-A4

Abstract

MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.

Tyrosinase-Catalyzed Peptide Macrocyclization for mRNA Display

“…We further deploy this enzymatic chemistry in the effective selection of new macrocyclic peptide inhibitors of the cancer-testes antigen MAGE-A4. 2,31 This work provides first evidence of the ability to use tyrosinase in peptide cyclization and uncovers key knowledge for the use of tyrosinase as well as other peptide-modifying enzymes in selection campaigns.…”

“… , Both regioisomers as well as cMCF01 and the respective linear precursors were tested in a previously developed TR-FRET assay to assess disruption of the MAGE-A4 binding interaction (Figures E and S8). All three macrocycles exhibited potent inhibitory activity with IC 50 values in the low nanomolar range, while linear versions were significantly less active. The major C5 regioisomer of MCF02 proved more potent than the minor C2 isomer by almost 5-fold, and the IC 50 of the linear precursor shows little or no inhibition up to 10 μM.…”

“…mRNA display is a powerful tool for discovery of macrocyclic peptide ligands against challenging protein targets. − Macrocyclization typically rigidifies and preorganizes peptide hits into a high-affinity binding conformation. Additionally, macrocyclization tends to contribute to improved stability and cell permeability for potential therapeutic leads or probes. − Key examples include the peptide macrocycles MK-0616 − from Merck and RA101495 from UCB Pharma, both of which are derived from mRNA display hits and have advanced in clinical trials.…”

Roles of trans-lesion synthesis (TLS) DNA polymerases in tumorigenesis and cancer therapy

“…Owing to its cancer cell-specific expression and requirement for DNA damage tolerance, the MAGEA4/RAD18 signaling axis is an appealing pathway for therapeutic inhibition. Fleming et al identified small cyclic peptide inhibitors that bind directly to MAGEA4 and inhibit RAD18 binding with nanomolar potency ( 246 ). That study validates the chemical tractability of MAGEA4/RAD18 as a cancer-specific vulnerability and justifies further work to identify more drug-like pharmacological inhibitors of pathological TLS in cancer.…”

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase

“…Notably, enrichment of the top peptide dramatically increased between rounds 2 and 3 (Figure C). Thus, unlike many reported mRNA display selections, , we observed rapid enrichment for largely one sequence and one family.…”