mRNA display-enabled discovery of proximity-triggered covalent peptide–drug conjugates

Peptide–drug conjugates (PDCs) have emerged as a promising modality in precision oncology, enabling targeted delivery of cytotoxic payloads while minimizing off-target toxicity. The integration of covalent warheads, such as those based on sulfur(VI) fluoride exchange (SuFEx) chemistry, enhances drug–target residence time and tumor accumulation. However, existing screening methods for covalent peptide (CP) libraries require post-translational warhead conjugation, limiting throughput. Here, we present an integrated mRNA display platform that incorporates covalent warheads during ribosomal synthesis, enabling efficient screening of ultra-diverse covalent macrocyclic peptide libraries (>10 13 variants). This approach, using site-specific incorporation of N- chloroacetyl-D-phenylalanine and fluorosulfate-L-tyrosine, accelerated the discovery of irreversibly binding ( K i = 3.58 μmol/L) Nectin-4-targeting peptide CP-N1-N 3 via proximity-triggered SuFEx. The peptide was further conjugated to cytotoxic payloads, yielding the covalent PDC CP-N1-MMAE with potent cytotoxicity (IC 50 ≈ 43 nmol/L) against MDA-MB-468 cells. This platform establishes a new paradigm for precision covalent drug discovery.