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.