Phage Display of Two Distinct Warheads to Inhibit Challenging Proteins

Falling in between traditional small molecules and antibodies in size, peptides are emerging as a privileged therapeutic modality, one that can harness the benefits of both small molecule and antibody drugs. To discover potential peptide therapeutics, it is highly desirable to have high throughput screening platforms that can assess peptides with diverse and non-natural functional motifs. With this contribution, we present a novel phage library that incorporates two distinct designer groups. As an example, a pair of reversible covalent warheads was installed onto phage-displayed peptides to target a cysteine and a lysine. The double modification is realized by sequential modification of an N-terminal cysteine and then an internal cysteine using chemoselective chemistry. Screening of this double-warhead-presenting library against TEV protease readily revealed peptide inhibitors with single-digit micromolar potency. Importantly, our structure-activity studies demonstrate that both covalent warheads make important contributions to TEV protease inhibition. We envision that our strategy of double phage modification can be readily extended to build phage libraries with diverse structural motifs, allowing facile expansion of the chemical space coverable by phage display.