A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides

Thiopeptides make up a group of structurally complex
peptidic natural
products holding promise in bioengineering applications. The previously
established thiopeptide/mRNA display platform enables de novo discovery
of natural product-like thiopeptides with designed bioactivities.
However, in contrast to natural thiopeptides, the discovered structures
are composed predominantly of proteinogenic amino acids, which results
in low metabolic stability in many cases. Here, we redevelop the platform
and demonstrate that the utilization of compact reprogrammed genetic
codes in mRNA display libraries can lead to the discovery of thiopeptides
predominantly composed of nonproteinogenic structural elements. We
demonstrate the feasibility of our designs by conducting affinity
selections against Traf2- and NCK-interacting kinase (TNIK). The experiment
identified a series of thiopeptides with high affinity to the target
protein (the best KD = 2.1 nM) and kinase
inhibitory activity (the best IC50 = 0.15 μM). The
discovered compounds, which bore as many as 15 nonproteinogenic amino
acids in an 18-residue macrocycle, demonstrated high metabolic stability
in human serum with a half-life of up to 99 h. An X-ray cocrystal
structure of TNIK in complex with a discovered thiopeptide revealed
how nonproteinogenic building blocks facilitate the target engagement
and orchestrate the folding of the thiopeptide into a noncanonical
conformation. Altogether, the established platform takes a step toward
the discovery of thiopeptides with high metabolic stability for early
drug discovery applications.