De Novo Discovery of Thiopeptide Pseudo-natural Products Acting as Potent and Selective TNIK Kinase Inhibitors

Abstract

Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥1012) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affinity selection against Traf2- and NCK-interacting kinase (TNIK), a protein involved in several cancers, which yielded a plethora of candidate thiopeptides. Of the 11 synthesized compounds, 9 had high affinities for the target kinase (best K D = 1.2 nM) and 10 inhibited its enzymatic activity (best K i = 3 nM). X-ray structural analysis of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive inhibition exhibited by two of the discovered compounds. The thiopeptides internalized to the cytosol of HEK293H cells as efficiently as the known cell-penetrating peptide Tat (4–6 μM). Accordingly, the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether, our platform enables the exploration of pseudo-natural thiopeptides with favorable pharmacological properties in drug discovery applications.

Solid-Phase-Based Synthesis of Lactazole-Like Thiopeptides

“…2.2.1). Additionally, our companion article reports the X-ray crystal structure of TP15 bound to its target protein TNIK (PDB 7xzr), further confirming the chemical structure of the synthesized thiopeptide …”

“…Additionally, our companion article reports the X-ray crystal structure of TP15 bound to its target protein TNIK (PDB 7xzr), further confirming the chemical structure of the synthesized thiopeptide. 20 Using these general synthetic protocols, we synthesized 11 target thiopeptides identified from the mRNA display selection, 20 all in multimilligram quantities sufficient for further biochemical characterization (Figure 3c). The overall synthetic yields, calculated based on the original resin loading, varied between 5 and 27%, and in most cases the macrocyclization, deprotection, and oxidation steps could be carried out without the purification of the peptide intermediates.…”

“…The resulting carboxylic acid, TP4 OH -Sec(Me)13, could then be coupled to various amines, such as a chloroalkane tag or rhodamine 6G-amine, and the remainder of the synthesis followed the established techniques. Following these protocols, we synthesized six functionalized thiopeptides (TP1 ct , TP4 ct , TP8 ct , TP14 ct , TP15 ct , and TP4 Rho ) for use in downstream biological assays (reported separately 20 ).…”

“…As part of our ongoing thiopeptide bioengineering efforts, we reconstituted the biosynthesis of lactazole A, a cryptic thiopeptide from Streptomyces lactacystinaeus (Figure ), in vitro and established an mRNA display platform that leveraged lactazole biosynthesis for the de novo discovery of pseudonatural thiopeptides with designed biological activities. − A companion report details the use of this platform for the discovery of potent and selective inhibitors of TNIK, a kinase implicated in several forms of cancer. , The discovered compounds share the central triheterocyclic motif (Oxz11-Py1/12-Thz13) with lactazole A but feature larger macrocycles (41- or 44-membered structures versus the 32-membered parent compound).…”

An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

“…Rather, it would give more diverse peptides if expanded functionalities were directly incorporated within the peptide libraries. Genetic code expansion has been successfully done in in vitro experiments that use mRNA display technologies, [5][6][7][8] but has been limited in phage displayed libraries. Recently, our group and others have developed techniques to incorporate noncanonical amino acids (ncAAs) into phage-displayed peptide libraries, which has resulted in two promising techniques: phage display of genetically-encoded cyclic peptides 9,10 and active site directed ligand evolution.…”