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.

Figure 1

Design of the selection platform. (a) Lactazole A is a thiopeptide whose biosynthesis involves five dedicated enzymes. Depicted are a schematic of laz biosynthetic gene cluster and the chemical structure of lactazole A. (b) The design of library v.t.2. The library is based on LazA^min; compared to the parent sequence, v.t.2 features an expanded and randomized insert in the core peptide region, C-terminal linker, and biotin-Phe as the translation initiator amino acid. (c) Selection scheme. The library precursor peptides are first displayed on cognate mRNA, and then converted to thiopeptides with Laz enzymes. Partially modified and shunt products maintain the N-terminal biotin tag during biosynthesis and are eliminated by a streptavidin pulldown. The resulting mRNA-displayed library of thiopeptides is panned against immobilized TNIK to enrich for high affinity protein ligands. The process can be repeated by recovering DNA and converting it to mRNA for the following round of selection.

Figure 2

Analysis of the selection results. (a) Recovery of cDNA barcodes during the selection as monitored by qPCR. Numerical values represent the ratio of cDNA recovery between TNIK pulldown and counterselection fractions. (b) t-Distributed stochastic neighbor (t-SNE) embedding of 1000 most abundant sequences (their random insert regions) obtained after 5 rounds of selection. Sequences chosen for further evaluation are highlighted in orange. The analysis illustrates library convergence to several well-defined sequences families. (c) Maturation of 15 selected precursor peptides by Laz enzymes. In vitro expressed TPP1-15 were treated with LazDEF/BCF under the selection conditions, and the outcomes were analyzed by LC/MS. Displayed are extracted ion current (EIC) chromatograms showing the formation of leader-NH₂ (one of the macrocyclization products) and linear forms; and composite EIC chromatograms for the identified thiopeptides. Ser/Thr/Cys, that is, the residues which can potentially be modified by LazDEF and LazBF, are highlighted in random insert sequences: unmodified Ser/Thr/Cys are in black, dehydroamino acids derived from Ser and Thr by LazBF—in green; Ser/Thr/Cys cyclodehydrated to azolines and azoles—in blue. *: peaks corresponding to linear precursor peptide forms. ‡: translation protein carryover. †: see Figures S8–S11 for annotations of individual thiopeptides in product mixtures.

Figure 3

Biochemical characterization of the synthesized compounds. (a) TP15 as the prototypical lactazole-like thiopeptide; the chemical structures of other compounds can be inferred by analogy based on their insert sequences. The common structural element is highlighted in grey. (b) Binding affinity to TNIK (K_D values) and its inhibition (IC₅₀) by the discovered thiopeptides. Half-life times in human serum (τ_1/2) are also shown. The values were obtained via non-linear regression of experimental data and are reported with standard errors of the fits. ^[a]: an S highlighted in green indicates a Dha residue; C in red—l,γShGln (the product of iodoacetamide alkylation of Cys); C in blue—Thz. ^[b]: no measurable binding/inhibition ^[c]: n. d.: not determined. ^[d]: for TP8, binding was measured in the presence of 1 mM ATP. ^[e]: for TP15, K_i was determined as 3 nM (Figure S16). (c) Lineweaver–Burk plot of the inhibition of TNIK-mediated MSTtide phosphorylation by TP15. The thiopeptide acts as a substrate-competitive inhibitor of the enzyme. (d) Color-coded kinase selectivity profiling outcomes for TP15 against a panel of 67 human kinases. Numerical values are listed in Table S4. The compound shows good selectivity for the target enzyme. Analogous data for TP1 is summarized in Figure S17.

Figure 4

Structural analysis of TNIK inhibition by TP15. See Figure S22 for analogous data for TP1. (a) Overview of the X-ray crystal structure of the TNIK·AMPPNP·TP15 complex (pdb 7xzr). TNIK protein surface is shown in grey; TP15 and AMPPNP are displayed as ball and stick models. The thiopeptide engages with the substrate-binding region of TNIK. (b) The interaction between αG-helix of TNIK and the heterocyclic fragment of TP15. Atoms within 4 Å distance are connected with blue dotted lines. (c) The interaction between αC-helix of TNIK and Arg5, 7, 9 of TP15. The thiopeptide makes multiples ionic contacts with the protein. (d,e) Folding of TNIK-bound TP15 (d) and TP1 (e). The protein and amino acid side-chains of the ligands are omitted for clarity. Comparison of the structures reveals that lactazole-like thiopeptides can fold into β-hairpins in two different ways, as determined by the orientation of Py1 carbonyl moiety.

Figure 5

TP15 inhibits TNIK in HCT116 carcinoma cell line. (a) The results of the chloroalkane penetration assay for TP1, 8, 14 and 15. The assay was performed in HEK293H cells as described in Supporting Information S2.11. Three out of four studied thiopeptides demonstrated cytosolic internalization at concentrations comparable to that of a well-known cell-penetrating peptide, Tat. CP₅₀: midpoint internalization concentration. (b,c) Inhibition of TNIK autophosphorylation and downregulation of Wnt target genes by TP15 in HCT116 cells. Subconfluent HCT116 cells were treated with the indicated compounds for 24 h, and the cellular levels of TNIK, TNIK pSer764 (autophosphorylation product), c-Myc and Axin2 were analyzed by immunoblotting (b) and RT-qPCR (c). NCB0846 is a small molecule TNIK inhibitor control.