Revolution in an RNA-packaging capsid: Artificial nucleocapsid proteins, which could be analogous to those used by viruses to package their genomes, are a promising way to protect and deliver RNAs. Using an escalating challenge by nucleases, Tetter et al. evolved a protein that forms multimeric, spherical cages into a highly efficient capsid that selectively packages its own encoding RNA. Cryo–electron microscopy of the final design and intermediates revealed a stepwise expansion in size, enabled by destabilizing amino acid substitutions and a domain swap that results in a change of oligomerization interfaces for the base units of the cage. In addition to altering the protein, directed evolution resulted in changes to the encoding RNA structure that enabled efficient uptake versus other cellular RNAs. Science , abg2822, this issue p. 1220

Significance: Viruses consist of a protective proteinaceous shell that packages a DNA or RNA genome. The critical step in the evolution of primitive viruses was presumably the emergence of protein cages that could load, protect, and transfer their own genetic information. Here, we show that a nonviral protein cage formed by Aquifex aeolicus lumazine synthase and its encoding mRNA can be engineered and evolved into virus-like nucleocapsids. The optimized proteins specifically recognize designed motifs on cognate mRNAs, and these RNA tags can also be employed for the encapsulation of other RNA molecules. These model systems have great potential for the investigation of nucleocapsid assembly and evolution and as tailored protein compartments for protective packaging and delivery of nucleic acids.

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.

γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ^2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (M^pro). Two kinds of cyclic γ^2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ¹) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ²), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent M^pro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ¹ at the fourth position, manifests a 5.2 nM dissociation constant. An M^pro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ¹ interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and M^pro enabled production of a variant with a 5-fold increase in potency.