Michael J. Wheadon

Nonribosomal peptide synthetases (NRPSs) are a family of multidomain enzymes dedicated to the production of peptide natural products. Central to NRPS function are condensation (C) domains, which catalyze peptide bond formation and a number of specialized transformations including dehydroamino acid and beta-lactam synthesis. Structures of C domains in catalytically informative states are limited due to a lack of dear strategies for stabilizing C domain interactions with their substrates and client domains. Inspired by a beta-lactam forming C domain, we report herein the synthesis and application of 1, which forms irreversible cross-links with engineered thiol nucleophiles in a C domain active site. Deployment of 1 demonstrates the synthetic tractability of trapping late-stage nascent peptides in C domains and provides a readily adaptable tactic for stabilizing C domain interactions in multidomain NRPS fragments.

Interferon-lambda (IFN-λ) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-λ receptor (Ifnlr1), and is associated with IFN-λ production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

Significance: By applying phylogenetic tools, we infer the ancestral origins of a nonribosomal peptide synthetase (NRPS) condensation (C) domain responsible for β-lactam (antibiotic) synthesis. Subsequent in vitro experiments suggest a mechanism for how this unique synthetic function likely evolved. β-Lactam synthesizing ability appears to have emerged along with an adjacent epimerization (E) domain deletion and in parallel with other C domains whose catalysis presumably share intermediates with β-lactam synthesis, including ᴅ-amino acid and dehydroamino acid synthesis. The implied mechanistic link between β-lactam formation and the latter two C-domain functions can be experimentally tested in the future to discern how evolutionary differences dictate synthetic outcome. These insights may constructively guide protein engineering efforts to introduce potentially valuable structural elements into NPRS products.

Targeting essential bacterial processes beyond cell wall, protein, nucleotide, and folate syntheses holds promise to reveal new antimicrobial agents and expand the potential drugs available for combination therapies. The synthesis of isoprenoid precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), is vital for all organisms; however, humans use the mevalonate pathway for production of IDP/DMADP while many pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis, use the orthogonal methylerythritol phosphate (MEP) pathway. Toward developing novel antimicrobial agents, we have designed and synthesized a series of phosphonyl analogues of MEP and evaluated their abilities to interact with IspD, both as inhibitors of the natural reaction and as antimetabolite alternative substrates that could be processed enzymatically to form stable phosphonyl analogues as potential inhibitors of downstream MEP pathway intermediates. In this compound series, the S-monofluoro MEP analogue displays the most potent inhibitory activity against Escherichia coli IspD and is the best substrate for both the E. coli and P. falciparum IspD orthologues with a K_m approaching that of the natural substrate for the E. coli enzyme. This work represents a first step toward the development of phosphonyl MEP antimetabolites to modulate early isoprenoid biosynthesis in human pathogens.

Src-family kinases (SFKs) play important roles in human biology and are key drug targets as well. However, achieving selective inhibition of individual Src-family kinases is challenging due to the high similarity within the protein family. We describe rhodium(II) conjugates that deliver both potent and selective inhibition of Src-family SH3 domains. Rhodium(II) conjugates offer dramatic affinity enhancements due to interactions with specific and unique Lewis-basic histidine residues near the SH3 binding interface, allowing predictable, structure-guided inhibition of SH3 targets that are recalcitrant to traditional inhibitors. In one example, a simple metallopeptide binds the Lyn SH3 domain with 6 nM affinity and exhibits functional activation of Lyn kinase under biologically relevant concentrations (EC₅₀ ∼ 200 nM).

Turning viral persistence on and off: Norovirus causes >90% of the world's gastroenteritis. Norovirus can establish persistent infections, which may contribute to its spread. How does norovirus establish itself as a permanentw resident of the gut and how can such persistent infections be cured (see the Perspective by Wilks and Golovkina)? Baldridge et al. studied mice persistently infected with norovirus and found that viral persistence required the gut microbiota: resident bacteria in the gastrointestinal tract. Antibiotics prevented persistent mouse norovirus infection in a way that depended on the secreted antiviral protein interferon λ (IFN-λ). Nice et al. report that IFN-λ can cure mice persistently infected with norovirus, independent of the adaptive immune system. Science , this issue p. 266 , p. 269 ; see also p. 233