Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) compromise epithelial HCO 3 − and Cl − secretion, reduce airway surface liquid (ASL) pH, and impair respiratory host defenses in people with cystic fibrosis (CF) 1 – 3 . Here we report that apical addition of an unselective ion channel-forming small molecule, amphotericin B (AmB), restored HCO 3 − secretion and increased ASL pH in cultured human CF airway epithelia. These effects required the basolateral Na + /K + ATPase, indicating that apical AmB channels functionally interfaced with this driver of anion secretion. AmB also restored ASL pH, viscosity, and antibacterial activity in primary cultures of airway epithelia from people with CF caused by different mutations, including ones that yield no CFTR, and increased ASL pH in CFTR -null pigs in vivo . Thus, unselective small molecule ion channels can restore CF airway host defenses via a mechanism that is CFTR-independent and therefore genotype-independent.

Aromatic amines in nature are typically installed with Glu or Gln as the nitrogen donor. Here we report a pathway that features glycyl-tRNA as the nitrogen donor. During the biosynthesis of pyrroloiminoquinone-type natural products such as ammosamide, pe ptide- a minoacyl t R NA l igases (PEARLs) append amino acids to the C-terminus of a ribosomally synthesized peptide. First, adds Trp in a Trp-tRNA dependent reaction, and the flavoprotein AmmC 1 then carries out three hydroxylations of the indole ring of Trp. After oxidation to the corresponding ortho -hydroxy para- quinone, attaches Gly to the indole ring in a Gly-tRNA dependent fashion. Subsequent decarboxylation and hydrolysis results in an amino-substituted indole. Similar transformations are catalyzed by orthologous enzymes from Bacillus halodurans . This pathway features three previously unknown biochemical processes using a ribosomally synthesized peptide as scaffold for non-ribosomal peptide extension and chemical modification to generate an amino acid derived natural product.

The preparation of protein–protein, protein–peptide, and protein–small molecule conjugates is important for a variety of applications, such as vaccine production, immunotherapies, preparation of antibody–drug conjugates, and targeted delivery of therapeutics. To achieve site-selective conjugation, selective chemical or enzymatic functionalization of proteins is required. We have recently reported biosynthetic pathways in which small, catalytic scaffold peptides are utilized for the generation of amino acid-derived natural products called pearlins. In these systems, peptide amino-acyl tRNA ligases (PEARLs) append amino acids to the C-terminus of a scaffold peptide, and tailoring enzymes encoded in the biosynthetic gene clusters modify the PEARL-appended amino acid to generate a variety of natural products. Herein, we investigate the substrate selectivity of one such tailoring enzyme, BhaC 1 , that participates in pyrroloiminoquinone biosynthesis. BhaC 1 converts the indole of a C-terminal tryptophan into an o -hydroxy- p -quinone, a promising moiety for site-selective bioconjugation. Our studies demonstrate that BhaC 1 requires a 20-amino acid peptide for substrate recognition. When this peptide was appended at the C-terminus of proteins, the C-terminal Trp was modified by BhaC 1 . The enzyme is sufficiently selective that only small changes to the sequence of the peptide are tolerated. An AlphaFold model for substrate recognition explains the selectivity of the enzyme, which may be used to install a reactive handle onto the C-terminus of proteins.

Many protein ion channels harness membrane potential to move ions in opposition to their chemical gradient. Deficiencies of such proteins cause several human diseases, including cystic fibrosis, Bartter Syndrome Type II, and proximal renal tubular acidosis. Using yeast as a readily manipulated eukaryotic model system, we asked whether, in the context of a deficiency of such protein ion channel function in vivo, small molecule channels could similarly harness membrane potential to concentrate ions. In yeast, Trk potassium transporters use membrane potential to move potassium ions from a compartment of relatively low concentration outside cells (~15mM) to one of >10 times higher concentration inside (150-500mM). trk1Δtrk2Δ yeast are missing these potassium transporters and thus cannot concentrate potassium or grow in standard media. Here we show that potassium permeable, but not potassium selective, small molecule ion channels formed by the natural product amphotericin B can harness membrane potential to concentrate potassium in trk1Δtrk2Δ cells and thereby restore growth. This finding expands the list of potential human channelopathies that might be addressed by a molecular prosthetics approach..

Many protein ion channels harness membrane potential to move ions in opposition to their chemical gradient. Deficiencies of such proteins cause several human diseases, including cystic fibrosis, Bartter Syndrome Type II, and proximal renal tubular acidosis. Using yeast as a readily manipulated eukaryotic model system, we asked whether, in the context of a deficiency of such protein ion channel function in vivo, small molecule channels could similarly harness membrane potential to concentrate ions. In yeast, Trk potassium transporters use membrane potential to move potassium ions from a compartment of relatively low concentration outside cells (~15mM) to one of >10 times higher concentration inside (150-500mM). trk1Δtrk2Δ yeast are missing these potassium transporters and thus cannot concentrate potassium or grow in standard media. Here we show that potassium permeable, but not potassium selective, small molecule ion channels formed by the natural product amphotericin B can harness membrane potential to concentrate potassium in trk1Δtrk2Δ cells and thereby restore growth. This finding expands the list of potential human channelopathies that might be addressed by a molecular prosthetics approach.