Pharmacological expansion of type 2 alveolar epithelial cells promotes regenerative lower airway repair.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.

M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome.

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.

A high throughput screen for pharmacological inhibitors of the carbohydrate response element.

A central regulator of metabolism, transcription factor carbohydrate response element binding protein (ChREBP) senses and responds to dietary glucose levels by stimulating the transcription of glycolytic and lipogenic enzymes. Genetic depletion of ChREBP rescues ß-cell dysfunction arising from high glucose levels, suggesting that inhibiting ChREBP might represent an attractive therapeutic approach to manage diabetes and other metabolic diseases. However, the molecular mechanisms governing ChREBP activation are poorly understood and chemical tools to probe the cellular activity of ChREBP are lacking. Here, we report a high-throughput pharmacological screen in INS-1E ß-cells that identified novel inhibitors of ChREBP-driven transcription at carbohydrate response element sites, including three putative covalent inhibitors and two likely non-covalent chemical scaffolds. This work affords a pharmacological toolkit to help uncover the signaling logic controlling ChREBP activation and may ultimately reveal potential therapeutic approaches for treating metabolic disease.

Employing 25-Residue Docking Motifs from Modular Polyketide Synthases as Orthogonal Protein Connectors.

The proteins of trans-acyltransferase modular polyketide synthases (PKSs) self-organize into assembly lines, enabling the multienzyme biosynthesis of complex organic molecules. Docking domains comprised of ∼25 residues at the C- and N-termini of these polypeptides (CDDs and NDDs) help drive this association through the formation of four-helix bundles. Molecular connectors like these are desired in synthetic contexts, such as artificial biocatalytic systems and biomaterials, to orthogonally join proteins. Here, the ability of six CDD/NDD pairs to link non-PKS proteins is examined using green fluorescent protein (GFP) variants. As observed through size-exclusion chromatography and Förster resonance energy transfer (FRET), matched but not mismatched pairs of Venus+CDD and NDD+mTurquoise2 fusion proteins associate with low micromolar affinities.