Development of Photolenalidomide for Cellular Target Identification.

The thalidomide analogue lenalidomide (Len) is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photolenalidomide (pLen), a Len probe with a photoaffinity label and enrichment handle, designed for target identification by chemical proteomics. pLen preserves the substrate degradation profile, phenotypic antiproliferative and immunomodulatory properties of Len, and enhances interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using pLen, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by pLen and forms a ternary complex with CRBN in the presence of Len across several epithelial cell lines but is itself not ubiquitylated or degraded. These data point to the existence of a broader array of targets induced by ligands to CRBN that may or may not be degraded, which can be identified by the highly translatable application of pLen to additional biological systems.

Sanglifehrin A mitigates multiorgan fibrosis by targeting the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-ß1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-ß1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.

Sympathetic neurons secrete retrogradely transported TrkA on extracellular vesicles.

Proper wiring of the peripheral nervous system relies on neurotrophic signaling via nerve growth factor (NGF). NGF secreted by target organs (i.e. eye) binds to the TrkA receptor expressed on the distal axons of postganglionic neurons. Upon binding, TrkA is internalized into a signaling endosome and retrogradely trafficked back to the soma and into the dendrites to promote cell survival and postsynaptic maturation, respectively. Much progress has been made in recent years to define the fate of the retrogradely trafficked TrkA signaling endosome, yet it has not been fully characterized. Here we investigate extracellular vesicles (EVs) as a novel route of neurotrophic signaling. Using the mouse superior cervical ganglion (SCG) as a model, we isolate EVs derived from sympathetic cultures and characterize them using immunoblot assays, nanoparticle tracking analysis, and cryo-electron microscopy. Furthermore, using a compartmentalized culture system, we find that TrkA derived from endosomes originating in the distal axon can be detected on EVs secreted from the somatodendritic domain. In addition, inhibition of classic TrkA downstream pathways, specifically in somatodendritic compartments, greatly decreases TrkA packaging into EVs. Our results suggest a novel trafficking route for TrkA: it can travel long distances to the cell body, be packaged into EVs, and be secreted. Secretion of TrkA via EVs appears to be regulated by its own downstream effector cascades, raising intriguing future questions about novel functionalities associated with TrkA+ EVs.

Sanglifehrin A mitigates multi-organ fibrosis in vivo by inducing secretion of the collagen chaperone cyclophilin B.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis by myofibroblasts have clinical potential as anti-fibrotic agents. Lysine hydroxylation by the prolyl-3-hydroxylase complex, comprised of cartilage associated protein, prolyl 3-hydroxylase 1, and cyclophilin B, is essential for collagen type I crosslinking and formation of stable fibers. Here, we identify the collagen chaperone cyclophilin B as a major cellular target of the macrocyclic natural product sanglifehrin A (SfA) using photo-affinity labeling and chemical proteomics. Our studies reveal a unique mechanism of action in which SfA binding to cyclophilin B in the endoplasmic reticulum (ER) induces the secretion of cyclophilin B to the extracellular space, preventing TGF-ß1-activated myofibroblasts from synthesizing collagen type I in vitro without inhibiting collagen type I mRNA transcription or inducing ER stress. In addition, SfA prevents collagen type I secretion without affecting myofibroblast contractility or TGF-ß1 signaling. In vivo, we provide chemical, molecular, functional, and translational evidence that SfA mitigates the development of lung and skin fibrosis in mouse models by inducing cyclophilin B secretion, thereby inhibiting collagen synthesis from fibrotic fibroblasts in vivo . Consistent with these findings in preclinical models, SfA reduces collagen type I secretion from fibrotic human lung fibroblasts and precision cut lung slices from patients with idiopathic pulmonary fibrosis, a fatal fibrotic lung disease with limited therapeutic options. Our results identify the primary liganded target of SfA in cells, the collagen chaperone cyclophilin B, as a new mechanistic target for the treatment of organ fibrosis.

Beyond Epitope Binning: Directed in Vitro Selection of Complementary Pairs of Binding Proteins.

Many biotechnological applications require the simultaneous binding of affinity reagents to nonoverlapping target epitopes, the most prominent example being sandwich immunoassays. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Here, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process, which enables the efficient identification of affinity reagents that function together as complementary pairs, from in vitro libraries of ∼109 variants. We used RAPIDS to develop highly specific affinity pairs against biomarkers of tuberculosis, Zika virus, and sepsis. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or nontargets, yielding diagnostic assays that are sensitive and specific by design.