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The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide 1 , therapeutic agents used in the treatment of haematopoietic malignancies 2 – 4 and as ligands for targeted protein degradation 5 – 7 . These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN. C-terminal cyclic imides are physiological degrons that enable the ubiquitin E3 ligase adapter protein cereblon to target substrates for degradation.

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-β1-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-β1 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.

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-β1-activated myofibroblasts from synthesizing collagen type I 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-β1 signaling. 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 . 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.

The coxibs are a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that primarily target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, mechanisms to inhibit other members of the prostaglandin signaling pathway may improve selectivity and reduce off-target toxicity. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), an enzyme downstream of COX-2 in the prostaglandin signaling pathway, using a cleavable chelation-assisted biotin probe 6. Evaluation of the multi-functional probe 6 revealed significantly improved tagging efficiencies attributable to the embedded picolyl functional group. Application of the probe 6 within the small molecule interactome mapping by photo-affinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified PTGES and other membrane proteins in the top eight enriched proteins from A549 cells. Carbonic anhydrase 12, a known protein target of celecoxib, was also enriched. Four binding sites to photo-celecoxib were additionally mapped by the probe 6, including a binding site with PTGES. The binding interaction with PTGES was validated by competitive displacement with celecoxib and known PTGES inhibitor licofelone. The binding site of photo-celecoxib on PTGES enabled the development of a structural model of the interaction and will inform the design of new selective inhibitors of the prostaglandin signaling pathway.

The thalidomide analog lenalidomide 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 photo-lenalidomide, a lenalidomide probe with a photo-affinity label and enrichment handle, for target identification by chemical proteomics. After evaluating a series of lenalidomide analogs, we identified a specific amide linkage to lenalidomide that allowed for installation of the desired functionality, while preserving the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties of lenalidomide. Photo-lenalidomide maintains these properties by enhancing binding interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using photo-lenalidomide, 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 photolenalidomide and forms a complex with CRBN in the presence of lenalidomide, but is itself not ubiquitylated or degraded. These data point to the potentially broader array of substrates induced by ligands to CRBN that may or may not be degraded, which can be revealed by the highly translatable application of photo-lenalidomide and chemical proteomics in additional biological settings.

Cereblon (CRBN) is an E3 ligase substrate adapter widely exploited for targeted protein degradation (TPD) strategies. However, achieving efficient and selective target degradation is a preeminent challenge with ligands that engage CRBN. Here, we report that the cyclimids, ligands derived from the C-terminal cyclic imide degrons of CRBN, exhibit distinct modes of interaction with CRBN and offer a facile approach for developing potent and selective bifunctional degraders. Quantitative TR-FRET-based characterization of 60 cyclimid degraders in binary and ternary complexes across different substrates revealed that ternary complex binding affinities correlated strongly with cellular degradation efficiency. Our studies establish the unique properties of the cyclimids as versatile warheads in TPD and a systematic biochemical approach for quantifying ternary complex formation to predict their cellular degradation activity, which together will accelerate the development of degraders that engage CRBN.

The omicron variant (B.1.1.529) of SARS-CoV-2 has demonstrated partial vaccine escape and high transmissibility, with early studies indicating lower severity of infection than that of the delta variant (B.1.617.2). We aimed to better characterise omicron severity relative to delta by assessing the relative risk of hospital attendance, hospital admission, or death in a large national cohort. Individual-level data on laboratory-confirmed COVID-19 cases resident in England between Nov 29, 2021, and Jan 9, 2022, were linked to routine datasets on vaccination status, hospital attendance and admission, and mortality. The relative risk of hospital attendance or admission within 14 days, or death within 28 days after confirmed infection, was estimated using proportional hazards regression. Analyses were stratified by test date, 10-year age band, ethnicity, residential region, and vaccination status, and were further adjusted for sex, index of multiple deprivation decile, evidence of a previous infection, and year of age within each age band. A secondary analysis estimated variant-specific and vaccine-specific vaccine effectiveness and the intrinsic relative severity of omicron infection compared with delta (ie, the relative risk in unvaccinated cases). The adjusted hazard ratio (HR) of hospital attendance (not necessarily resulting in admission) with omicron compared with delta was 0·56 (95% CI 0·54–0·58); for hospital admission and death, HR estimates were 0·41 (0·39–0·43) and 0·31 (0·26–0·37), respectively. Omicron versus delta HR estimates varied with age for all endpoints examined. The adjusted HR for hospital admission was 1·10 (0·85–1·42) in those younger than 10 years, decreasing to 0·25 (0·21–0·30) in 60–69-year-olds, and then increasing to 0·47 (0·40–0·56) in those aged at least 80 years. For both variants, past infection gave some protection against death both in vaccinated (HR 0·47 [0·32–0·68]) and unvaccinated (0·18 [0·06–0·57]) cases. In vaccinated cases, past infection offered no additional protection against hospital admission beyond that provided by vaccination (HR 0·96 [0·88–1·04]); however, for unvaccinated cases, past infection gave moderate protection (HR 0·55 [0·48–0·63]). Omicron versus delta HR estimates were lower for hospital admission (0·30 [0·28–0·32]) in unvaccinated cases than the corresponding HR estimated for all cases in the primary analysis. Booster vaccination with an mRNA vaccine was highly protective against hospitalisation and death in omicron cases (HR for hospital admission 8–11 weeks post-booster vs unvaccinated: 0·22 [0·20–0·24]), with the protection afforded after a booster not being affected by the vaccine used for doses 1 and 2. The risk of severe outcomes following SARS-CoV-2 infection is substantially lower for omicron than for delta, with higher reductions for more severe endpoints and significant variation with age. Underlying the observed risks is a larger reduction in intrinsic severity (in unvaccinated individuals) counterbalanced by a reduction in vaccine effectiveness. Documented previous SARS-CoV-2 infection offered some protection against hospitalisation and high protection against death in unvaccinated individuals, but only offered additional protection in vaccinated individuals for the death endpoint. Booster vaccination with mRNA vaccines maintains over 70% protection against hospitalisation and death in breakthrough confirmed omicron infections. Medical Research Council, UK Research and Innovation, Department of Health and Social Care, National Institute for Health Research, Community Jameel, and Engineering and Physical Sciences Research Council.

Molecular glues are a unique class of molecules that induce novel protein–protein interactions. This class of compounds, which includes the macrocyclic immunosuppressants cyclosporin A, rapamycin, and sanglifehrin A and the immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide, has found extensive use in medicine, and study of these compounds has revealed not only their mechanisms of action, but also related biological phenomena. However, molecular glues have largely been discovered and characterized serendipitously. Here, I use modern chemical biology approaches, including photo-affinity labeling and chemical proteomics, to study the interactions and mechanisms of action of molecular glues towards an increased understanding of their protein targets and biological effects.First, I discuss use of the FKBP12–rapamycin–FRB ternary complex as a model system for obtaining structural information from photo-affinity labeling mass spectrometry of ternary complexes. After establishing that photo-rapamycin, in which rapamycin is functionalized with a diazirine- and alkyne-containing handle, retains the ability to form a ternary complex with FKBP12 and FRB, we proceeded to irradiate the complex and installed a tag with an imbedded isotopic code on photo-rapamycin–conjugated peptides. Mass spectrometry analysis revealed that specific regions on each protein were labeled with a photo-rapamycin fragment; mutagenesis studies confirmed that a significant amount of labeling occurred on a single acidic residue on each protein. Molecular dynamics simulations revealed a 5.0 Å minimum distance between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photo-activation. This study demonstrates that photo-affinity labeling mass spectrometry can be used to profile both protein components of a molecular glue–induced ternary complex and to gain structural insight into small molecule–induced protein–protein interactions.Next, I discuss the use of photo-affinity labeling and chemical proteomics to characterize targets of the immunosuppressive natural product sanglifehrin A. This study led to the discovery of cyclophilin B as a primary target of sanglifehrin A in cells, a finding with applications in the development of anti-fibrotic therapies. Initial studies of sanglifehrin A and sanglifehrin B highlighted that the sanglifehrin A–induced cyclophilin A–IMPDH2 interaction is not required for the immunosuppressive activity of the sanglifehrins, suggesting that additional interactions may play roles in sanglifehrin activity. Profiling of the sanglifehrin A interactome by photo-affinity labeling in live cells revealed cyclophilin B as a primary cellular target of sanglifehrin A. Cyclophilin B is an endoplasmic reticulum–resident peptidyl-prolyl cis-trans isomerase that is critical in collagen folding. Treatment of cells with sanglifehrin A promoted secretion of cyclophilin B, resulting in depletion of intracellular cyclophilin B and an increase in extracellular cyclophilin B. The depletion of intracellular cyclophilin B results in impaired collagen production in TGF-β1–activated myofibroblasts, in a mouse model of fibrosis, and in primary fibroblasts isolated from patients with idiopathic pulmonary fibrosis. SfA also attenuates the innate immune response in the mouse model, suggesting a dual mechanism of anti-fibrotic action. These results suggest induction of cyclophilin B secretion as a novel mechanistic target for the development of anti-fibrotic therapies.Finally, I discuss the identification of C-terminal cyclic imides as degrons for the E3 ligase substrate recognition factor cereblon. Cereblon is a target of the immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide, which act as molecular glues to promote the recruitment of new substrates to cereblon for ubiquitylation and degradation. While several native substrates of cereblon have been identified, a conserved protein recognition sequence, termed a degron, utilizing the same binding site as the immunomodulatory drugs has remained elusive. First, I describe the use of photo-affinity labeling, thermal shift assays, and mass spectrometry to characterize the interaction of cereblon with metabolites in vitro. Then, I describe the successful use of bifunctional degraders for degron discovery. By substituting the thalidomide moiety in a bifunctional degrader with candidate degrons, we identified peptides with C-terminal cyclic imides resulting from cyclization of glutamine or asparagine as degrons for cereblon. These degrons are functional when installed on the C-termini of proteins and can be observed in the proteome. These findings suggest new directions of inquiry into the characterization of these overlooked post-translational modifications and the role that cereblon-mediated degradation of cyclic imide–modified proteins plays in biology.