Structural and mechanistic studies of molecular glues and their targets

Abstract

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.