David Adam Spiegel

Targeted protein degradation (TPD) has emerged as an effective therapeutic strategy for a wide range of diseases; however, the blood-brain barrier (BBB) limits access of degraders into the central nervous system (CNS). Here, we present a new class of bifunctional small molecules, called TransMoDEs (Transcytosis-inducing molecular degraders of extracellular proteins), capable of both (1) removal of target protein via lysosomal proteolysis and (2) transcytosis of protein targets across brain endothelial cells. TransMoDEs are derived from Angiopep-2, a peptide motif previously employed as a covalent tag to facilitate receptor-mediated transcytosis across the BBB. We demonstrate that TransMoDEs containing either a biotin or chloroalkane ligand can trigger endocytosis of streptavidin or HaloTag protein, respectively. Interestingly, although low-density lipoprotein receptor-related protein 1 (LRP1) has been reported as the primary receptor for Angiopep-2, TransMoDE-mediated target uptake does not rely exclusively on this pathway. Furthermore, TransMoDE-mediated endocytosis of streptavidin in a bEnd.3 BBB model occurs in a clathrin-mediated mechanism and results in both lysosomal localization and transcytosis of the target protein. This study demonstrates that TransMoDEs can recruit, transcytose, and degrade proteins of interest in cells relevant to the CNS, supporting their further development for the removal of pathogenic neuroproteins.

Autoantibodies are the causative agents in a wide range of autoimmune diseases. Existing approaches to the depletion of such autoantibodies include therapies that deplete the whole antibody repertoire or immunoadsorption, which although antigen-specific is costly and time-consuming. Here, we report the first pharmacological strategy for the antigen-specific depletion of autoantibodies. Specifically, we synthesized a synthetic bifunctional molecule (β-MoDE-A) capable of selectively depleting antibodies against the beta-1 adrenergic receptor (β1AR) in vitro and in vivo. β-MoDE-A contains a motif that mimics the anti-β1AR binding epitope linked to a ligand for the asialoglycoprotein receptor, which re-routes pathogenic autoantibodies to the liver for degradation. We demonstrate that β-MoDE-A is effective in mediating ternary complex formation and endocytosis of anti-β1AR antibodies in vitro and depletes these antibodies in vivo in a mouse model. The novel drug platform represented by this approach provides the first example of an antigen-specific extracellular degrader of an individual pathogenic IgG species of any isotype or subclass, but without any potential for broad-based immunosuppression.

We present the application of Angiopep-2 as a molecular degrader of extracellular proteins (MoDE) promoting target protein removal by both transcytosis and lysosomal degradation mechanisms in brain endothelial cells. The accumulation of pathogenic proteins is a hallmark of many neurodegenerative diseases, and removal of these species is a promising avenue for the development of novel therapies. Targeted protein degradation technologies are emerging as efficacious therapeutic strategies in a wide range of diseases. However, there are no existing methods for the degradation of extracellular proteins in the central nervous system (CNS). Angiopep-2, a brain-targeting peptide derived from aprotinin, has previously been employed as a covalent tag to facilitate receptor-mediated transcytosis of therapeutics across the blood brain barrier (BBB). MoDEs/LyTACs consisting of Angiopep-2 modified with biotin or a chloroalkane ligand triggered endocytosis of streptavidin and HaloTag protein, respectively. Interestingly, uptake occurred independently of LRP-1, which is the reported receptor for Angiopep-2. MoDE-mediated endocytosis of streptavidin in a bEnd.3 BBB model resulted in two mechanisms of protein removal: both bi-directional transcytosis and lysosomal degradation. This study demonstrates that Angiopep-2-based MoDEs can recruit, endocytose, and degrade proteins of interest in CNS cells, supporting their further development as molecular degraders of pathogenic neuroproteins.

Pentosinane is a structurally complex nonenzymatic post-translational modification of proteins believed to be present in all living things. It falls into the category of advanced glycation end products (AGEs) and is structurally related to the other AGEs pentosidine and glucosepane. Although pentosidine and glucosepane have been widely studied for their role in wide-ranging conditions (e.g., diabetes mellitus, Alzheimer’s disease, and human aging), relatively little is known about pentosinane. Interestingly, previous reports have suggested that pentosidine may derive from pentosinane. The (patho)physiological significance of pentosinane in humans is largely unexplored. As a first step to address this knowledge gap, we report herein the first total synthesis of pentosinane. Our synthesis is high yielding (1.7% over seven steps), concise, and enantioselective, and it leverages a strategy for synthesizing 2,5-diaminoimidazoles previously developed by our lab. Access to synthetic pentosinane has allowed us to perform additional studies showing that its oxidation to pentosidine is both pH and oxygen dependent and is substantially slower under physiological conditions than previously believed. Additionally, pentosinane rapidly decomposes under harshly acidic conditions typically employed for pentosidine isolation. Taken together, these results suggest that pentosinane is likely to be more abundant in vivo than previously appreciated. We believe these results represent a critical step toward illuminating the role(s) of pentosinane in human biology.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin–proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

Post‐translational modifications (PTMs) play roles in both physiological and pathophysiological processes through the regulation of enzyme structure and function. We recently identified a novel PTM, lactoylLys, derived through a nonenzymatic mechanism from the glycolytic by‐product, lactoylglutathione. Under physiologic scenarios, glyoxalase 2 prevents the accumulation of lactoylglutathione and thus lactoylLys modifications. What dictates the site‐specificity and abundance of lactoylLys PTMs, however, remains unknown. Here, we report sirtuin 2 as a lactoylLys eraser. Using chemical biology and CRISPR‐Cas9, we show that SIRT2 controls the abundance of this PTM both globally and on chromatin. These results address a major gap in our understanding of how nonenzymatic PTMs are regulated and controlled.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch’s membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

Targeted protein degradation (TPD) has emerged as a promising and exciting therapeutic strategy. The majority of existing TPD technologies rely on the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modularly designed, bifunctional synthetic molecules called MoDE-As (Molecular Degraders of Extracellular proteins through the Asialoglycoprotein receptor (ASGPR)), which are capable of mediating the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and the ASGPR, which is expressed primarily on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing bifunctional molecules that induce the degradation of both antibody and pro-inflammatory cytokine proteins. To our knowledge, these data represent the first experimental evidence that non-proteinogenic, synthetic molecules can be employed for the TPD of extracellular proteins both in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

Targeted protein degradation (TPD) has emerged as a promising and exciting therapeutic strategy. The majority of existing TPD technologies rely on the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modularly designed, bifunctional synthetic molecules called MoDE-A s ( Mo lecular D egraders of E xtracellular proteins through the A sialoglycoprotein receptor (ASGPR)), which are capable of mediating the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and the ASGPR, which is expressed primarily on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing bifunctional molecules that induce the degradation of both antibody and pro-inflammatory cytokine proteins. To our knowledge, these data represent the first experimental evidence that non-proteinogenic, synthetic molecules can be employed for the TPD of extracellular proteins both in vitro and in vivo . We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.