David M. McDonald

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.

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.

Deaths from coronavirus disease (COVID-19) have exceeded 300,000 persons globally, calling for rapid development of mobile diagnostics that can assay widespread prevalence and infection rates. Data provided in this study supports the utility of a newly-designed lateral flow immunoassay (LFA) for detecting SARS-CoV-2 IgM and IgG antibodies. We employed a clinical cohort of 1,892 SARS-CoV-2 patients and controls, including individuals diagnosed by RT-qPCR at Yale New Haven Hospital, The First Affiliated Hospital of Anhui Medical University, the Chinese Center for Disease Control and Prevention of Hefei City (Hefei CDC), Anhui Province (Anhui Province CDC), and Fuyang City (Fuyang CDC). The LFA studied here detects SARS-CoV-2 IgM and IgG antibodies with a specificity of 97.9–100% for IgM, 99.7–100% for IgG, and sensitivities ranging from 94.1–100% for patients >14-days post symptom onset. Sensitivity decreases in patients <14-days post symptom onset, which is likely due to lower IgG/IgM antibody levels in this population. Finally, we developed a visual intensity reporting system that we believe will be suitable for laboratory and point-of-care settings, and will provide granular information about antibody levels. Overall our results support the widespread utility of this and other LFAs in assessing population-level epidemiological statistics.

Tuberculosis (TB) remains a staggering burden on global public health. Novel preventative tools are desperately needed to reach the targets of the WHO post-2015 End-TB Strategy. Peptide or protein-based subunit vaccines offer potential as safe and effective generators of protection, and enhancement of local pulmonary immunity may be achieved by mucosal delivery. We describe the synthesis of a novel subunit vaccine via native chemical ligation. Two immunogenic epitopes, ESAT6_1–20 and TB10.4_3–11 from Mycobacterium tuberculosis (Mtb), were covalently conjugated to the TLR2-ligand Pam₂Cys to generate a self-adjuvanting lipopeptide vaccine. When administered mucosally to mice, the vaccine enhanced pulmonary immunogenicity, inducing strong Th17 responses in the lungs and multifunctional peripheral T-lymphocytes. Mucosal, but not peripheral vaccination, provided substantial protection against Mtb infection, emphasizing the importance of delivery route for optimal efficacy.

Access to lipopeptide-based vaccines for immunological studies remains a significant challenge owing to the amphipathic nature of the molecules which makes them difficult to synthesize and purify to homogeneity. Here, we describe the application of a new peptide ligation technology – the diselenide-selenoester ligation (DSL) – to access self-adjuvanting glycolipopeptide vaccines. We show that rapid ligation of glycopeptides and lipopeptides is possible via DSL in mixed organic solvent-aqueous buffer and, when coupled with deselenization chemistry, affords rapid and efficient access to a vaccine candidate possessing a MUC1 glycopeptide epitope and the lipopeptide adjuvant Pam2Cys. This construct was shown to elicit MUC1 specific antibody and cytotoxic T lymphocyte responses in the absence of any other injected lipids or adjuvants. The inclusion of the T cell helper epitope PADRE boosted both the antibody response and resulted in elevated cytokine production.

Due to changes in glycosyltransferase expression during tumorigenesis, the glycoproteins of cancer cells often carry highly truncated carbohydrate chains compared to those on healthy cells. These glycans are known as tumor-associated carbohydrate antigens, and are prime targets for use in vaccines for the prevention and treatment of cancer. Herein, we review the state-of-the-art in targeting the immune system towards tumor-associated glycopeptide antigens via synthetic self adjuvanting vaccines, in which the antigenic and adjuvanting moieties of the vaccines are present in the same molecule. The majority of the self-adjuvanting glycopeptide cancer vaccines reported to date employ antigens from mucin 1, a protein which is highly over-expressed and aberrantly glycosylated in many forms of cancer. The adjuvants used in these vaccines predominantly include lipopeptide- or lipoamino acid-based TLR2 agonists, although studies investigating stimulation of TLR9 and TLR4 are also discussed. Most of these adjuvants are highly lipophilic, and, upon conjugation to antigenic peptides, provide amphiphilic vaccine molecules. The amphiphilic nature of these vaccine constructs can lead to the formation of higher-order structures by vaccines in solution, which are likely to be important for their efficacy in vivo.

Naive T cell activation is normally restricted to the lymphoid organs, in part because of their limited ability to migrate into the parenchyma of peripheral tissues. The liver vasculature is unique, however, and circulating leukocytes within the hepatic sinusoids have direct access to liver-resident cells, which include an abundant population of Kupffer cells. It is well accepted that recognition of cognate Ag within the liver leads to naive CD8⁺ T cell activation in situ, but it is unclear whether the liver also supports naive CD4⁺ T cell activation. In this study, we show that naive CD4⁺ T cells can be activated to proliferate in the liver when cognate Ag expression is induced in hepatocytes by recombinant adeno-associated viral vectors. Ag-specific retention and activation of naive CD4⁺ T cells within the liver are independent of lymphoid tissues but dependent on a clodronate liposome–sensitive population of liver-resident phagocytic cells. To our knowledge, this study provides the first unequivocal evidence that naive CD4⁺ T cells can be activated in a nonlymphoid organ. It also gives critical insight into how CD4⁺ T cells specific for Ag expressed in the liver are recruited to participate in protective or pathological responses during hepatotropic infections and autoimmune liver disease.