Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs.

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.

Targeting the deubiquitinase USP7 for degradation with PROTACs.

Targeting deubiquitinating enzymes (DUBs) has emerged as a promising therapeutic approach in several human cancers and other diseases. DUB inhibitors are exciting pharmacological tools but often exhibit limited cellular potency. Here we report PROTACs based on a ubiquitin-specific protease 7 (USP7) inhibitor scaffold to degrade USP7. By investigating several linker and E3 ligand types, including novel cereblon recruiters, we discovered a highly selective USP7 degrader tool compound that induced apoptosis of USP7-dependent cancer cells. This work represents one of the first DUB degraders and unlocks a new drug target class for protein degradation.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium.

Atherosclerosis is the foundation of potentially fatal cardiovascular diseases and it is characterized by plaque formation in large arteries. Current treatments aimed at reducing atherosclerotic risk factors still allow room for a large residual risk; therefore, novel therapeutic candidates targeting inflammation are needed. The endothelium is the starting point of vascular inflammation underlying atherosclerosis and we could previously demonstrate that the chemokine axis CXCL12-CXCR4 plays an important role in disease development. However, the role of ACKR3, the alternative and higher affinity receptor for CXCL12 remained to be elucidated. We studied the role of arterial ACKR3 in atherosclerosis using western diet-fed Apoe-/- mice lacking Ackr3 in arterial endothelial as well as smooth muscle cells. We show for the first time that arterial endothelial deficiency of ACKR3 attenuates atherosclerosis as a result of diminished arterial adhesion as well as invasion of immune cells. ACKR3 silencing in inflamed human coronary artery endothelial cells decreased adhesion molecule expression, establishing an initial human validation of ACKR3's role in endothelial adhesion. Concomitantly, ACKR3 silencing downregulated key mediators in the MAPK pathway, such as ERK1/2, as well as the phosphorylation of the NF-kB p65 subunit. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in ACKR3-deficient mice. Lack of smooth muscle cell-specific as well as hematopoietic ACKR3 did not impact atherosclerosis in mice. Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways.

Apolipoprotein Mimetic Peptide Inhibits Neutrophil-Driven Inflammatory Damage via Membrane Remodeling and Suppression of Cell Lysis.

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP1. X-ray structural studies and theoretical calculations show that apoMP1 induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and "turn off" diverse membrane-permeating molecules besides H4n. In vitro experiments show an apoMP1 dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP1 inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage.

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

Chrono-pharmacological Targeting of the CCL2-CCR2 Axis Ameliorates Atherosclerosis.

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.

Protective Aptitude of Annexin A1 in Arterial Neointima Formation in Atherosclerosis-Prone Mice-Brief Report.

OBJECTIVE: Restenosis as a consequence of arterial injury is aggravated by inflammatory pathways. Here, we investigate the role of the proresolving protein annexin A1 (AnxA1) in healing after wire injury. APPROACH AND RESULTS: Apoe-/- and Apoe-/-Anxa1-/- mice were subjected to wire injury while fed a high-cholesterol diet. Subsequently, localization of AnxA1 and AnxA1 plasma levels were examined. AnxA1 was found to localize within endothelial cells and macrophages in the neointima. Levels of AnxA1 in the plasma and its lesional expression negatively correlated with neointima size, and in the absence of AnxA1, neointima formation was aggravated by the accumulation and proliferation of macrophages. In contrast, reendothelialization and smooth muscle cell infiltration were not affected in Apoe-/-Anxa1-/- mice. CONCLUSIONS: AnxA1 is protective in healing after wire injury and could, therefore, be an attractive therapeutic compound to prevent from restenosis after vascular damage.

Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment.

BACKGROUND: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment. METHODS: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe-/-and CatG-deficient mice (Apoe-/-Ctsg-/-) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy. RESULTS: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice. CONCLUSIONS: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.

Resolving Lipid Mediators Maresin 1 and Resolvin D2 Prevent Atheroprogression in Mice.

RATIONALE: Atheroprogression is a consequence of nonresolved inflammation, and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore, we hypothesized that lesional lipid mediator imbalance favors atheroprogression. OBJECTIVE: To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on the delivery of resolving lipid mediators. METHODS AND RESULTS: Aortic lipid mediator profiling of aortas from Apoe-/- mice fed a high-fat diet for 4 weeks, 8 weeks, or 4 months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 and Prostaglandin E2, and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic Leukotriene B4 and Prostaglandin E2 levels correlated with traits of plaque instability, whereas RvD2 and MaR1 levels correlated with the signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile toward a reparative phenotype, which secondarily stimulated collagen synthesis in smooth muscle cells. CONCLUSIONS: We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression, suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammation.