During stroke, the chronic release of inflammatory and oxidative mediators causes blood-brain barrier rupture, peripheral immune cell invasion, activation of cell adhesion molecules, and neuroglial cell activation, resulting in more brain damage. The last several years have seen more and more effort being put into designing therapies to regulate the inflammatory and oxidative conditions following stroke.

Bromodomain and extra terminal domain (BET) proteins are essential to inflammatory gene transcription. BRD2/4 are of particular interest to stroke-induced neuroinflammation that contributes to delayed cell death as they are required for NF-κB-dependent gene transcription. As a result, BRD4 blockade has attracted increasing attention in the treatment of stroke. The ideal approach to block multi-domain protein BRD4 function is to delete BRD4 completely by either targeting protein degradation with proteolysis-targeting chimeras (PROTACs) technique or genetic manipulation. PROTAC dBET1 is a promising therapeutic approach for stroke and other diseases caused by neuroinflammation.

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• Neuroprotective Agent Development for Stroke

Structure and Function of BET

Members of the BET family include BRD2, BRD3, BRD4, and BRDT. BRDT is testis-specific and, although commonly expressed, is expressed in very low abundance in BRD3, whereas BRD2 and BRD4 are abundant and commonly expressed. BET proteins are epigenetic readers that recognize acetylated proteins, including histones and non-histone proteins (e.g., transcription factors). The multiple functions of the BET family are associated with RNA translation, epigenetic modification, and transcriptional regulation, as well as many other roles in cell proliferation, apoptosis, and immune responses.

PROTAC Technology

PROTAC technology is centered on the targeted degradation of proteins, using intracellular UPS to remove mutated, denatured, and deleterious proteins by recruiting E3 ligases to the target proteins, leading to neighborhood-induced ubiquitination, and consequently, degradation of the proteins.

PROTAC technology combines the advantages of small molecule compounds and small molecule nucleic acids to effectively target and degrade specific proteins, including proteins that are difficult to recognize and bind. PROTAC is therefore of paramount importance for drug discovery and the treatment of neurological diseases.

Unlike conventional small molecules that merely block protein activity, PROTACs promote complete protein degradation by directing proteins to the proteasome. Key advantages of PROTACs include the ability to selectively degrade a variety of proteins in the same gene without the need for large amounts of drug. They do not need to bind to the target proteins for long or strongly, thus enabling effects that are difficult to achieve with conventional drugs. The technology can target proteins that are difficult to modulate with small molecules or antibodies. In addition, the PROTAC mechanism is similar to a catalytic reaction, allowing for reuse of drugs and the creation of highly active drugs without the need for molar amounts of drugs for inhibition.

PROTAC dBET1

dBET1 is a novel and potent BRD4 degrader achieved by the PROTAC strategy. dBET1 selectively binds the JQ1 portion of BRD4 and the phthalimide portion of the E3 ubiquitin ligase. This design allows BRD4 to be ubiquitin-labeled for subsequent proteasomal degradation.

Fig.1. Schematic illustration of BRD4 PROTAC dBET1 mechanism-induced BRD4 protein degradation through the ubiquitin–proteasome system. (Liu, et al., 2022)

Targeted BRD4 Protein Degradation by dBET1 Ameliorates Ischemic Stroke

The BRD4 degrader dBET1 counteracts ischemia-induced neurological dysfunction and brain damage by modulating inflammation and oxidative stress and protecting the integrity of the blood-brain barrier (BBB).

Neuroprotective Effects of dBET1 in Stroke

Since the PROTAC molecule dBET1 contains the BET bromodomain-binding part JQ1, which has much higher selectivity and affinity for BRD4 than other BET members, BRD4 function regulation is the main role of dBET1. Studies have shown that dBET1 significantly improves outcomes in mouse models of permanent and transient ischemic stroke. Treated animals exhibited reduced neurological deficits and shrinkage of brain damage, and protective effects were observed even when treatment was initiated four hours after reperfusion, consistent with the clinically relevant therapeutic window. This study enhances the understanding of how BRD4 degradation via dBET1 provides protection against ischemic stroke.

BRD4 protein degradation by dBET1 is associated with reduced neuroinflammation and oxidative stress

Inflammation and oxidative stress are pathomechanisms of stroke that influence brain injury, impairment, and survival. Researchers have found that dBET1 therapy decreases many of the pro-inflammatory cytokines and chemokines IL-1, IL-6, TNF-, CCL2, CXCL1, and CXCL10. Furthermore, dBET1 decreased oxidative damage, and increased expression of the antioxidant enzymes SOD2 and GPx1.

BRD4 protein degradation by dBET1 is associated with preservation of BBB integrity

The excessive accumulated inflammatory and oxidative mediators following stroke cause endothelial dysfunction, which leads to BBB breakdown, indicated by increased BBB permeability and the degradation of BBB structural components, including the endothelial tight junction proteins ZO-1 and occludin. Treatment with dBET1 reduces the BBB permeability increased by IgG absorption and loss of tight junction proteins ZO-1 and occludin. Acute post-ischemic dBET1 also suppresses post-stroke MMP-9, the colonization of the ischemic brain by neutrophils, and the dysregulation of cell adhesion molecules (e.g., ICAM-1).

BRD4 protein degradation by dBET1 is associated with reduced reactive gliosis

Microglia and astrocytes are the major cell types that regulate ischemia-induced neuroinflammation and oxidative stress, and they are the most rapidly responsive to brain injury through morphologic changes and proliferation, as well as enhanced release of a variety of inflammatory mediators. dBET1 attenuates the destructive progression of reactive gliosis in microglia and astrocytes in peri-infarct regions, thus protecting against ischemic injury.

Fig.2. Schematic summarizing the molecular mechanisms underlying the protection of PROTAC dBET1 in ischemic stroke. (Liu, et al., 2022)

1. Liu, L., et al. (2022). Targeted BRD4 protein degradation by dBET1 ameliorates acute ischemic brain injury and improves functional outcomes associated with reduced neuroinflammation and oxidative stress and preservation of blood–brain barrier integrity. Journal of neuroinflammation, 19(1), 168.