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Development of Peptide Inhibitors of Nuclear Factor KappaB as Therapeutics for Ischemic Stroke

Ischemic strokes occur when a major cerebral artery or its branches are occluded and result in activation of inflammatory processes, causing secondary tissue injury, breakdown of the blood‐brain barrier, edema, or hemorrhage. Treatments that inhibit inflammatory processes may thus be highly beneficial, but are not currently available. A key inflammatory process is the nuclear factor kappa B (NF‐κB) pathway. In its active form, NF‐κB is translocated to the nucleus of the cell where it regulates expression of proinflammatory and proapoptotic genes. The molecules that interact with NF‐κB, and the subunits that compose NF‐κB itself, allow for therapeutic targets to decrease inflammatory processes by inhibiting the nuclear localization of NF‐κB. Our goal is to develop such NF‐κB‐interacting peptides attached to the drug carrier elastin‐like polypeptide (ELP). ELP has a long plasma half‐life, can be attached to cell penetrating peptides, and has been shown to be an effective drug carrier in other models of disease. Here, we cloned five peptide inhibitors of the NF‐κB pathway into vectors to generate chimeric fusion proteins with ELP which were subsequently expressed recombinantly in E. coli and purified by inverse transition cycling. The ELP‐peptide fusions were screened using an in vitro luciferase assay in human embryonic kidney 293 cells transiently transfected with a reporter plasmid containing NF‐κB response elements driving the luciferase gene. One peptide fusion protein, targeted to the p50 subunit of NF‐κB (ELP‐p50i) resulted in attenuation of luminescence and thereby NF‐κB activity. Cellular localization of ELP‐p50i was assessed using fluorescently labeled protein after in vitro incubation with human umbilical vein endothelial cells (HUVEC) and mouse macrophages (RAW264.7) by confocal microscopy. Inhibition of NF‐κB activation by ELP‐p50i protein or a control protein lacking the NF‐κB inhibitory peptide was also confirmed in HUVECs stimulated with TNF‐a and RAW 264.7 cells stimulated with lipopolysaccharide by immunolabeling the NF‐κB complex and observing its nuclear translocation. ELP‐p50i was internalized by both HUVECs and RAW264.7 cells and localized to punctate cytoplasmic structures, likely endosomes and lysosomes, and diffusely in the cell cytoplasm. Pretreatment of cells with ELP‐p50i prevented NF‐κB nuclear localization after pro‐inflammatory stimuli. Similar analyses of the other NF‐κB inhibitory constructs are currently ongoing to determine which construct is most effective at inhibiting NF‐κB nuclear translocation. Biodistribution and pharmacokinetics comparing intravenous and intraarterial injection of these ELP‐NF‐κB peptides have been determined using healthy Wistar rats and those subjected to the middle cerebral artery occlusion (MCAO) model of stroke. In conclusion, we developed a peptide to inhibit NF‐κB nuclear translocation that we hypothesize will inhibit inflammatory processes following stroke. Future studies will test the efficacy in reducing infarct size using MCAO in rats.