Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria

Abstract

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

Effectiveness of Analogs of the GS-Nitroxide, JP4-039, as Total Body Irradiation Mitigators

“…These compounds have proven significant and differential affinity for mitochondria in vivo . XJB-5-131 has a longer peptide-targeting sequence and results in a 600-fold enrichment in the mitochondria compared to cytoplasm, while JP4-039 contains a shortened alkene dipeptide isostere moiety attached to the nitroxide, which results in a 32-fold enrichment of nitroxide to the mitochondria compared to the cytoplasm (23). …”

“…Previous studies have demonstrated a 30- to 33-fold concentration of 4-amino-Tempo nitroxide at the mitochondria when linked to JP4-039 (14, 15, 23). Another GS-nitroxide, XJB-5-131, has been shown to have a 600-fold mitochondrial concentration in the mitochondria (24), attributable to the longer mitochondrial localization sequence, which was also derived from the natural antibiotic GS.…”

A Topical Mitochondria-Targeted Redox-Cycling Nitroxide Mitigates Oxidative Stress-Induced Skin Damage

“…Further, detection of the nitroxide signal by EPR verified its biologic activity in these cells. To visually confirm the presence of JP4-039 in the mitochondria, JP4-039-Bodipy was synthesized (Bernard et al, 2011; Frantz et al, 2013) and applied to mouse skin as described above. Skin sections (Fig 1c) and isolated keratinocytes (Fig 1d) were labeled with Mitotracker Red-CMXROS and DAPI and analyzed by confocal microscopy (600X).…”

“…JP-Bodipy was synthesized as described previously (Bernard et al, 2011; Frantz et al, 2013) and incorporated into liposomes as described earlier. Liposomes containing 4 mg/mL of Bodipy conjugated JP4-039 were applied to the shaved backs of mice for 30 min, and skin was processed and biopsied as described above.…”

Addressing the Symptoms or Fixing the Problem? Developing Countermeasures against Normal Tissue Radiation Injury

“…For example, the Pittsburgh Center for Medical Countermeasures against Radiation (CMCR) has developed a series of novel TEMPOL-derived mitochondria-targeted GS-nitroxides (65) that inhibit cyto-chrome c peroxidase activity, blocking acute apoptosis and reducing early lethality after cytotoxic doses of both chemotherapy and radiation. Of note, the most promising results published to date for the lead candidate, JP4-039, when administered alone, were seen when the agent was delivered in close temporal proximity to irradiation [within minutes to hours either pre- or postirradiation (66)], limiting its utility after a large accidental or deliberate radiological event.…”