Preclinical Development and Screening of a Quinone-Nitroalkene Molecule for Sickle Cell Anemia

Abstract

Background: Sickle cell anemia (SCA) is a hereditary disorder caused by the formation of hemoglobin S (HbS). HbS undergoes polymerization and generates sickled red blood cells (RBCs). Along with having reduced oxygen carrying capacity, sickled RBCs are also prone to frequent hemolysis releasing the pro-oxidant heme in the circulation. Oxidative stress created by the free heme leads to sterile inflammation, vaso-occlusion and cardiovascular complications. The prevalence of SCA is greatest in the poorer regions of Sub-Saharan Africa and there are around 100,000 new incidents of SCA every year in the USA alone. Although a number of therapeutic options are currently FDA-approved for the treatment of SCA patients, there is considerable interpatient variability in terms of their efficacy and safety, along with high morbidity and mortality rates. We have developed a novel quinone-nitroalkene hybrid molecule called CP50 (figure 1), which has been shown to activate two important proteins: 1) nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of cellular resistance to oxidants and 2) cytochrome b5 reductase 3 (CYB5R3), an anti-stress enzyme in the cardiovascular system. Methodology and results: Primary human aortic endothelial cells (HAECS) were incubated with 5 µM CP50 for 6 hours to perform a genomic screening. The mRNA expression profile revealed that CP50 differentially regulated pathways associated with oxidative balance, inflammation, and erythropoiesis. CP50 was associated with upregulation of genes responsible for counteracting stress, including heat shock protein family, HSPA6, HSPA1A, HSPA1B and heme-oxygenase 1 (HMOX-1) (n=5, p<0.05). CP50 downregulated genes that inhibit effective erythropoiesis, such as ZFP36 Ring Finger Protein Like 2 (ZFP36L2) and Growth/differentiation factor 15 (GDF15), thus imparting a positive effect on the process (n=5, p<0.05). Immunoblotting for heme-oxygenase 1 (HO-1) using wild type and Nrf2-knockdown HAECs demonstrated that HO-1 was induced by 5 µM CP50 by 5 fold (±1.36) after 24 hours of exposure, in a Nrf2 dependent manner (n=3, p<0.0001). CYB5R3 activity measured by its impact on myoglobin reduction rate, was also found to increase dose-dependently by CP50 (n=3, p<0.001). Afterwards, CD34+ hematopoietic stem cells (HSCs, n=3) were cultured with 5 µM CP50 and 50 µM hydroxyurea (HU) to observe the effect on erythropoiesis, using CD71, CD235a and fetal hemoglobin (HbF) as markers for flow cytometry. CP50 augmented the percentage of HbF-expressing F cells by 1.7 fold (±0.54) in comparison to 2.21 fold (±0.89) by HU on day 9. F cells are known to impart resilience against sickling and hemolysis in sickle cell anemia. We then observed the effect of CP50 administered by osmotic pump implantation on hematopoiesis in Townes HbSS mice (vehicle, 5mg/kg/day, 15mg/kg/day, n=3/4 in each group). After a period of 4 weeks, we collected whole bone marrow from the mice and using flow cytometry checked for HSCs. CP50 (15 mg/kg/day) significantly increased (p<0.001) the HSC pool by 59% in comparison to vehicle. Additionally, it also enhanced the differentiation of megakaryocyte-erythroid progenitors (MEP) in bone marrow cells by 11% (p<0.05). In a separate experiment, peripheral blood samples from Townes HbSS mice were collected and incubated with 5 µM CP50 before chemically inducing oxidative hemolysis. CP50 reduced hemolysis by 73.68% in comparison to vehicle. Conclusion: Together, our studies indicate that CP-50 has the potential to correct oxidative imbalance by activating antioxidant genes, reverse anemia by inducing hematopoiesis, and inhibit hemolysis by increasing F cell percentage. Thus, we can conclude that CP50 is a prospective drug candidate with the promise to satisfy the unmet need for additional safe and effective therapeutic options for patients with sickle cell anemia. Legend (figure 1): The quinone head group in CP50 serves as an antioxidant, whereas the nitro group in the fatty acid side chain acts as an electrophile. The electrophile modifies the cysteine maintaining the Keap1-Nrf2 interaction and releases the Nrf2. Nrf2 enters the nucleus and initiates the expression of genes that counteract oxidative stress. Keap1: Kelch-like ECH-associated protein 1 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal