Category: Preclinical Development
Purpose: Leukemia stem and progenitors cells are central to relapse in Acute Myelogenous Leukemia by maintaining and initiating the leukemic cell population. The sesquiterpene lactone (SL) parthenolide (PTL) has raised interest as a potential anti-leukemic compound because its ability to target leukemia stem cells. Unfortunately PTL’s poor water solubility and relatively low potency limit its in vivo effectiveness. In our continued effort to identify SLs with a better pharmaceutical profile.
Methods: Parthenin (PRT), a SL from Parthenium hysterophorus L. and Tetraneurin-E, a SL from Parthenium confertum var. lyratum (A. Gray) Rollins were isolated and tested in comparison with PTL against a battery comprised of 12 leukemia cell lines, 4 primary leukemia cell samples, and 3 normal peripheral mononuclear blood cell (PMBC) samples at concentrations of 1.25-20uM. Flow Cytometry was to count apoptotic cells. QSAR was used to test for fold changes of NFkB1 and HOMX1. Western blot was used to study the phosphorylated levels of NFkB (S536) and HMOX1 after 2 hours incubation of cells with all three compounds.Molecular modeling (SYBYL 2.0) was used to construct hydrophobic surfaces for the compounds using their crystal structures.
Results: Compared to PTL, PRT kills AML at lower doses with a mean LD50 of 6.81 μM vs. 11.56 μM for all cell lines and 6.80 μM vs 7.91μM for primary cells. No cellular death was seen for TET at the highest concentration of 20uM used. PRT did not have any detectable toxicity when tested with PMBCs at 20uM. PRT decreases the fold change of NF-κB1 transcript approximately 4x more than PTL. PRT does not increase the fold change of HOMOX-1, indicating that it does not induce the Nrf2 cytoprotective response – a limiting factor in the effectiveness of PTL. HMOX-1 is increased 2x and 3x more than the control for TET and PTL, respectively. PRT decreases active NF-κB (pS536) and HMOX-1 protein levels better than PTL. Mechanistically, it is believed a major mechanism of SLs is the induction of apoptosis through covalent modification of C-38 on NF-κB. Molecular modeling analysis suggests that both PRT and PTL can bind very well to NF-κB because they possess a large hydrophobic surface formed by C-8, C-9, C-13 and C- 14 that can interact with a complementary hydrophobic surface surrounding C-38 on NF-κB. Although Tetraneurin-E (TET) contains the same pharmacophore of PTL and PRT, its smaller hydrophobic surface is incompatible with the hydrophobic surface surrounding C-38 of Nf-kB. Therefore, it’s pharmacophore is most likely not able to interact with C-38 which is necessary for targeting NF-kB.
Conclusion: These results support the evidence that a major mechanism of potent sesquiterpene lactones against AML involve targeting NF-kB. Until now, it has not been rationally discussed why certain closely related sesquiterpene lactones bearing the a-methylene-gamma-lactone pharmacophore do not show specificity for NF-kB while others inhibit it very effectively. It is our theory, using in vitro results and molecular modeling that hydrophobic surfaces are critically important for sesquiterpene lactone potency against AML.
David Mery– Little Rock, Arkansas
David Mery– Little Rock, Arkansas
Rachel Yang– New York, New York
Cesar Compadre– Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, Tocol Pharmaceuticals, LLC, Little Rock, AR 77205, USA, Little Rock, Arkansas
Monica Guzman– New York, New York
Paola Ordóñez– Sede Ibarra, Imbabura, Ecuador