Graduation Year
2024
Document Type
Master's Thesis
Degree
Master of Science
Program
Biological Science
Program Director
Meredith Protas, PhD
First Reader
Tyler Johnson, PhD
Second Reader
Kenneth Frost, PhD
Abstract
Marine natural products from sponges of the Indo-Pacific have proven to be a source of structurally diverse secondary metabolites that are biologically active against a variety of targets. In the first chapter of this thesis, the stability of the nanomolar-potent, irreversible microtubule stabilizer, (-)-zampanolide (1), the cytotoxicity of its N-acyl hemiaminal side chain (2), and the activity of the more stable related compound (-)-dactylolide (3) were investigated. Following the discovery of 1 from extracts of Cacospongia mycofijiensis, in a previous study, the first in vivo evaluation of 1 in MDA-MB-231 tumor bearing mice was performed, demonstrating it had potent and persistent antitumor efficacy but only when targeted to the tumor site due to a narrow therapeutic index. It was hypothesized that 1 is thermally unstable at body temperature (37°C, 98°F) and may decompose into the toxicant Michael acceptor, 2, and covalent microtubule stabilizing agent, 3. Thermolysis at 80°C of 1 reveals the decomposition of 1 into 2 and 3 as keto (3k)/enol (3e) tautomers shown by HPLC and NMR. HR-LCMS analysis revealed incubation at 37°C (body temperature) of 1 for 24 hrs generated 2 and 3 similar to 80°C thermolysis. Antiproliferative and biochemical assays reveal low micromolar cytotoxicity of 2 against selected triple negative breast cancer (TNBC) cell lines HCC1806, MDA-MB-453, BT-549, HCC70, and MDA- MB-231 (IC50 = 1.9 ~ 10.6 μM) and no microtubule binding affinity. Compound 3k displayed nanomolar potency against BT-549 (IC50 = 0.37 μM), MDA-MB-231 (IC50 = 0.62 μM), HCC70 (IC50 = 0.65 μM), with selectivity for MDA-MB-453 (IC50 = 0.054 μM) and HCC1806 (IC50 = 0.080 μM) and delayed binding mode to microtubules compared to 1. These results show that 1 is thermally unstable at body temperature, that 2 may be responsible, at least partially, for the in vivo toxicity of 1 in MDA-MB-231 tumor bearing mice, and that 3 may serve as a more stable and selective therapeutic lead against MDA-MB-453 and HCC1806 cell lines.
In the second chapter of this thesis, a panel of secondary metabolites from the marine sponges C. mycofijiensis, Jaspis splendens, and Jaspis coriacea were evaluated against the most notorious malaria parasite Plasmodium falciparum in a drug response assay. Due to the rise in resistance to standard antimalarials such as chloroquine (1), dihydroartemisinin (2), and lumefantrine (3), there is a critical need to determine novel lead therapeutics with unique mechanisms of action (MOA) that can circumvent forms of drug resistance. Scale-up isolation by HPLC was conducted to purify latrunculin A (4), fijianolide A/isolaulimalide (5), fijianolide B/laulimalide (6), (-)-zampanolide (7), (-)-dactylolide (8), (+)-(5Z)-(8S)-(14Z)-mycothiazole (9), (+)-(5Z)-(8S)-8-O-acetyl-(14Z)- mycothiazole (10), (-)-(5E)-(8R)-(14Z)-mycothiazole (11), jasplakinolide (12), bengamide B (13), and fascaplysin (14). Both microtubule stabilizing agents, 6 and 7, from C. mycofijiensis displayed the greatest efficacy against P. falciparum proliferation on par with standard antimalarials (1-3). To identify the importance of the C-15 and C-20 chiral centers of 6, semi-synthesis was performed to produce the di-oxo fijianolide L (15). The malaria parasite drug response assay demonstrated a 2000-fold reduction in potency in 15 compared to 6, suggesting C-15 and C-20 to be key functional groups in the activity of 6. Overall, these results revealed that 41% of the selected sponge-derived secondary metabolites were comparable to frontline standard antimalarials used today, suggesting there is potential in developing marine natural products as antimalarial therapeutics.