The Mode of Action and Possible Target of Artemisinin

1. The Mode of Action and Possible Targetof Artemisinin Mike Van Linn Chemistry 496 23 April 2004

2. Outline • Introduction • Malaria • Artemisinin • Rationale for Research • Modes of Action • Iron-Oxo route • Epoxidation reactions • Alkylation reactions • The Target of Artemisinin

3. Introduction • Malaria • Four species of Plasmodium • Infects 200 million people annually • 1 million lethal • Resistance to current drugs

4. Introduction • Artemisinin • Natural product extracted from sweet wormwood, Artemisia annua • Used by Chinese for over 2000 years • A. absinthium used to make absinthe

5. Rationale for Research • Anti-malarial Activity of Artemisinin • Artemisinin Derivatives • Used currently for life threatening cases

6. Rationale for Research • Anti-malarial Activity of Artemisinin • Artemisinin Derivatives • Used currently for life threatening cases • Drug Resistance of Plasmodium • Malaria spreading • Synthesis of new drugs

7. Possible Modes of Action • Iron-Oxo Route • Epoxidation Reactions • Alkylation Reactions

8. Iron-Oxo Route • Donation of Oxygen from Peroxide Bridge to Iron • Generate Fe(IV)=O • No Support from Raman Resonance Data • Signal/Noise < 2 • Should be ~10 or 20

9. Epoxidation Reactions MnIITPP or FeCl2 NO EPOXIDE FORMATION + ARTEMISININ OR MnIITPP or FeCl2 + Na+-OCl EPOXIDE FORMATION

10. Robert, et al

11. Cazelles, et al

12. Cazelles, et al

13. 1,5 H Shift Possible??? • Critical Distance Calculated to be 2.1Å • Exceeded in Stable Conformation • Boat-like Conformation (High energy state) • Houk

14. Comparing Route 1 and 2 • Route 1 Dominant to Route 2 • 90/10 ratio from isolated products • Artemisinin + MnIITPP • 1,5 H shift? • Route 1 Biologically Active • Route 2 Inactive • Stereochemistry Effecting Alkylation

15. Robert, et al Cazelles, et al

16. Mode of Action • Route 1 Dominant • Alkyl radical formation from reduction of peroxide bridge • Derivatives Used • Observe correlation of alkylating ability to drug activity • Alkylate MnIITPP Pharm. active

17. The Target • Alkylation of Heme within Infected Erythrocytes (RBC’s) • Free heme in food vacuole of erythrocyte • Cleavage of peroxide bond • Alkylation of heme or specific parasite proteins can occur • Too General…

18. The Target, More Specifically • Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Enzyme • PfATP6 gene sequence • Testing the hypothesis • Heme Not Required? • Free heme blocked with Ro 40-4388 protease inhibitor • Localized in the Food Vacuole? • Fluorescent labeled artemisinin

20. Conclusions • Malaria Remains as a Problem • Resistant strains • Anti-malarial Activity of Artemisinin • Mode of Action is Now Understood • Alkylation via route 1 • A Specific Target Found • PfATP6 gene sequence of the SERCA enzyme • Fe2+ is required • Activity not localized in the food vacuole

21. References • Robert, Anne, et al. “From Mechanistic Studies on Artemisinin Derivatives to New Modular Antimalarial Drugs.” Accounts of Chemical Research, 2002,Vol. 35, pp. 167-174. • Cazelles, Jerome, et al. “Alkylating Capacity and Reaction Products of Antimalarial Trioxanes after Activation by a Heme Model.” The Journal of Organic Chemistry, 2002, Vol. 67, Number 3, pp. 609-619. • Wu, Wen-Min, et al. “Unified Mechanistic Framework for the Fe(II)-Induced Cleavage of Qinghaosu and Derivatives/Analogues. The First Spin-Trapping Evidence for the Previously Postulated Secondary C-4 Radical.” J. Am. Chem. Soc., 1998, Vol. 120, pp. 3316-3325. • Biot, Christophe, et al. “Synthesis and Antimalarial Activity in Vitro and in Vivo of a New Ferrocene-Chloroquine Analogue.” J. of Medicinal Chemistry, 1997, Vol. 40, pp. 3715-3718. • Yarnell, Amanda; “Rethinking How Artemisinin Kills,” Chemical and Engineering News, Aug. 25, 2003, Vol. 81 (24), pp. 6. • Eckstein-Ludwig, Ursula, et al. “Artemisinins Target the SERCA of Plasmodium falciparum,” Nature, 2003, Vol. 424, pp.957.

22. Questions