The Mode of Action and Possible Target of Artemisinin Mike Van Linn Chemistry 496 23 April 2004

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

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

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

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

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

Possible Modes of Action Iron-Oxo Route Epoxidation Reactions Alkylation Reactions

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

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

Robert, et al

Cazelles, et al

Cazelles, et al

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

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

Robert, et al Cazelles, et al

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

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…

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

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

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.

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