Glutathione Reductase of the Malarial Parasite Plasmodium falciparum: Crystal Structure and Inhibitor Development G. N. Sarma 1 , S. N. Savvides 1 , K. Becker 2 , M. Schirmer 2,3 R. H. Schirmer 3 * and P. A. Karplus 1 * 1 Department of Biochemistry and Biophysics, Oregon State University, 2011 AG Life Sciences Bldg, Corvallis, OR 97331-7305, USA 2 Interdisciplinary Research Center, Giessen University Heinrich-Buff-Ring 26-32 D-35392 Giessen, Germany 3 Biochemistry Center Heidelberg University, Im Neuenheimer Feld 328 D-69120 Heidelberg, Germany The malarial parasite Plasmodium falciparum is known to be sensitive to oxidative stress, and thus the antioxidant enzyme glutathione reductase (GR; NADPH þ GSSG þ H þ Y NADP þ þ 2 GSH) has become an attrac- tive drug target for antimalarial drug development. Here, we report the 2.6 A ˚ resolution crystal structure of P. falciparum GR. The homodimeric flavoenzyme is compared to the related human GR with focus on struc- tural aspects relevant for drug design. The most pronounced differences between the two enzymes concern the shape and electrostatics of a large (450 A ˚ 3 ) cavity at the dimer interface. This cavity binds numerous non- competitive inhibitors and is a target for selective drug design. A 34-residue insertion specific for the GRs of malarial parasites shows no density, implying that it is disordered. The precise location of this inser- tion along the sequence allows us to explain the deleterious effects of a mutant in this region and suggests new functional studies. To comple- ment the structural comparisons, we report the relative susceptibility of human and plasmodial GRs to a series of tricyclic inhibitors as well as to peptides designed to interfere with protein folding and dimerization. Enzyme-kinetic studies on GRs from chloroquine-resistant and chloro- quine-sensitive parasite strains were performed and indicate that the structure reported here represents GR of P. falciparum strains in general and thus is a highly relevant target for drug development. q 2003 Elsevier Science Ltd. All rights reserved Keywords: Plasmodium falciparum glutathione reductase; crystal structure; drug design; 10-arylisoalloxazines; malaria *Corresponding authors Introduction Tropical malaria represents an increasing threat to human health and welfare. 1,2 The disease is caused by the multiplication of the protozoan para- site Plasmodium falciparum in human erythrocytes. 3 The emerging resistance of Plasmodium to chloro- quine and other antimalarials underlines the need for the development of new chemotherapeutic agents with different modes of action. 4–6 During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress pro- duced by activated macrophages of the host and also by toxic heme and other decomposition pro- ducts of hemoglobin. We are pursuing enhanced oxidative stress as an attractive avenue for drug development, because a number of lines of evi- dence suggest that this can effectively inhibit para- site growth (reviewed by Schirmer et al. 7 ). Indeed the toxic effects of chloroquine and other quinoline antimalarials that inhibit b-hematin formation 8–12 may be partly due to increased levels of O 2 -activat- ing heme, and peroxide antimalarials such as artemisinin are believed to be activated by heme 0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved Present address: S. N. Savvides, Dienst Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium. E-mail addresses of the corresponding authors: heiner.schirmer@gmx.de; karplusp@ucs.orst.edu Abbreviations used: hGR, human glutathione reductase; PfGR, Plasmodium falciparum glutathione reductase; EcGR, Escherichia coli glutathione reductase; GSSG, oxidized glutathione; arilloxazines, 10- arylisoalloxazines; TLS, translation, libration and screw- rotation; r rms , root-mean-square electron density of map, often reported as s. doi:10.1016/S0022-2836(03)00347-4 J. Mol. Biol. (2003) 328, 893–907