The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas' disease. As a first step in the analysis of the parasite enzyme we report its purification and characterization.1. 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells. The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45%.2. The enzyme is a dimer with a subunit M , of 50000. Using NADPH (K, = 5 pM) and trypanothione disulfide (K, = 45 pM) as substrates, a turnover number of 14200 min-' was estimated. Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates.3. When screening cell cultures or column eluates for the presence of trypanothione reductase, a microassay based on Ellman's reagent as indicator was used. A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay.4. Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions E,, + NADPH + H' + EH2 + NADP', and EH, + trypanothione disulfide + E,, + dihydrotrypanothione. This is also true for the crystallized enzyme in the presence of 2 M (NH&S04. 5. The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electronreduced form (EH,) closely resemble those of human glutathione reductase. Both proteins contain a flavin and a redox-active disulfide at the catalytic site. After reduction of E,, to EH2, trypanothione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.Trypanosomes and leishmanias are unicellular eukaryotes which belong to the family Trypanosomatidue and to the order Kinetoplastida [l, 21. Diseases of man and domestic animals caused by these protozoa include sleeping sickness (Trypanosoma rhodesiense, T . gambiense), Chagas' disease ( T . cruzi), nagana (T. brucei, T . vivax, T . congolense), oriental sore (Leishmania tropica) and kala-azar (L. donovani). Most parasitic Kinetoplastida are thought to be more sensitive to oxidative stress than their hosts; indeed recent evidence suggests that the host's defense system and the action of certain drugs is largely based on reactive oxygen species [3 -51. In addition, there is a qualitative difference: the major antioxidant enzyme system of the parasites needs trypanothione [6] and possibly other glutathionylspermidines Abbreviations. E,,, enzyme in oxidized form; EH2, two-electronreduced enzyme; GSH, reduced glutathione; GSSG, glutathione disulfide; Nbsz, 5,5'-dithiobis(2-nitrobenzoate), Ellman's reagent; trypanothione disulfide, N1,N8-bis(L-y-glutamyl-L-hemicystinylglycy1)spermidine; dihydrotrypanothione, N' ,N8-bis(L-y-glutamyl-Lcysteinyl-glycy1)spermidine.

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On the basis of investigations of the malarial blood-stage antigens SERP, HRPII, and MSAI from Plasmodium fakciparum, we chose two Escherichia coli-expressed hybrid proteins containing selected partial sequences of these antigens. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding P. fakciparum polypeptides. In two independent trials with 13 animals, immunization of Aotus monkeys with either of the two hybrid proteins administered in a well-tolerated oil-based formulation protected the animals from an experimental P. falciparum infection.

Deoxyfloxacrine derivatives (1-hydrazone: S 83 0083; 1-imine: S 84 7277) and floxacrine derivatives (10-methoxy-floxacrine: L 84 7667; 1-imine: L 84 7693) selected from a series of newly synthesized 3-aryl-7-chloro-3,4-dihydro-1,9(2H,10H)-acridinediones were evaluated for blood schizontocidal activities in mice infected with asexual stages of various drug-resistant lines of P. berghei and in New World monkeys infected with blood schizonts of different chloroquine-resistant strains of P. falciparum. All compounds tested showed high activity against drug-resistant lines of P. berghei (ED50: 1.0-4.4 mg/kg x 5, per os) and were distinctly superior in their antimalarial potency to floxacrine. Compounds L 84 7667 and L 84 7693 proved to be highly active against the FCBR strain of P. falciparum in vitro (IC50: 0.73-1.78 nmol); they effected temporary clearance of parasitemias due to the Palo Alto strain of P. falciparum in squirrel monkeys at oral doses of 15 mg/kg given daily for 5 consecutive days. Compounds S 83 0083 and S 84 7277, showing moderate in vitro effects (12.9-24.8 nmol), cleared parasitemias of the FCBR strain of P. falciparum in owl monkeys at oral doses of 20 mg/kg (S 84 7277) given daily for 5 or 7 consecutive days (follow-up period, 17 and 30 days, respectively) or at doses of 20 mg/kg (x 4) (S 83 0083) followed by doses of 40 mg/kg (x 3) within a follow-up period of 30 days. These observations suggest that the range of doses required for the cure of established P. falciparum infections is probably too large to cover infections with strains of the least susceptibility and might evoke toxic reactions by the potential candidates tested.

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