ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1988, p. 793-798 0066-4804/88/060793-06$02.

00/0 Copyright 1988, American Society for Microbiology

Vol. 32, No. 6

MINIREVIEWS
Antimalarial Agents: Mechanisms of Action
PAUL H. SCHLESINGER,' DONALD J. KROGSTAD,2"3* AND BARBARA L. HERWALDT2 Departments of Medicine2 and Pathology,3 Washington University School of Medicine, 660 South Euclid Avenue, and Department of Biomedical Research, Washington University School of Dental Medicine,' St. Louis, Missouri 63110 INTRODUCTION
Global impact of malaria. The data available suggest that there are 200 million to 300 million cases of malaria, with more than 2 million deaths, each year (70). Most deaths result from Plasmodium falciparum infection in Africa and are among children less than 5 years of age. The numbers of malaria cases and deaths dwarf those of virtually all other infectious diseases, including human immunodeficiency virus infection. Plasmodia that infect humans. Four species of malaria parasites infect humans: P. falciparum, P. vivax, P. ovale, and P. malariae. Of these, only P. falciparum poses a significant risk of death in the nonimmune patient, because of its ability to invade erythrocytes of any age and thus to produce overwhelming parasitemias (>106%a.l; 13, 43). The other species produce less morbidity and mortality because they are able to invade only young (P. vivax or P. ovale) or old (P. malariae) erythrocytes (43). Use of antimalarial agents. Antimalarial agents have been used since the time of Hippocrates (30). However, antimalarial treatment has traditionally been empirical, and the mechanisms of antimalarial action have been unknown. Effect of parasite stage. Only asexual erythrocytic parasites produce clinical illness. Neither persistent exoerythrocytic hepatic parasites (hypnozoites) nor sexual parasites (gametocytes) produce recognizable clinical symptoms. Therefore, antimalarial agents that inhibit the replication of asexual erythrocytic parasites (chloroquine, mefloquine, quinine, and quinidine) produce the most rapid clinical improvement. Another antimalarial agent, primaquine, is used to treat the other parasite stages (exoerythrocytic and sexual erythrocytic parasites) when treatment is indicated. In this review, we examine the present knowledge of antimalarial action. Elsewhere, we examine resistance (37) and the regimens most widely used for antimalarial chemoprophylaxis (B. L. Herwaldt, D. J. Krogstad, and P. H. Schlesinger, Antimicrob. Agents Chemother., in press) and treatment (D. J. Krogstad, B. L. Herwaldt, and P. H. Schlesinger, Antimicrob. Agents Chemother., in press). Investigational antimalarial agents and their likely mechanisms of action are shown in Table 1.
DRUGS ACTIVE AGAINST ASEXUAL ERYTHROCYTIC PARASITES The agents used most widely against asexual erythrocytic parasites are chloroquine, mefloquine, quinine, and quinidine. Other important drugs include inhibitors of protein synthesis (such as tetracycline and doxycycline) and antifo*

lates, such as the pyrimethamine plus sulfadoxine combination (Fansidar).

MECHANISM OF CHLOROQUINE ACTION Although chloroquine has been used as an antimalarial agent for more than 40 years, the ability of chloroquine and other weak bases to raise the pH of acid vesicles has been appreciated only in the last 10 to 20 years (8-10, 12, 16, 25, 35, 52-54, 60, 62, 69). These studies suggest that the ability of chloroquine to raise intravesicular pH may play a major role in its antimalarial action. Chloroquine as a weak base. More than a decade ago, Homewood and her colleagues suggested that chloroquine might inhibit the degradation of hemoglobin ingested by the parasite by raising the pH of the food vacuole of the parasite (28). The ability of weak bases to raise intravesicular pH was first postulated by de Duve et al. to explain their inhibition of protein catabolism by mammalian lysosomes. They proposed that weak bases (compounds with pKs from 7 to 11) inhibited the hydrolytic activity of lysosomes by raising intravesicular pH above the pH optima of their acid proteases (8, 9). Effects of weak bases on intravesicular pH and on parasite growth. The driving force for the intravesicular accumulation of monoprotic weak bases, such as NH4CI, is proportional to the hydrogen ion gradient (100 for an intravesicular pH of 5 and an extracellular pH of 7). For a diprotic weak base, such as chloroquine, the driving force is proportional to the square of the hydrogen ion gradient (10,000 for an intravesicular pH of 5 with an extracellular pH of 7) (51). For this reason, chloroquine is concentrated more than simple monoprotic weak bases within acid vesicles. As a result, chloroquine raises lysosomal pH in mammalian cells at lower extracellular concentrations than NH4Cl (1 to 2 FM for chloroquine versus 2 to 3 mM for NH4Cl) (34, 36, 46, 50). Our results and those of Yayon et al. demonstrate that chloroquine raises intravesicular pH in P. falciparum (36, 71, 72). Our recent studies have shown also that weak bases which are not antimalarial agents (NH4CI, methylamine, propylamine, benzylamine) raise intravesicular pH in mammalian cells and P. falciparum and inhibit parasite growth at those same concentrations (36; D. J. Krogstad, P. H. Schlesinger, I. Y. Gluzman, and C. M. Koziol, Clin. Res. 34:523A, 1986). The effects of these weak bases on vesicular pH can be predicted purely on physicochemical grounds for both parasites and mammalian cells (from their pKs and the A pH between the acid vesicle and the extracellular medium) (51). In contrast, several weak bases which are antimalarial agents (such as chloroquine and mefloquine) raise intravesicular pH in the parasite and inhibit parasite growth at concentrations one one-hundredth (or less) of those neces793

Corresponding author.

794

MINIREVIEWS
TABLE 1. Investigational antimalarial agents
Agent Presumed mode of action

ANTIMICROB. AGENTS CHEMOTHER.

Reference(s)

Cyclosporin A
Sinefungin Aminopterin
Bredinin

Unknown Inhibition of methylation

Inhibition of purine salvage pathway

44

61 67
19

Chlorpromazine Calmidazolium Trifluoperazine Clindamycin Pirlimycin Tetracycline Chloramphenicol Erythromycin Rifampin

Inhibition of calmodulin

Inhibition of protein synthesis by 70S ribosomes

11, 21, 56

its properties as a weak base). Inhibition of the parasite vesicle proton pump seems unlikely because the buffering capacity measurements performed with chloroquine tested instantaneous buffering capacity (36) and thus permitted only a limited contribution from the vesicle proton pump. Physical disruption of the parasite vesicle seems unlikely because removal of chloroquine from the system by washing permitted vesicular pH to return to baseline (P. H. Schlesinger and D. J. Krogstad, unpublished observations). For these reasons, we believe that the first two potential explanations are unlikely and have focused our attention on the ability of the parasite vesicle to concentrate chloroquine. Chloroquine accumulation by mammalian ceils and by P. falciparum. The concentration of chloroquine accumulated in the vesicles of mammalian cells is the same as that predicted by its properties as a weak base. In contrast, the concentra-

Inhibition of DNA-dependent RNA polymerase

11 11

lonophores
Rhodamine 123 Janus green Antimycin A

Disruption of ion gradients

Inhibition of mitochondrial function

11, 22

Riboflavin analogs

Antagonism of flavin adenine dinucleotide

Unknown Increased erythrocyte permeability HMG' coenzyme A reductase inhibition Antoxidant enzyme inhibition

18 48 48
32 32

Imidazoles
Amphotericin B
Mevinolin

Aminotriazole Diethyldithiocarbamate

BCNUb
a HMG, 3-Hydroxy-3-methyl glutaryl. b BCNU, 1,3-bis(2-Chloroethyl)-l-nitrosourea.
sary to raise intravesicular pH in mammalian cells. These results cannot be explained on physicochemical grounds. They indicate that there is a specific interaction between the parasite vesicle and antimalarial agents such as chloroquine (or mefloquine). This interaction is specific because it does not occur between the parasite vesicle and weak bases which are not antimalarial agents or between the mammalian vesicle and chloroquine (or mefloquine). The ability of chloroquine (or mefloquine) to raise intravesicular pH in the parasite at concentrations one one-hundredth (or less) of those expected (according to their properties as weak bases) is thought to be responsible for the inhibition of parasite growth at extracellular chloroquine concentrations which do not affect mammalian cells. We have called this phenomenon the non-weak base effect (33, 34). Non-weak base effect. Mechanisms potentially responsible for the non-weak base effect of antimalarial agents on intravesicular pH in susceptible parasites include (i) inhibition of the parasite vesicle proton pump, (ii) physical disruption of acid intracellular vesicles, and (iii) a concentrating mechanism which results in excess accumulation of chloroquine within the parasite vesicle (beyond that predicted by

tion of chloroquine accumulated in the parasite vesicle is more than 100-fold greater than predicted by its properties as a diprotic weak base (34, 35). (This 100-fold discrepancy is consistent with the ability of chloroquine to raise parasite vesicular pH at extracellular concentrations less than one one-hundredth of those predicted by its properties as a weak base; see above.) These results suggest that the vesicle of the susceptible parasite has a chloroquine-concentrating mechanism which concentrates enough excess chloroquine (beyond that due to its properties as a weak base) to explain the non-weak base effect of chloroquine on parasite vesicular pH. The nature of this chloroquine-concentrating mechanism is unknown (Fig. 1). Potential biological consequences of raising vesicular pH. The role of an acid intravesicular pH in lysosomal proteolysis (8), receptor-mediated endocytosis (59), and the intracellular targeting of lysosomal enzymes (7, 27, 57) in mammalian cells has been well described. However, mammalian cells survive for days and P. falciparum survives for at least 12 h after the pH of their acid intracellular vesicles has been raised to .6.0 (27, 57). Thus, the mechanism by which raising intravesicular pH and/or concentrating chloroquine within the parasite vesicle inhibits parasite growth and ultimately kills the parasite is not defined. Although the parasite is dependent on the digestion of hemoglobin for essential amino acids, recent studies indicate that the acid proteases of the parasite have broad pH optima (26, 63-65). Therefore, raising intravesicular pH may inhibit the activity of acid proteases in the parasite lysosome (food vacuole)

only modestly.

Alternative explanations for the antimalarial action of chloroquine include the potential effects of raising intravesicular pH on the intracellular transport of macromolecules and membranes (59) and on phospholipase activity. In mammalian cells, intracellular transport of macromolecules and membranes is necessary for differentiation and development and is inhibited by raising intravesicular pH (2, 31, 41, 57). If the parasite employs similar receptor-mediated intracellular targeting of newly synthesized lysosomal enzymes and requires an intact acid vesicle system for differentiation, those processes are likely to be inhibited by raising the pH of the intracellular vesicles of the parasite. As noted by Ginsburg and his colleagues, phospholipase activity may be necessary for the transfer of hemoglobin from endocytic vesicles to the food vacuole of the parasite (23, 40, 73-75). Thus, raising vesicular pH may kill the parasite by inhibiting intracellular targeting and membrane movement sufficiently to inhibit maturation irreversibly, by inhibiting the transfer of hemoglobin to the food vacuole of the parasite or by other mechanisms.

VOL. 32, 1988

MINIREVIEWS

795

TRANSPORTER

INTRAVESICULAR BINDING SITE

FIG. 1. Concentration of chloroquine by vesicle of susceptible parasite. The vesicle of the susceptible parasite concentrates more chloroquine than can be explained by its pKs and the A pH between the parasite vesicle and the extracellular medium (33, 35). Potential explanations for this anomalous behavior of chloroquine with the parasite include a transporter (which could be located at the external surface of the erythrocyte, at the parasite-erythrocyte interface, and/or at the cytoplasmic surface of the parasite vesicle) (left panel) or a high-affinity chloroquine-binding site within the parasite vesicle (shown diagrammatically as the reverse image of CQ) (right panel).

Other theories of chloroquine action. In addition to the theory described above based on its ability to raise vesicular pH, two other theories of chloroquine action have been prominent: (i) the intercalation of chloroquine into DNA, and (ii) the binding of chloroquine to ferriprotoporphyrin IX (FP) produced by the degradation of hemoglobin by the parasite. In our opinion, neither of these hypotheses satisfactorily explains the effects of chloroquine on P. falciparum (34). Although chloroquine binds to DNA and other polyanions, the extracellular chloroquine concentrations necessary for chloroquine to intercalate into DNA (7 ,uM to 1 mM) (1, 5, 47; S. Meshnick, Annu. Meet. Am. Soc. Trop. Med. Hyg., 1987) or to inhibit DNA or RNA polymerase (0.1 to 2.0 mM) (6, 45) are several orders of magnitude greater than those which inhibit the growth of susceptible plasmodia in vitro (1 to 10 nM) (20, 24, 36). In addition, chloroquine inhibits nucleic acid synthesis in virtually all cells, in contrast to its exquisite specificity against plasmodia in vivo. Although chloroquine binds to FP with high affinity (4, 14, 15), the levels of free FP available within the parasite to which chloroquine can bind are not defined. Furthermore, although micromolar concentrations of the chloroquine-FP complex added to the extracellular medium lyse both parasites and erythrocytes (4, 14, 15), the concentrations of this complex achieved within the parasite are unknown.

phologic observations of Jacobs et al. (29), who have reported that the most striking ultrastructural change produced by mefloquine is swelling of the secondary lysosome (food vacuole) of the parasite. Although the nature of the interaction of mefloquine with the parasite vesicle is less well characterized than that of chloroquine, the parasite can become resistant to chloroquine alone, to mefloquine alone, or to both drugs (3, 20, 36, 58). Because the non-weak base effect (33-35) is observed with mefloquine in chloroquineresistant parasites, it is likely that the mechanism of chloroquine resistance is independent of the non-weak base effect.
MECHANISM OF QUININE AND QUINIDINE ACTION Like chloroquine and mefloquine, quinine and quinidine are diprotic weak bases that can raise the pH of mammalian and parasite acid vesicles (36). Because their more acidic pK is 5.1 (versus 8.2 for chloroquine), quinine and quinidine are concentrated in acid vesicles as monoprotic weak bases at physiologic pH (Fig. 2). This decreases their ability to raise vesicular pH by 10- to 100-fold. Studies of vesicle-buffering capacity indicate that quinine has little or no non-weak base activity (33) and suggest that the antimalarial activity of quinine results primarily from its properties as a weak base. Of interest but unexplored are recent observations which suggest that quinidine may be more effective than quinine against P. falciparum in vitro and in vivo (49, 68). Because quinine and quinidine are diastereochemical isomers, they are likely to have similar pKs and thus similar weak base properties. If quinidine has a greater ability to raise parasite vesicular pH (greater non-weak base activity), the stereochemical differences between quinine and quinidine may provide important clues to the structural prerequisites for non-weak base activity. Such a result would be consistent with the hypothesis that non-weak base activity requires a highly specific interaction between the parasite vesicle and the drug in question. Non-weak base activity and toxicity of antimalarial agents. The non-weak base effects of chloroquine and mefloquine on parasite vesicular pH and/or their concentration in the parasite vesicle permit them to inhibit and kill the parasite without producing undesirable side effects secondary to raising vesicular pH in human cells. This hypothesis is consistent with the relatively low incidence of side effects

MECHANISM OF MEFLOQUINE ACTION The effect of mefloquine on the intravesicular pH of the acid vesicles of the parasite is similar to that of chloroquine (36). Although the uptake of mefloquine by P. falciparum has not been studied quantitatively, the low nanomolar concentrations (.10 nM) of mefloquine which raise vesicular pH in the parasite are inconsistent with its pKs and indicate that it cannot be acting only as a weak base. In contrast, the micromolar concentrations of mefloquine (0.5 to 10 ,uM) necessary to raise vesicular pH in mammalian cells are consistent with its properties as a weak base (51). As discussed above for chloroquine, these findings indicate that mefloquine has non-weak base effects on vesicular pH in the mefloquine-susceptible parasite. These findings suggest that the action of mefloquine against P. falciparum results from a specific interaction between mefloquine and the acid vesicles of the parasite. This hypothesis is consistent with the mor-

796

MINIREVIEWS
CHLOROQUINE

ANTIMICROB. AGENTS CHEMOTHER.

<+~~"'r~PrH+
QUININE

pKI :8.3 1

3 H0+ J P]K2 1,02

=

pK2=9.7
,
I

pH 5 Vesicle pH

7 Medium pH

FIG. 2. pKs of chloroquine and quinine in relation to vesicular and medium pH. The pKs of chloroquine permit it to act as a diprotic weak base at physiologic pH because they are more than 1 and 3 pH units above 7, respectively. In contrast, the lower pK of quinine is 5.1, which is low enough that quinine behaves as a monoprotic weak base at physiologic pH.

observed with these drugs. In contrast, quinine, which has little or no non-weak base activity, raises the pH of mammalian acid vesicles at concentrations similar to those used therapeutically, has a narrow therapeutic ratio, and produces some toxicity in virtually all patients who receive it. These observations are consistent with the hypothesis that antimalarial agents with little or no non-weak base activity may be more toxic for mammalian cells. SULFONAMIDES AND DIHYDROFOLATE REDUCTASE INHIBITORS Sulfonamides and dihydrofolate reductase inhibitors are thought to act against the parasite by inhibiting the synthesis of folate, as they do in bacteria, although few studies have been performed to determine whether this is truly their mechanism of action. The ability of p-aminobenzoic acid, folic acid, or folinic acid to reverse the effects of sulfonamides on parasite growth suggests that these compounds do indeed act by inhibiting the folate pathway (17, 42). The gene coding for dihydrofolate reductase and thymidylate synthetase in P. falciparum was cloned and sequenced recently (2a). The antifolate combination used most frequently is pyrimethamine plus sulfadoxine. Proguanil (Paludrine), which has been used for chemoprophylaxis by a number of travelers, is a dihydrofolate reductase inhibitor (66).
INHIBITORS OF PROTEIN SYNTHESIS Some agents are thought to act against the parasite by inhibiting protein synthesis, as they do in bacteria. Several studies suggest that tetracycline, clindamycin, chloramphenicol, and other agents which act primarily against 70S ribosomes are more active against the parasite than compounds which act primarily against 80S ribosomes. Geary and his colleagues have suggested that these agents produce their antiplasmodial effects by acting against 70S ribosomes in the parasite mitochondrion (11, 21).
INVESTIGATIONAL ANTIMALARIAL AGENTS The wide variety of compounds active against the parasite in vitro suggests that there are a number of pathways

amenable to inhibition by drugs (Table 1). This information is included because agents which are active against the parasite in vitro or in animal models are likely to be important for the development of future antimalarial agents.
DRUGS ACTIVE AGAINST EXOERYTHROCYTIC STAGES: 8-AMINOQUINOLINES The mechanisms by which primaquine and other similar 8-aminoquinolines eradicate hypnozoite forms from the liver and kill gametocytes in the peripheral blood are unknown. The concentrations of primaquine necessary to raise intravesicular pH in mammalian cells and to inhibit sporozoite entry into liver cells in vitro are substantially greater than those achieved in vivo (55). Despite the inconclusive nature of these laboratory data, the ability of primaquine to prevent late relapse due to dormant hypnozoite forms in the liver is supported by liver biopsy studies in primates, which demonstrate eradication of hypnozoites after treatment with primaquine (38, 39).
ACKNOWLEDGMENTS These investigations received the financial support of the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases. They were supported also in part by Public Health Service grants Al 18911 and Al 07766 from the National Institute of Allergy and Infectious Diseases and HL 26300 from the National Heart, Lung, and Blood Institute. We thank Ilya Y. Gluzman for his many contributions to our studies, John S. Wolfson for his thoughtful review of the manuscript, and Hagai Ginsburg for his constructive suggestions.
LITERATURE CITED 1. Allison, J., R. L. O'Brien, and F. Hahn. 1965. DNA: reaction with chloroquine. Science 149:1111-1113. 2. Bonner, J. T., H. B. Suthers, and G. M. Odell. 1986. Ammonia orients cell masses and speeds up aggregating cells of slime moulds. Nature (London) 323:630-632. 2a.Bzik, D. J., W. B. Li, T. Horii, and J. Inselburg. 1987. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthetase gene. Proc. Natl. Acad. Sci. USA 84:8360-8364. 3. Centers for Disease Control. 1985. Revised recommendations for

VOL. 32, 1988

MINIREVIEWS

797

preventing malaria in travelers to areas with chloroquine-resistant Plasmodiumfalciparum. Morbid. Mortal. Weekly Rep. 34: 185-195. 4. Chou, A. C., R. Chevii, and C. D. Fitch. 1980. Ferriprotoporphyrin IX fulfills the criteria for identification as the chloroquine receptor of malaria parasites. Biochemistry 19:1543-1549. 5. Ciak, J., and F. Hahn. 1966. Chloroquine: mode of action. Science 151:347-349. 6. Cohen, S. N., and K. L. Yielding. 1965. Inhibition of DNA and RNA polymerase reactions by chloroquine. Proc. Natl. Acad. Sci. USA 54:521-527. 7. Creek, K. E., and W. S. Sly. 1984. The role of the phosphomannosyl receptor in the transport of acid hydrolases to lysosomes, p. 63-82. In J. T. Dingle, R. T. Dean, and W. S. Sly (ed.), Lysosomes in biology and pathology, vol. 7. Elsevier Biomedical Press, Amsterdam. 8. de Duve, C. 1963. General properties of lysosomes: the lysosome concept, p. 1-31. In M. P. Cameron, A. V. S. de Rueck, and C. de Duve (ed.), Lysosomes, CIBA Foundation Symposium. Little, Brown, & Co., Boston. 9. de Duve, C., T. de Barsy, B. Poole, A. Trouet, P. Tulkens, and F. van Hoof. 1974. Lysosomotropic agents. Biochem. Pharmacol. 24:2495-2531. 10. de Duve, C., and R. Wattiaux. 1966. Functions of lysosomes. Annu. Rev. Physiol. 28:435-492. 11. Divo, A. A., T. G. Geary, and J. B. Jensen. 1985. Oxygen- and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture. Antimicrob. Agents Chemother. 27:21-27. 12. Farquhar, M. G., and G. E. Palade. 1981. The Golgi apparatus (complex)-(1954-1981)-from artifact to center stage. J. Cell Biol. 91(Suppl.):77S-103S. 13. Field, J. W. 1949. Blood examination and prognosis in acute falciparum malaria. Trans. R. Soc. Trop. Med. Hyg. 43:33-56. 14. Fitch, C. D. 1983. Mode of action of antimalarial drugs. CIBA Found. Symp. 94:222-232. 15. Fitch, C. D., R. Chevii, H. S. Banyal, G. Phillips, M. A. Pfaller, and D. J. Krogstad. 1982. Lysis of Plasmodium falciparum by ferriprotoporphyrin IX and a chloroquine-ferriprotoporphyrin IX complex. Antimicrob. Agents Chemother. 21:819-822. 16. Forgac, M., L. Cantley, B. Wiedenmann, L. Altstiel, and D. Branton. 1983. Clathrin-coated vesicles contain an ATP-dependent proton pump. Proc. Natl. Acad. Sci. USA 80:1300-1303. 17. Geary, T. G., A. A. Divo, L. C. Bonanni, and J. B. Jensen. 1985. Nutritional requirements of Plasmodium falciparum in culture: III. Further observations on essential nutrients and antimetabolites. J. Protozool. 32:608-613. 18. Geary, T. G., A. A. Divo, N. L. Davis, and J. B. Jensen. 1985. Nutritional requirements of Plasmodium falciparum in culture: II. Effects of antimetabolites in a semidefined minimal culture medium. J. Protozool. 32:65-69. 19. Geary, T. G., A. A. Divo, and J. B. Jensen. 1986. Effect of calmodulin inhibitors on viability and mitochondrial potential of Plasmodiumfalciparum in culture. Antimicrob. Agents Chemother. 30:785-788. 20. Geary, T. G., and J. B. Jensen. 1983. Lack of cross-resistance to 4-aminoquinolines in chloroquine-resistant Plasmodiumfalciparum in vitro. J. Parasitol. 69:97-105. 21. Geary, T. G., and J. B. Jensen. 1983. Effects of antibiotics on Plasmodium falciparum in vitro. Am. J. Trop. Med. Hyg. 32: 221-225. 22. Ginsburg, H., A. A. Divo, T. G. Geary, M. T. Boland, and J. B. Jensen. 1986. Effects of mitochondrial inhibitors on intraerythrocytic Plasmodium falciparum in in vitro cultures. J. Protozool. 33:121-125. 23. Ginsburg, H., and T. G. Geary. 1987. Current concepts and new ideas on the mechanism of action of quinoline-containing antimalarials. Biochem. Pharmacol. 36:1567-1576. 24. Gluzman, I. Y., P. H. Schlesinger, and D. J. Krogstad. 1987. Inoculum effect with chloroquine and Plasmodium falciparum. Antimicrob. Agents Chemother. 31:32-36. 25. Griffiths, G., and K. Simons. 1986. The trans Golgi network: sorting at the exit site of the Golgi complex. Science 234:

438-443. 26. Gyang, F. N., B. Poole, and W. Trager. 1982. Peptidases from Plasmodium falciparum cultured in vitro. Mol. Biochem. Parasitol. 5:263-273. 27. Hasilik, A., and K. von Figura. 1984. Processing of lysosomal enzymes in fibroblasts, p. 3-16. In J. T. Dingle, R. T. Dean, and W. S. Sly (ed.), Lysosomes in biology and pathology, vol. 7. Elsevier Biomedical Press, Amsterdam. 28. Homewood, C. A., D. C. Warhurst, W. Peters, and V. C. Baggaley. 1972. Lysosomes, pH and the antimalarial action of chloroquine. Nature (London) 235:50-52. 29. Jacobs, G. H., M. Aikawa, W. K. Milhous, and J. R. Rabbege. 1987. An ultrastructural study of the effects of mefloquine on Plasmodium falciparum. Am. J. Trop. Med. Hyg. 36:9-14. 30. Jones, W. H. S. 1909. Malaria and Greek history. University Press, Manchester, United Kingdom. 31. Kent, C. 1982. Inhibition of myoblast fusion by lysosomotropic amines. Dev. Biol. 90:91-98. 32. Krogstad, D. J., and M. A. Pfaller. 1984. Chemotherapy of malaria, p. 187-191. In L. Leive and D. Schlessinger (ed.), Microbiology-1984. American Society for Microbiology, Washington, D.C. 33. Krogstad, D. J., and P. H. Schlesinger. 1986. A perspective on antimalarial action: effects of weak bases on Plasmodium falciparum. Biochem. Pharmacol. 35:547-552. 34. Krogstad, D. J., and P. H. Schlesinger. 1987. The basis of antimalarial action: non-weak base effects of chloroquine on vesicle pH. Am. J. Trop. Med. Hyg. 36:213-220. 35. Krogstad, D. J., and P. H. Schlesinger. 1987. Acid vesicle function, intracellular pathogens and the action of chloroquine against Plasmodiumfalciparum. N. Engl. J. Med. 317:542-549. 36. Krogstad, D. J., P. H. Schlesinger, and I. Y. Gluzman. 1985. Antimalarials increase vesicle pH in Plasmodiumfalciparum. J. Cell Biol. 101:2302-2309. 37. Krogstad, D. J., P. H. Schlesinger, and B. L. Herwaldt. 1988. Antimalarial agents: mechanism of chloroquine resistance. Antimicrob. Agents Chemother. 32:799-801. 38. Krotoski, W. A., W. E. Collins, R. S. Bray, P. C. C. Garnham, F. B. Cogswell, R. W. Gwadz, R. Killick-Kendrick, R. Wolf, R. Sinden, L. C. Koontz, and P. S. Stanffil. 1982. Demonstration of hypnozoites in sporozoite transmitted Plasmodium vivax infection. Am. J. Trop. Med. Hyg. 31:1291-1293. 39. Krotoski, W. A., D. M. Krotoski, P. C. C. Garnham, R. S. Bray, R. Killick-Kendrick, C. C. Draper, G. A. T. Targett, and M. W. Guy. 1980. Relapses in primate malaria: discovery of two populations of exoerythrocytic stages. Preliminary note. Br. Med. J. 280:153-154. 40. Krugliak, M., Z. Waldman, and H. Ginsburg. 1987. Gentamicin and amikacin repress the growth of Plasmodium falciparum in culture, probably by inhibiting a parasite acid phospholipase. Life Sci. 40:1253-1257. 41. Lot, T. Y., and T. Bennett. 1982. The effects of chronic chloroquine administration in growing chicks. Med. Biol.

60:210-216.
42. Milhous, W. K., N. F. Weatherly, J. H. Bowdre, and R. E. Desjardins. 1985. In vitro activities of and mechanisms of resistance to antifol antimalarial drugs. Antimicrob. Agents Chemother. 27:525-530. 43. Neva, F. A. 1977. Looking back for a view of the future: observations on immunity to induced malaria. Am. J. Trop. Med. Hyg. 26(Suppl):211-215. 44. Nickell, S. P., L. W. Scheibel, and G. A. Cole. 1982. Inhibition by cyclosporin A of rodent malaria in vivo and human malaria in vitro. Infect. Immun. 37:1093-1100. 45. O'Brien, R. L., J. G. Olenick, and F. E. Hahn. 1966. Reactions of quinine, chloroquine, and quinacrine with DNA. Proc. Natl. Acad. Sci. USA 55:1511-1517. 46. Ohkuma, S., and B. Poole. 1978. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. USA 75:3327-3331. 47. Parker, F. S., and J. L. Irvin. 1952. The interaction of chloroquine with nucleic acids and nucleoproteins. J. Biol. Chem.

798

MINIREVIEWS

ANTIMICROB. AGENTS CHEMOTHER.

199:897-909. 48. Pfaller, M. A., and D. J. Krogstad. 1981. Imidazole and polyene activity against chloroquine-resistant Plasmodium falciparum. J. Infect. Dis. 144:372-375. 49. Phillips, R. E., D. A. Warrell, N. J. White, S. Looareesuwan, and J. Karbwang. 1985. Intravenous quinidine for the treatment of severe falciparum malaria: clinical and pharmacokinetic studies. N. Engl. J. Med. 312:1273-1278. 50. Poole, B., and S. Ohkuma. 1981. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J. Cell Biol. 90:665-669. 51. Roos, A., and W. F. Boron. 1981. Intracellular pH. Physiol. Rev. 61:296-434. 52. Roth, T. F., and K. R. Porter. 1964. Yolk protein uptake in the oocyte of the mosquito Aedes aegypti L. J. Cell Biol. 20:313-332. 53. Rothman, J. E. 1981. The Golgi apparatus: two organelles in tandem. Science 213:1212-1219. 54. Rothman, J. E., and R. E. Fine. 1980. Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages. Proc. Natl. Acad. Sci. USA 77:780-784. 55. Schwartz, A. L., and M. R. Hollingdale. 1985. Primaquine and lysosomotropic amines inhibit malaria sporozoite entry into human liver cells. Mol. Biochem. Parasitol. 14:305-311. 56. Seaberg, L. S., A. R. Parquette, I. Y. Gluzman, G. W. Phillips, Jr., T. F. Brodasky, and D. J. Krogstad. 1984. Clindamycin activity against chloroquine-resistant Plasmodium falciparum. J. Infect. Dis. 150:904-911. 57. Sly, W. S., and H. D. Fischer. 1982. The phosphomannosyl recognition system for intracellular transport of lysosomal enzymes. J. Cell. Biochem. 18:67-85. 58. Smrkovski, L. L., R. L. Buck, A. K. Alcantara, C. S. Rodriguez, and C. V. Uylangco. 1985. Studies of resistance to chloroquine, quinine, amodiaquine and mefloquine using Philippine strains of Plasmodium falciparum. Trans. R. Soc. Trop. Med. Hyg. 79: 37-41. 59. Stahl, P., and A. L. Schwartz. 1986. Receptor-mediated endocytosis. J. Clin. Invest. 77:657-662. 60. Stone, D. K., X.-S. Xie, and E. Racker. 1983. An ATP-driven proton pump in clathrin-coated vesicles. J. Biol. Chem. 258: 40594062. 61. Trager, W., M. Tershakovec, P. K. Chiang, and G. L. Cantoni. 1980. Plasmodiumfalciparum: antimalarial activity in culture of sinefungin and other methylation inhibitors. Exp. Parasitol. 50:83-89.

62. Tycko, B., and F. R. Maxfield. 1983. Rapid acidification of endocytic vesicles containing a2-macroglobulin. Cell 28: 643-651. 63. Vander Jagt, D. L., B. R. Baack, and L. A. Hunsaker. 1984. Purification and characterization of an aminopeptidase from Plasmodium falciparum. Mol. Biochem. Parasitol. 10:45-54. 64. Vander Jagt, D. L., L. A. Hunsaker, and N. M. Campos. 1986. Characterization of a hemoglobin-degrading, low molecular weight protease from Plasmodium falciparum. Mol. Biochem. Parasitol. 18:389-400. 65. Vander Jagt, D. L., L. A. Hunsaker, and N. M. Campos. 1987. Comparison of proteases from chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Biochem. Pharmacol. 36:3285-3292. 66. Warhurst, D. C. 1986. Antimalarial drugs: mode of action and resistance. J. Antimicrob. Chemother. 18(Suppl. B):51-59. 67. Webster, H. K., and J. M. Whaun. 1982. Antimalarial properties of bredinin: prediction based on identification of differences in human host-parasite purine metabolism. J. Clin. Invest. 70: 461-469. 68. White, N. J., S. Looareesuwan, D. A. Warrell, T. Chongsuphajaisiddhi, D. Bunnag, and T. Harinasuta. 1981. Quinidine in falciparum malaria. Lancet ii:1069-1071. 69. Willingham, M. C., and I. H. Pastan. 1980. The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts. Cell 21:67-77. 70. Wyler, D. J. 1983. Malaria-resurgence, resistance, akad research. N. Engl. J. Med. 308:875-878, 934-940. 71. Yayon, A., Z. I. Cabantchik, and H. Ginsburg. 1984. Identification of the acidic compartment of Plasmodium falciparuminfected human erythrocytes as the target of the antimalarial drug chloroquine. EMBO J. 3:2695-2700. 72. Yayon, A., Z. I. Cabantchik, and H. Ginsburg. 1985. Susceptibility of human malaria parasites to chloroquine is pH-dependent. Proc. Natl. Acad. Sci. USA 82:2784-2788. 73. Yayon, A., R. Timberg, S. Friedman, and H. Ginsburg. 1984. Effects of chloroquine on the feeding mechanism of the intraerythrocytic malarial parasite Plasmodium falciparum. J. Protozool. 31:367-372. 74. Yayon, A., J. A. Vande Waa, M. Yayon, T. G. Geary, and J. B. Jensen. 1983. Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro. J. Protozool. 30:642-647. 75. Zarchin, S., M. Krugliak, and H. Ginsburg. 1986. Host cell digestion by intraerythrocytic malarial parasites is the primary target for quinoline-containing antimalarials. Biochem. Pharmacol. 35:2435-2442.