New mechanisms of drug resistance in parasitic protozoa.

KEY WORDS: P-glycoproteins, drug transport, chemotherapeutic targets, parasitic diseases

CONTENTS

INTRODUCTION AN OVERVIEW OF DRUG RESISTANCE IN PARASITIC PROTOZOA P-GLYCOPROTEINS AND RELATED TRAFFIC ATPases

Mammalian P-Glycoproteins

P-Glycoproteins and Related Transport Proteins in Parasites RESISTANCE TO CHLOROQUINE, MEFLOQUINE, QUININE, AND HALOFANTRINE RESISTANCE TO ARSENICALS AND ANTIMONIALS

Resistance to Arsenicals in Trypanosomes

Resistance to Oxyanions in Leishmania spp

A Speculative Model for Oxyanion Resistance in Leishmania spp RESISTANCE TO ANTIFOLATES

Mutations in DHFR and DHPS

DHFR Overproduction

Transport Mutations in Leishmania spp

Alternative Pathway for Folate Synthesis in Leishmania spp MISCELLANEOUS RECENTLY DESCRIBED MECHANISMS OF DRUG RESISTANCE

Resistance to Purine Analogues

Resistance to Metronidazole

Resistance to Ornithine Decarboxylase Inhibitors

Resistance to Emetine

Resistance to Drugs Used in the Laboratory but Not in Patients So Far OUTLOOK

Resistance mechanisms highlighted in this review include:

1. Decrease of drug uptake because of the loss of a transporter required for uptake. This decrease contributes to resistance to arsenicals and diamidines in African trypanosomes.

2. The export of drugs from the parasite by P-glycoproteins and other traffic ATPases. This export could potentially be an important mechanism of resistance, as these proteins are richly represented in the few protozoa analyzed. There are indications that such transmembrane transporters can be involved in resistance to emetine in Entamoeba spp., to mefloquine in Plasmodium spp., and to antimonials in Leishmania spp.

3. The possible involvement of the P-glycoprotein encoded by the Plasmodium falciparum pfmdr1 gene in chloroquine resistance. We present the available data that lead to the conclusion that overproduction of the wildtype version of this protein results in chloroquine hypersensitivity rather than resistance.

4. The involvement of the PgpA P-glycoprotein of Leishmania spp. in low-level resistance to arsenite and antimonials. We raise the possibility that this protein transports glutathione conjugates of arsenite and antimonials rather than the compounds themselves.

5. Loss of drug activation as the main mechanism of metronidazole resistance in Trichomonas and Giardia spp. Recent evidence indicates that a decrease of the proximal cellular electron donor for metronidazole activation, ferredoxin, is the main cause of resistance in Trichomonas.

6. Resistance arising through alteration of drug targets. The amino acid substitutions in the dihydrofolate reductase-thymidylate synthase of Plasmodium spp. are good examples of this mechanism.

We show here that the field of drug resistance in parasitic protozoa is currently very active and holds considerable future opportunity. With the tools now available, progress should be rapid in the coming years.

INTRODUCTION

Parasitic protozoa are responsible for some of the most devastating and prevalent diseases of humans and domestic animals. Malaria (Plasmodium spp.), the various forms of (muco)cutaneous and visceral leishmaniasis (Leishmania spp.), African sleeping sickness (Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense), South-American Chagas' disease (Trypanosoma cruzi), amoebic dysentery (Entamoeba spp.), and toxoplasmosis (Toxoplasma spp.) are serious diseases that threaten the lives of nearly one quarter of the human population worldwide. In addition, the parasitic diseases caused by Trichomonas vaginalis (vaginitis, urethritis) and Giardia duodenalis (diarrhea) are very widespread and unpleasant, even though not life-threatening. Protozoal parasites also result in enormous losses of life and productivity of domesticated animals, both mammals and fowl.

Drugs and prevention are the two major weapons now available against protozoan parasites. There are high hopes that effective vaccines may eventually be available (171), but such vaccines have been slow in coming (30), and one sometimes wonders whether the vaccine aficionados are not underestimating the ability of protozoa to elude the mammalian immune system. Most protozoa! diseases are chronic and occur in immunocompetent patients. Is the competence of immunologists really sufficient to boost an immune system that fails to deal with the invader in a natural fashion? We hope the answer is yes, but for the moment drugs are the mainstay in cases when preventive measures fail or prove impractical.

Even though protozoa are eukaryotes and usually contain many of the organelles and metabolic pathways of their hosts, the differences in biochemistry between parasite and host are great enough to leave a large window for the development of parasite-specific drugs. It is not always appreciated that the protozoa! parasite differs much more from a human cell than from cells of fungi or plants. On an evolutionary scale deduced from differences in small-subunit ribosomal RNAs (58, 167), protozoa such as Giardia lamblia and Trypanosoma brucei are nearly as similar to Escherichia cold as to humans. Hence, it is not surprising that fairly effective drugs are available for many parasitic diseases. It is even embarrassing that there are still major parasites, such as the American trypanosomes, that cannot be safely and effectively tackled by chemotherapy. Had the same amount of effort and money been invested in the development of antiprotozoal drugs as in the refinement of anticancer drugs, chemotherapy of protozoa! diseases would be in a much better position today. It should be easier to develop drugs against trypanosomatids with their exotic biochemistry than against cancer cells, which differ in less than 0.1% of their gene products from normal cells.

As it stands, however, the arsenal of antiprotozoal drugs is limited, and the effectiveness of these drugs is being eroded by drug resistance. For example, there is widespread resistance to some of the most effective drugs ever developed, chloroquine in malaria and metronidazole in anaerobic parasites. With effective vaccines not yet in sight and development of new drugs proceeding slowly, the rising tide of drug resistance is menacing the frail dikes of disease management of protozoa! infections.

Studies of resistance mechanisms cannot reverse the tide, but can help to handle the threat more rationally at three levels:

1. by developing tools to recognize resistance early in infection and prevent the loss of time with useless (and often toxic) chemotherapy

2. by pointing the way to more rational use of drugs and drug combinations to minimize development of resistance

3. by pinpointing intracellular drug targets and parasite defense mechanisms allowing the rational development of drug analogues not affected by the most common defenses.

Most of the studies on drug resistance in protozoa have been done with laboratory strains under conditions that do not mimic the normal parasite-host relation. Moreover, most of the studies have been correlative, i.e. the drug-resistance mechanisms have been studied by determining alterations in drug handling or in cellular metabolism of resistant mutants. If an alteration is consistently found in multiple mutants, if it is lost in revertants, and if it provides a plausible mechanism for resistance, then the resistance mechanism is usually thought to be solved. This may be the case, but there are pitfalls in this approach (20). Therefore it is important to reconstruct the resistance mechanism by transforming wild-type cells with the relevant genes. For gain-of-function mutants, such a reconstruction may only require the introduction of additional copies of suitably altered genes. For loss-of-function mutants, the reconstruction may require the directed disruption of endogenous genes. Only if the complete resistance phenotype can be reconstructed in sensitive cells by such directed genetic modifications can one be sure that the resistance mechanism has been solved. This reconstruction requires a convenient procedure for stable transformation, which is now available for a rapidly increasing number of parasitic protozoa. After Bellofatto & Cross (15) and Laban & Wirth (108) showed that electroporation can be used to obtain transient transformation of Leptomonas seymouri and Leishmania enriettii, respectively, this procedure soon resulted in stable transformation of Leishmania spp. (40, 41, 97, 107), T. brucei (49, 50, 111, 172), and L. seymouri (16). More recently, stable transformation was obtained in T. cruzi (35, 79, 100, 131), Toxoplasma gondii (48, 102), and Plasmodium berghei (MR Van Dijck, AP Waters & CJ Jansen, personal communication), and transient transformation was obtained in Entamoeba histolytica (126, 152). In most protozoa studied thus far, exogenous DNA preferentially integrates into host DNA by homologous recombination. Gene disruption experiments, so laborious in mammals, are therefore relatively simple in these protozoa. With the tools in hand it should be possible to reconstruct resistance mechanisms by transformation.

In this review we concentrate on some of the newer mechanisms of resistance that have emerged in the past five years. We indicate those proposed mechanisms that have been proven by transfection, and we emphasize the rare cases in which solid information is available for resistance mechanisms in field isolates. As this review is selective, we refer the reader to other recent overviews for more complete reviews of specific drugs or parasites. A concise overview of modern parasitology with a clear description of the major parasitic protozoa and their metabolic peculiarities that can be targeted by drugs can be found in Cox (39). A special issue of Science (June 24, 1994) devoted to parasitology also highlights the impact of parasites on developing countries, the (lack of) funding for parasite control and research, and the cost to society of unchecked parasites. Special issues of Parasitology Today (May 1993) and Acta Tropica (March 1994) address drug resistance.

AN OVERVIEW OF DRUG RESISTANCE IN PARASITIC PROTOZOA

To interfere with parasite multiplication, a drug must find the parasite (often in a host cell) and reach its target within the parasite. Usually me drug must pass the parasite membrane, and often it must be activated inside. After the drug hits the target, the parasite must be sufficiently incapacitated to be killed by me host defense or to die spontaneously. Each of these steps provides the parasite with opportunities to interfere with drug action, resulting in drug resistance Resistance mechanisms are most easily studied in parasites made resistant in the laboratory. One can then work with genetically defined, cloned populations, and by a detailed comparison of resistant mutants and the parental strain from which they were derived, one can usually find the gene(s) involved in resistance.

Resistance in clinical samples is less easily defined in biochemical terms. Parasite populations are often heterogeneous; the exact parental strain is often not available for comparison, and the culturing of the parasites--often necessary to get sufficient material for analysis--may change resistance. Nevertheless, it should now be possible to verify whether resistance mechanisms defined in the laboratory play a role in the field, as the polymerase chain reaction and monoclonal antibodies allow me study of genes and proteins in a few cells. Studying resistance mechanisms induced under laboratory conditions has been fruitful in the study of clinically relevant forms of drug resistance in cancer (20), and we are optimistic that the same approach will also allow a precise molecular characterization of clinically relevant forms of resistance in protozoal diseases, even if we are still far from that ideal today.

The main biochemical mechanisms responsible for drug resistance are illustrated in Figure 1. Parasites may evade drug action by hiding in sanctuaries such as me brain (e.g. many drugs do not pass the blood-brain barrier); drug uptake may be thwarted by loss of uptake systems or alteration of membrane composition; once inside, drugs may be inactivated, excreted, modified and excreted, or routed into vacuoles (the latter mechanism is still speculative); drug activation mechanisms may be suppressed or lost; the interaction of drug with target may be made less effective by increasing the level of competing substrates or by altering the target to make it less sensitive to me drug; the parasite may learn to live with a blocked target by circumventing the block; and finally the parasite may become more proficient in repairing drug damage. Concrete examples that illustrate the scheme in Figure 1 are discussed in the relevant sections of this review.

[Figure 1 ILLUSTRATION OMITTED]

P-GLYCOPROTEINS AND RELATED TRAFFIC ATPases

Mammalian P-Glycoproteins

The importance of P-glycoproteins (Paps) and related transmembrane transporters for drug resistance in organisms ranging from bacteria to human tumor cells has been amply demonstrated since this class of proteins was discovered by Juliano & Ling (96) in multidrug-resistant hamster tumor cells. Pgps belong to the family of Adenine nucleotide Binding Cassette (ABC) transporters (84), also known as traffic ATPases (47). A detailed discussion of the structure and function of these transporters and their possible role in drug resistance can be found in recent reviews (21,31,47, 72, 84, 123, 127, 136, 158,161, 176). The classical Pgps responsible for multidrug resistance (MDR) in mammalian cancer cells are large plasma membrane glycoproteins consisting of two similar halves, each containing six putative transmembrane segments and an ATP-binding site. Drug resistance is caused by the ability of Pgps to extrude drugs against a concentration gradient, resulting in a decrease of the intracellular drug concentration in contact with the drug target.

An interesting new member of the traffic ATPase family is the MDR-associated protein MRP, the second type of mammalian transporter involved in MDR (34, 73). Similar to Pgp, this protein is predominantly present in the plasma membrane and causes drug resistance by lowering the intracellular drug concentration by drug extrusion (195). MRP is not just another member of the Pgp family, however. The sequence identity between human MDR1 Pgp and MRP is only 23%, and MRP seems to have a more asymmetric structure than Pgp (34). Recent experiments indicate that there may also be fundamental differences between MDR1 Pgp and MRP in substrate specificity, even though the two proteins confer resistance to a similar spectrum of natural product drugs. These experiments show that an increase in cellular MRP is associated with an increase in the activity of the elusive GS-X pump (91, 114, 124). This pump, also known as the leukotriene [C.sub.4] ([LTC.sub.4]) transporter (114) or the multispecific anion transporter (132), is known to transport complex molecules with a hydrophobic organic …