Книги фарма 2 / Bertram G. Katzung-Basic & Clinical Pharmacology(9th Edition)

will permit selective inhibition in the parasite and not in the host. One might expect that the parasite would have many deficiencies in its metabolism associated with its parasitic nature. This is true of many parasites—the oversimplified metabolic pathways are usually indispensable for survival of the parasite and thus represent points of vulnerability. However, oversimplified metabolic pathways are not the only opportunity for attack. Although the parasite lives in a metabolically luxurious environment and may become "lazy," the environment is not entirely friendly and the parasite must have defense mechanisms in order to survive—ie, to defend itself against immunologic attack, proteolytic digestion, etc, by the host. In some instances, necessary nutrients are not supplied to the parasite from the host, though the latter can obtain the same nutrients from the diet. In this situation, the parasite will have acquired the synthetic activity needed for its survival. Finally, the great evolutionary distance between host and parasite has in some cases resulted in sufficient differences among individual enzymes or functional pathways to allow selective inhibition of the parasite. Thus, there can be three major types of potential targets for chemotherapy of parasitic diseases: (1) unique essential enzymes found only in the parasite; (2) similar enzymes found in both host and parasite but indispensable only for the parasite; and (3) common biochemical functions found in both parasite and host but with different pharmacologic properties. Examples of specific targets and drugs that act on them are summarized in Table 52–1. This chapter discusses antiparasitic mechanisms based upon these examples and provides background information for the drugs described in Chapter 53: Antiprotozoal Drugs and Chapter 54: Clinical Pharmacology of the Anthelmintic Drugs.

Table 52–1. Identified Targets for Chemotherapy in Parasites.

Katzung PHARMACOLOGY, 9e > Section VIII. Chemotherapeutic Drugs > Chapter 52. Basic Principles of Antiparasitic Chemotherapy >

Essential Enzymes Found Only in Parasites

These enzymes would appear to be the cleanest targets for chemotherapy. Like enzymes involved in the synthesis of bacterial cell walls (see Chapter 43: Beta-Lactam Antibiotics & Other Inhibitors of Cell Wall Synthesis), inhibition of these enzymes should have no effect on the host. Unfortunately, only a few of these enzymes have been discovered among the parasitic protozoa. Furthermore, their usefulness as chemotherapeutic targets is sometimes limited because of the development of drug resistance. Examples of important target enzymes in this category are discussed in the following pages.

Enzymes for Dihydropteroate Synthesis

Intracellular protozoa of the phylum Apicomplexa such as plasmodium, toxoplasma, and eimeria have long been known to respond to sulfonamides and sulfones. This has led to the assumption that Apicomplexa must synthesize their own folate in order to survive. The reaction of 2-amino-4- hydroxy-6-hydroxymethyl-dihydropteridine diphosphate with p-aminobenzoate to form 7,8- dihydropteroate has been demonstrated in cell-free extracts of the human malaria parasite Plasmodium falciparum. 2-Amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophosphokinase and 7,8-dihydropteroate synthase have also been identified. Sulfathiazole, sulfaguanidine, and sulfanilamide act as competitive inhibitors of p-aminobenzoate. It has not been possible to demonstrate dihydrofolate synthase activity in the parasites, which raises the possibility that 7,8-dihydropteroate may have substituted for dihydrofolate in malaria parasites. Similar lack of recognition of folate as substrate was also observed in the dihydrofolate reductase of Eimeria tenella, a parasite of chickens.

However, lack of utilization of exogenous folate may not fully explain the apparently indispensable nature of the synthesis of 7,8-dihydropteroate in plasmodium, toxoplasma, and eimeria. It is known that most of the folate molecules in mammalian cells are linked with polyglutamates in the cytoplasm and are transported across cell membranes with difficulty. It may compound the problem of obtaining 7,8-dihydropteroate or dihydrofolate for the parasite and makes all of the enzymes

involved in their synthesis attractive targets for anti-Apicocomplexa chemotherapy.

The gene encoding 7,8-dihydropteroate synthase was cloned from P falciparum and found to encode a bifunctional enzyme that includes the pyrophosphokinase at the amino terminal of the protein. Discrepancies were observed in the sequences of 7,8-dihydropteroate synthase portion of the genes from sulfadoxine-sensitive versus sulfadoxine-resistant P falciparum, thus confirming that this enzyme is the target for the antimalarial sulfonamide drugs.

The sulfones and sulfonamides synergize with the inhibitors of dihydrofolate reductase, and the combinations have been effective in controlling malaria, toxoplasmosis, and coccidiosis. Fansidar, a combination of sulfadoxine and pyrimethamine, has been successful in controlling some strains of chloroquine-resistant Plasmodium falciparum malaria (see Chapter 53: Antiprotozoal Drugs). However, reports of Fansidar resistance have increased in recent years. New inhibitors effective against the sulfonamide-resistant 7,8-dihydropteroate synthase are needed.

The pharmacologic properties of parasite 7,8-dihydropteroate synthases may differ from those of the bacterial enzymes. For instance, metachloridine and 2-ethoxy-p-aminobenzoate are both ineffective against sulfonamide-sensitive bacteria, but the former has antimalarial activity and the latter is effective against infection by the chicken parasite Eimeria acervulina; both activities can be reversed by p-aminobenzoate.

Pyruvate:Ferredoxin Oxidoreductase

Certain anaerobic protozoal parasites lack mitochondria and mitochondrial activities for generating ATP and disposing of electrons. They possess, instead, ferredoxin-like or flavodoxin-like low- redox-potential electron transport proteins. In trichomonad flagellates and rumen ciliates, the process takes place in a membrane-limited organelle called the hydrogenosome. By the actions of pyruvate:ferredoxin oxidoreductase (PFOR) and hydrogenase in the hydrogenosome, H2 is produced by these organisms under anaerobic conditions as the major means of electron disposition. Although entamoeba species and Giardia lamblia have no hydrogenosome, a ferredoxin has been isolated from Entamoeba histolytica, and the genes encoding ferredoxin and pyruvate:ferredoxin oxidoreductase have been isolated from E histolytica and G lamblia.

Pyruvate:ferredoxin oxidoreductase has no counterpart in mammalian systems. In contrast to the mammalian pyruvate dehydrogenase complex, pyruvate: ferredoxin oxidoreductase is incapable of reducing pyridine nucleotides because of its low redox potential (approximately –400 mV). However, this low potential can also transfer electrons from pyruvate to the nitro groups of metronidazole and other 5-nitroimidazole derivatives to form cytotoxic reduced products that bind to DNA and proteins. This is apparently why anaerobic protozoal species are highly susceptible to drugs such as metronidazole. Despite the recent development of drug resistance in some Trichomonas vaginalis strains and the possibility of carcinogenic properties (see Chapter 53: Antiprotozoal Drugs), metronidazole remains the drug of choice for controlling anaerobic protozoal parasite infections. Nitazoxanide, an agent recently approved for use against G lamblia and Cryptosporidium parvum, apparently acts through an active metabolite, tizoxanide, which inhibits the pyruvate:ferredoxin oxidoreductase pathway.

Though pyruvate:ferredoxin oxidoreductase activates the prodrug metronidazole, it may not perform an essential function in the anaerobic parasites. The enzyme activity was significantly lower in metronidazole-resistant G lamblia. Some aerobic metronidazole-resistant T vaginalis strains have no detectable pyruvate:ferredoxin oxidoreductase activity but are viable. A knockdown of expression of this enzyme in G lamblia by ribozyme-antisense chimeric RNA resulted in

facilitated cell growth under aerobic condition and metronidazole resistance.

Pyruvate Phosphate Dikinase

Glycolysis provides the main source of ATP in Trypanosoma brucei, E histolytica, and G lamblia, which possess pyruvate kinase as well as a pyruvate phosphate dikinase for converting phosphoenolpyruvate (PEP) to pyruvate and generating ATP. Pyruvate phosphate dikinase is not a homolog of pyruvate kinase but is closely related to PEP synthase from bacteria. The enzyme catalyzes conversion of PEP to pyruvate accompanied by the synthesis of ATP from AMP and pyrophosphate. Genes encoding the enzyme have been isolated from E histolytica and G lamblia and have demonstrated considerable structural divergences. No specific inhibitor of this enzyme has yet been identified.

Nucleoside Phosphotransferases

All the protozoal parasites studied thus far are deficient in de novo synthesis of purine nucleotides. The various purine salvage pathways in these parasites are thus essential for their survival and growth. Among the leishmania species, a unique salvage enzyme has been identified—purine nucleoside phosphotransferase—that can transfer the phosphate group from a variety of monophosphate esters, including p-nitrophenylphosphate, to the 5' position of purine nucleosides. This enzyme also phosphorylates purine nucleoside analogs such as allopurinol riboside, formycin B, 9-deazainosine, and thiopurinol riboside, converting them to the corresponding nucleotides. These nucleotides are either further converted to triphosphates and eventually incorporated into nucleic acid of leishmania or become inhibitors of other essential enzymes in purine metabolism. Consequently, allopurinol riboside, formycin B, 9-deazainosine, and thiopurinol riboside all act as antileishmanial agents both in vitro and in vivo. Allopurinol riboside is particularly interesting because it is remarkably nontoxic to the mammalian host.

The mechanism of action of these purine nucleoside analogs is thus not by blocking purine salvage in leishmania because there are many other major purine salvage enzymes in the organism that remain unaffected. The nucleoside transferase is a minor enzyme, but it recognizes a number of false substrates and converts them to products toxic to leishmania. This approach of identifying "subversive substrates" for a purine salvage enzyme as potential antiparasitic targets does not depend on the lack of de novo purine nucleotide synthesis in these parasites. It could be applied—in any parasite—to any enzyme that happens to recognize a subversive substrate.

Trichomonad flagellates appear to be deficient in de novo synthesis of both purines and pyrimidines. Their salvage thus becomes indispensable for these parasites. In T vaginalis, for instance, the supply route for thymidine 5'-monophosphate (TMP) is provided by a single salvage pathway that converts exogenous thymidine to TMP by the action of a thymidine phosphotransferase. The enzyme activity is not affected by thymidine kinase inhibitors such as acyclovir but is inhibitable by guanosine or 5-fluorodeoxyuridine; both compounds also inhibit the in vitro growth of the parasites. This enzyme is an attractive target for chemotherapeutic treatment of the anaerobic protozoal parasites.

Purine Nucleoside Kinase

Exogenous adenosine is the precursor of the entire purine nucleotide pool in T vaginalis through its partial conversion to inosine and the action of purine nucleoside kinase, a unique enzyme in the organism, which converts adenosine and inosine to the corresponding nucleotides. It performs a critical role in T vaginalis purine salvage and has a unique substrate specificity suitable as a target

of chemotherapy.

Trypanothione Synthase, Reductase, & Peroxidase

Protozoa with kinetoplasts are unusual in that a considerable proportion of their intracellular spermidine and glutathione is found in the unique conjugate N1-N8-(glutathionyl)spermidine, which has been assigned the name trypanothione. Trypanothione synthase, reductase, and peroxidase activities have been detected in these parasites. A knockout of the gene encoding trypanothione reductase from the African trypanosome Trypanosoma brucei resulted in apparent cessation of growth of the organism. Nifurtimox, a nitrofuran derivative effective in treating Chagas' disease (caused by Trypanosoma cruzi), has been found to be a potent inhibitor of trypanothione reductase, and other inhibitors are under study. No extensive studies of trypanothione synthetase or peroxidase have been performed. The antitrypanosomal trivalent arsenicals are taken up by the African trypanosome Trypanosoma brucei and complex with trypanothione, forming a product that is also an effective inhibitor of trypanothione reductase.

Glycolipid Synthetic Enzymes

The variant surface glycoprotein on the plasma membranes of bloodstream African trypanosomes provides the organisms with the means of evading host immune responses. The glycoprotein is anchored to the cell surface by a glycosyl phosphatidylinositol that contains myristate as its only fatty acid component. Thus, the introduction of a subversive substrate to replace myristate from the glycolipid anchor could result in loss of the variant surface glycoprotein, which might suppress development of trypanosomes in mammalian blood. A myristate analog, 10-(propoxy)decanoic acid, was found incorporated into the glycolipid and also active against T brucei in in vitro tests. However, further validation of this approach to antitrypanosomal chemotherapy must await results of in vivo tests.

Shikimate Pathway Enzymes

The availability of P falciparum genome database led to the identification of shikimate pathway enzymes in this organism. The pathway is known to exist in bacteria, fungi, algae, and plants but not in mammals. A herbicide, glyphosate, known to inhibit the enzyme 5'-enolpyruvylshikimate 3- phosphate synthase, was found to inhibit growth of P falciparum. However, it is not known if the in vitro antimalarial action of glyphosate is by inhibiting this enzyme. Furthermore, the shikimate pathway leads to biosynthesis of aromatic amino acids. Since P falciparum grows on digested hemoglobin, it is not clear if biosynthesis of aromatic amino acids plays an essential role in this organism.

Isoretinoid Biosynthetic Enzymes

A mevalonate-independent isoprenoid biosynthetic pathway occurring only among bacteria, algae, and plants was also identified in P falciparum and T gondii.Fosmidomycin, known to inhibit 1- deoxy-D-xylulose-5-phosphate isomerase in this pathway, was found to also inhibit in vitro growth of P falciparum and to cure P vinckei infection in mice. However, the same questions about whether the pathway plays an indispensable role in this parasitic organism and whether fosmidomycin inhibits the parasites by inhibiting the particular enzyme remain to be answered.

Katzung PHARMACOLOGY, 9e > Section VIII. Chemotherapeutic Drugs > Chapter 52. Basic Principles of Antiparasitic Chemotherapy >

Enzymes Indispensable Only in Parasites

Because of the many metabolic deficiencies among parasites resulting from the unique environments in which parasites live in their hosts, there are enzymes whose functions may be essential for the survival of the parasites, but the same enzymes are not indispensable to the host— ie, the host may be able to survive the complete loss of these enzyme functions by achieving the same result through alternative pathways. This discrepancy opens up opportunities for antiparasitic chemotherapy, though insufficiently selective inhibition of parasite enzymes remains an important safety concern.

Lanosterol C-14 Demethylase

T cruzi and leishmania contain ergosterol as the principal sterol in plasma membranes. The azole antifungal agents (eg, ketoconazole, miconazole, itraconazole), which are known to act by inhibiting the cytochrome P450-dependent C-14 demethylation of lanosterol in the ergosterol biosynthetic pathway, also inhibit growth of T cruzi and leishmania by blocking C-14 demethylation of lanosterol in these parasites. Recently, an antifungal bistriazole, D0870, demonstrated encouraging in vivo anti-T cruzi activity in mouse infection models. It is thus likely that lanosterol C-14 demethylase plays an essential role in ergosterol synthesis and therefore qualifies as a target for chemotherapy against T cruzi and leishmania.

The same C-14 demethylation of lanosterol is also required for cholesterol synthesis in mammals. As rather nonselective inhibitors of lanosterol C-14 demethylases, the azoles may exert a variety of endocrine side effects by inhibiting this enzyme in the adrenal glands and gonads while remaining acceptable as systemic antifungal agents. Because of its excessive toxicity, D0870 was not developed as an antifungal or antiparasitic agent. However, since human and yeast lanosterol C- 14 demethylases share only 38–42% sequence identities, there may be a good chance of designing inhibitors that are more selective against the fungal or parasitic enzymes.

Purine Phosphoribosyl Transferases

The absence of de novo purine nucleotide synthesis in protozoal parasites as well as in the trematode Schistosoma mansoni is reflected in the relative importance of purine phosphoribosyl transferases in many parasite species.

G lamblia has an exceedingly simple scheme of purine salvage. It possesses only two pivotal enzymes: the adenine and guanine phosphoribosyl transferases, which convert exogenous adenine and guanine to the corresponding nucleotides. There is no salvage of hypoxanthine, xanthine, or any purine nucleosides and no interconversion between adenine and guanine nucleotides in the parasite. Functions of the two phosphoribosyl transferases are thus both essential for the survival and development of G lamblia (Figure 52–1). The guanine phosphoribosyl transferase is an interesting enzyme because it does not recognize hypoxanthine, xanthine, or adenine as substrate. This substrate specificity distinguishes the giardia enzyme from the mammalian enzyme, which uses hypoxanthine, and the bacterial one, which uses xanthine as substrate. Design of a highly specific inhibitor of this enzyme is thus possible. The crystal structures of both guanine and adenine phosphoribosyltransferases from G lamblia have been solved recently, which should provide good opportunities for specific inhibitor design.

Figure 52–1.

Katzung PHARMACOLOGY, 9e > Section VIII. Chemotherapeutic Drugs > Chapter 52. Basic Principles of Antiparasitic Chemotherapy >

Indispensable Biochemical Functions Found in Both Parasite & Host But with Different Pharmacologic Properties

In the parasite, these functions have differentiated sufficiently to become probable targets for antiparasitic chemotherapy, not because of the parasitic nature of the organism or its unique environment but, more likely, because of the long evolutionary distances separating the parasite and the host. It is thus difficult to discover these targets through studying metabolic deficiency or special nutritional requirements of the parasite. They have usually been found by investigating the modes of action of some well-established antiparasitic agents discovered by screening methods in the past. More recently, comparison of genome databases between the host and the parasite has become practical. The target may not be a single well-defined enzyme but may include transporters, receptors, cellular structural components, or other specific functions essential for survival of the parasite.

Dihydrofolate Reductase-Thymidylate Synthase Bifunctional Enzyme

Dihydrofolate reductase (DHFR), a classic target in antimicrobial and anticancer chemotherapy, has been shown to be a useful therapeutic target in plasmodium, toxoplasma, and eimeria species. Pyrimethamine is the prototypical DHFR inhibitor, exerting inhibitory effects in all three groups. However, pyrimethamine resistance in P falciparum has become widespread in recent years. This is largely attributable to specific point mutations in P falciparum DHFR that have rendered the enzyme less susceptible to the inhibitor.

A highly unusual feature of DHFR in Apicomplexa and Kinetoplastida is its association with thymidylate synthase in the same protein. DHFR activity is always located at the amino terminal portion, while the thymidylate synthase activity resides in the carboxyl terminal. The two enzyme functions do not appear to be interdependent; eg, the DHFR portion of the P falciparum enzyme molecule was found to function normally in the absence of the thymidylate synthase portion. It is likely that since the protozoan parasites do not perform de novo synthesis of purine nucleotides, the primary function of the tetrahydrofolate produced by DHFR is to provide 5,10methylenetetrahydrofolate only for the thymidylate synthase-catalyzed reaction. Physical association of the two enzymes may improve efficiency of TMP synthesis. If an effective means of disrupting the coordination between the two activities can be developed, this bifunctional protein may qualify as a target for antiparasitic therapy.

Thiamin Transporter

Carbohydrate metabolism provides the main energy source in coccidia. Diets deficient in thiamin, riboflavin, or nicotinic acid—all cofactors in carbohydrate metabolism—result in suppression of parasitic infestation of chickens by E tenella and E acervulina. A thiamin analog, amprolium—1- [(4-amino-2-propyl-5-pyrimidinyl)-methyl]-2-picolinium chloride—has long been used as an effective anticoccidial agent in chickens and cattle with relatively low host toxicity. The antiparasitic activity of amprolium is reversible by thiamin and is recognized to involve inhibition of thiamin transport in the parasite. Unfortunately, amprolium has a rather narrow spectrum of antiparasitic activity; it has poor activity against toxoplasmosis, a closely related parasitic infection.

Mitochondrial Electron Transporter

Mitochondria of E tenella appear to lack cytochrome c and to contain cytochrome o—a cytochrome oxidase commonly found in the bacterial respiratory chain—as the terminal oxidase. Certain 4- hydroxyquinoline derivatives such as buquinolate, decoquinate, and methyl benzoquate that have long been known to be relatively nontoxic and effective anticoccidial agents have been found to act on the parasites by inhibiting mitochondrial respiration. Direct investigation on isolated intact E tenella mitochondria indicated that the 4-hydroxyquinolines have no effect on NADH oxidase or succinoxidase activity but that they are extremely potent inhibitors of NADHor succinate-induced mitochondrial respiration. On the other hand, the ascorbate-induced E tenella mitochondrial respiration was totally insusceptible to these 4-hydroxyquinolines. The block by the anticoccidial agents thus may be located between the oxidases and cytochrome b in the electron transport chain. A certain component at this location must be essential for mediating the electron transport and would appear to be highly sensitive to the 4-hydroxyquinolines. This component must be a very specific chemotherapeutic target in eimeria species, since the 4-hydroxyquinolines have no effect on chicken liver and mammalian mitochondrial respiration and no activity against any parasites other than eimeria.

Many 2-hydroxynaphthoquinones have demonstrated therapeutic activities against Apicomplexa. Parvaquinone and buparvaquinone have been developed for the treatment of theileriosis in cattle and other domestic animals. Atovaquone is an antimalarial drug and is also used in the treatment of Pneumocystis jiroveci and P carinii infections. The 2-hydroxynaphthoquinones are analogs of ubiquinone. The primary site of action of atovaquone in Plasmodium is the cytochrome bc1 complex, where an apparent drug-binding site is present in cytochrome b. In plasmodium, ubiquinone also plays an important role as an electron acceptor for dihydroorotate oxidase. Consequently, pyrimidine biosynthesis in plasmodium is also inhibited by atovaquone. This chemical compound has also been found to be active against T gondii cysts in the brains of infected mice.

A major problem in the use of the antiparasitic 4-hydroxyquinolones and 2- hydroxynaphthoquinones is resistance that can develop with extraordinarily high frequency. It may be attributable to their mitochondrial site of action, which is known to have a high frequency of mutation.

Microtubules

Microtubules are an important part of the cytoskeleton and the mitotic spindle and consist of - and -tubulin subunit proteins. Recent comparisons of - and -tubulins from several species of parasitic nematodes indicated the presence of multiple isoforms with varying isoelectric points. This variation is interesting not only in pointing out the evolutionary relations among eukaryotic cells but useful also in classifying the tubulins in parasites as potential targets for antiparasitic chemotherapy. A group of benzimidazole derivatives have long been established as highly effective anthelmintics. Mebendazole was among the first such anthelmintics found to act primarily by blocking transport of secretory granules and movement of other subcellular organelles in the parasitic nematode Ascaris lumbricoides. This inhibition coincides with the disappearance of cytoplasmic microtubules from the intestinal cells of the worm. The microtubular systems of the host cells are unaffected by the treatment. Mebendazole and fenbendazole, another anthelmintic benzimidazole, have also been shown to compete with colchicine binding to A lumbricoides embryonic tubulins with 250–400 times higher potencies than the binding competition to bovine brain tubulins. These differential binding affinities may explain the selective toxicity of benzimidazoles toward parasitic nematodes.

Synaptic Transmission