Advances in Mode of Action of Antimalarials and Resistance Mechanisms Part 1 of 2 Prof. David Warhurst

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Advances in Mode of Action of Antimalarials and Resistance Mechanisms Part 1 of 2 1 Life

malariae no relapse Sporogony Mid-gut Schizogony Blood 2 Blood schizont & developing

Plasma 3 Endoplasmic reticulum RBC Double membrane Compartments Nucleus Cytoplasm

1 Advances in Mode of Action of Antimalarials and Resistance Mechanisms Part 1 of 2 1 Life cycle stage as drug target Salivary gland Liver P. falciparum, P. malariae no relapse Sporogony Mid-gut Schizogony Blood 2 Blood schizont & developing gametocyte Malaria parasite in red blood cell 3 genomes: Nuclear Apicoplast Mitochondrial Plasma 3 Endoplasmic reticulum RBC Double membrane Compartments Nucleus Cytoplasm Mitochondrion Apicoplast Cytostome Digestive vacuole where hemoglobin is digested (Blood stages only) Heme is detoxified to malaria pigment (hemozoin) 1

4 Action of antiplasmodial drugs Blood schizontocides: life-saving drugs for an acute infection need to attack multiplying blood stages which are responsible for all symptoms and pathology; They are

hematin, which can cause irreversible membrane damage and parasite death; In the DV it is rapidly detoxified by dimerization to malaria pigment, hemozoin, non-enzymatically While heme will activate

2 4 Action of antiplasmodial drugs Blood schizontocides: life-saving drugs for an acute infection need to attack multiplying blood stages which are responsible for all symptoms and pathology; They are used in treatment and in suppressive prophylaxis Blood stages digest hemoglobin (HGB) in the digestive vacuole (DV) and this results in the release of iron porphyrin - heme, rapidly oxidized to hematin, which can cause irreversible membrane damage and parasite death; In the DV it is rapidly detoxified by dimerization to malaria pigment, hemozoin, non-enzymatically While heme will activate artemisinins (ARTs), to give toxic free radicals and adducts, hematin is the target for 4-aminoquinolines: chloroquine (CQ) amodiaquine desethyl metabolite (DAQ) and piperaquine (PIP) (see e.g. Warhurst et al., 2007) Probable target for arylamino alcohols quinine (QN), mefloquine (MQ), halofantrine (HF) and lumefantrine (LF), which bind to it to prevent dimerization Chloroquine base is trapped in acidic DV in both plasmodia and mammalian cells (e.g. CQ + 2H + = CQ2H + ) (e.g. de Duve et al. Biochem Pharmacol. 1974; 23: ) Fitch s group showed that the target in blood stages was toxic hematin released during HGB digestion; Binding CQ prevented its detoxification to malaria pigment (hemozoin) Biochemistry 1980; 19: Hemozoin 5 Targets other than DV Drug-targets except hematin are essential parasite enzymes; Drugs associated with these targets have more or less effect on all growing stages of the parasite Cytosol Antifolates (sulfonamides) Pyrimethamine Cycloguanil ?Artemisinins (PfATP6) RBC Apicoplast Azithromycin Doxycycline ther antibacterials 6 Mitochondrion Atovaquone/proguanil Primaquine metabolites (artemisinins?) 2

Cytoplasm has a microbial-type folate synthesis cycle; Sulfonamides and anti-folate drugs target sequential enzymes DHPS and DHFR to prevent the production of folinic acid co-factors essential for

Pteridine Dihydropteroate synthase: DHPS Thymidylate synthase TS Dihydrofolate reductase: DHFR 7 Tetrahydrofolate Resistance to sulfadoxine and pyrimethamine (S/P) Alteration of two parasite target

3 Cytoplasm has a microbial-type folate synthesis cycle; Sulfonamides and anti-folate drugs target sequential enzymes DHPS and DHFR to prevent the production of folinic acid co-factors essential for synthesis; Sulfadoxine (SDX) and pyrimethamine (PYR) synergise in S/P to attack blood stages and (in experimental animals at least) growing liver stages (treatment, causal prophylaxis, sporogony) Pteridine Dihydropteroate synthase: DHPS Thymidylate synthase TS Dihydrofolate reductase: DHFR 7 Tetrahydrofolate Resistance to sulfadoxine and pyrimethamine (S/P) Alteration of two parasite target enzymes by mutation Resistance to the most active drug in the combination, the diamino-pyrimidine pyrimethamine, mimicking substrate dihydrofolate, is developed through mutations in the gene for enzyme DHFR Where there is high level resistance to S/P in Africa we see 3 DHFR mutations (108N, 51I, 59R) and 2 in DHPS: 437G and 540E This type spread from SE Asia (see Roper et al., Science 2004); Dihydrotriazines cycloguanil or chlorcycloguanil, (metabolites of paludrine or lapudrine) can replace pyrimethamine Already cycloguanil resistance (further mutations in PfDHFR) has developed in S. E. Asia and S. America These strains are resistant to LapDap (lapudrine/dapsone) (now withdrawn) introduced after resistance to replace S/P e.g. S-108-N and I-164-L together (this is seen but not yet established in Africa) 8 As in other eukaryotes, a primary endosymbiotic plastid derived from the ancestral ingested bacterium: (2 membranes, vs. small genome encoding only 3 proteins, Cox1 Cox3 and CyB); Most mitochondrial proteins, as also seen for the apicoplast (see next) are now encoded in nuclear DNA and imported into the organelle from the cytoplasm In Apicomplexa, drastic reduction in the mt-genome may have followed the later acquisition of the apicoplast since some functions among the 2 organelles were initially duplicated, now shared or exchanged, accounting for their close ultastructural association (Vaidya & Mather Ann Rev Microbiol. 2009; 63: 249) Drug class: quinone analogues of coenzyme Q affecting electron transport Attack all growing stages Treatment drugs, and causal prophylactics Atovaquone Primaquine metabolites Primaquine metabolites are redox intermediates which also kill dormant stages such as mature P. f. gametocytes in blood and P. v. hypnozoites in the liver, preventing relapses Blood schizontocidal effects are weak but there is marked inhibition of growing liver stage 9 Compartment: mitochondrion 3

Ubiquinone 8, (CoQ8) oxido-reduction by mitochondrial CyB/C1 complex (phytyl) 8 10

Malarone (atovaquone/proguanil) 11 Apicoplast This is a secondary endosymbiotic plastid

& circular genome; Apicoplast functions include synthesis of type 2 fatty acids and

prevents apicoplast development in growing stages, is a slow acting treatment drug;

mainly second generation (progeny of drug-treated parasites die); This accounts for

) apparent sporontocidal action See rapid screen of Ekland et al., FASEB J.

4 Ubiquinone 8, (CoQ8) oxido-reduction by mitochondrial CyB/C1 complex (phytyl) 8 10 Atovaquone: CoQ8 mimic TYR268 ATV ASN268 ATV α-16 α-16 WT cytochrome b Mutant cytochrome b Fivelman et al., Mal J 2002; First report of atovaquone (ATV) resistance in routine treatment with Malarone (atovaquone/proguanil) 11 Apicoplast This is a secondary endosymbiotic plastid (cf chloroplast) derived from ingestion of a primitive alga; It has 4 limiting membranes & circular genome; Apicoplast functions include synthesis of type 2 fatty acids and synthesis of ~50 proteins (affected by antibacterials) Azithromycin for example, prevents apicoplast development in growing stages, is a slow acting treatment drug; Since schizonts develop containing merozoites which lack apicoplasts, its effects are mainly second generation (progeny of drug-treated parasites die); This accounts for causal prophylactic effects and (?) apparent sporontocidal action See rapid screen of Ekland et al., FASEB J. 2011; 25, Localization of apicoplast labelled with GFP next to MitoRed-labelled mitochondrion in trophozoite and schizont of P. falciparum in RBC photo: Waller et al., EMB J 2000; 19:1794 See Lim & McFadden,2010; Phil Trans. B 365: 749 (review) 4

Cultured pre-erythrocytic (liver) schizont of P. berghei Blue (Hoechst) nuclei Green (GFP) apicoplasts See review: Borrmann and Matuschewski Trends Parasitol.

Science 2012 13 14 Action of CQ, QN, mefloquine, artemisinin and analogues Used in treatment and in suppressive prophylaxis Blood stages digest hemoglobin (HGB) in the digestive vacuole (DV) and this

malaria pigment, hemozoin, non-enzymatically While heme will activate artemisinins (ARTs), to give toxic free radicals and adducts, hematin is the target for 4-aminoquinolines: chloroquine (CQ)

5 Cultured pre-erythrocytic (liver) schizont of P. berghei Blue (Hoechst) nuclei Green (GFP) apicoplasts See review: Borrmann and Matuschewski Trends Parasitol EE growth not inhibited by Azithromycin but merozoites do not survive in RBC Stanway et al., Biol. Cell 2009 (photo) For High-throughput screen on liver stages see Meister et al. Science Action of CQ, QN, mefloquine, artemisinin and analogues Used in treatment and in suppressive prophylaxis Blood stages digest hemoglobin (HGB) in the digestive vacuole (DV) and this results in the release of iron porphyrin heme, rapidly oxidized to hematin, which can cause irreversible membrane damage and parasite death; In the DV it is rapidly detoxified by dimerization to malaria pigment, hemozoin, non-enzymatically While heme will activate artemisinins (ARTs), to give toxic free radicals and adducts, hematin is the target for 4-aminoquinolines: chloroquine (CQ) amodiaquine desethyl metabolite (DAQ) and piperaquine (PIP) (see e.g. Warhurst et al., 2007), probable target for arylamino alcohols quinine (QN), mefloquine (MQ), halofantrine (HF) and lumefantrine (LF), which bind to it to prevent dimerization Activity of most of these drugs depends on Hgb digestion taking place Around the Thai-Myanmar and Thai-Cambodia border and parts of Vietnam, QN, MQ and antifolate resistance accompany resistance to CQ The best treatment proved to be artemisinin, from Chinese herbal remedy qinghao, or a derivative, with longer acting MQ or its analogue LF This ACT (artemisinin combination therapy) approach showed success in world wide trials wherever there was a CQ -resistance problem 15 Sweet wormwood Artemisia annua (Qinghao) 5

Chemical synthesis is complicated Percentage of drug in Artemisia annua is low ral bioavailability of

toxicity on erythroblasts is shown in human trials resulting in lowered reticulocyte counts Lowered primitive

period, day 21 to wk 9 perhaps avoids foetotoxicity; Clark Reproductive Toxicology 2009 28 Advantages of

stages The short plasma half-life reduces the selection of resistant clones from new infections, and

PIP, MQ or analogue LF in artemisinin combination treatment (ACT) This is now widely being applied, although

artemisinin derivatives Artemisinin Dihydroartemisinin Artesunate (semisynthetic) C Trioxane H Water-soluble

metabolism to dihydroartemisinin and metabolic inactivation to deoxy derivatives Semisynthetic derivatives from

oxygen species (RS) and heme/drug adducts: fits with blood stage restriction and activity of enantiomers 2.

restriction and activity of enantiomers; Though drug may have a site of action in the cytoplasm, the target is

6 Chemical synthesis is complicated Percentage of drug in Artemisia annua is low ral bioavailability of unmodified natural product is low (highly lipophilic) Plasma half life is about 45 mins in man Mild to moderate toxicity on erythroblasts is shown in human trials resulting in lowered reticulocyte counts Lowered primitive erythroblast production is foetotoxic in animals; In humans, the long primitive erythroblast circulation period, day 21 to wk 9 perhaps avoids foetotoxicity; Clark Reproductive Toxicology Advantages of artemisinins include their rapid action in clearing parasites from the blood, acting even on the smallest ring stages The short plasma half-life reduces the selection of resistant clones from new infections, and persistence of a few viable parasites can be prevented by including a longer half-life companion drug like AQ, PIP, MQ or analogue LF in artemisinin combination treatment (ACT) This is now widely being applied, although less success has been seen if resistance to some companion drugs is already present 16 Artemisia and artemisinin derivatives Artemisinin Dihydroartemisinin Artesunate (semisynthetic) C Trioxane H Water-soluble Deoxydihydroartemisinin & glucuronides (inactive) 17 Artemether (semisynthetic) Structure of artemisinin, metabolism to dihydroartemisinin and metabolic inactivation to deoxy derivatives Semisynthetic derivatives from dihydroartemisinin Peroxide group essential, only acts in blood-stage parasites Mirror image drugs (enantiomers) are active 1. In the DV, heme iron 2+ released during HGB digestion activates (reduces) the peroxide: producing reactive oxygen species (RS) and heme/drug adducts: fits with blood stage restriction and activity of enantiomers 2. Drug perhaps attacks cytoplasmic SERCA ATP-ase (PfATP6) by blocking active site; Doesn t fit with blood stage restriction and activity of enantiomers; Though drug may have a site of action in the cytoplasm, the target is unclear 3. Mitochondrial electron transport chain can also reduce the peroxide (heme also involved?); Wang et al. 2010; Fits with activity of enantiomers, possibly also blood stage restriction since blood stage mitochondria not fully functional 18 Artemisinin mode of action 6

7 Evidence for drug activation (reduction) by heme or by mitochondrial electron transport, but against action through SERCA PfATP-ase6 H H F F H 3 C CH 3 Identical antiplasmodial activity of enantiomeric (mirror image) trioxanes suggests they are not binding to active site of an enzyme molecule like pfatpase 6 (Wang et al. PLS ne 5: 2010)

Anas Raed. - Zaid Emad. - Malik Zuhlof

Anas Raed. - Zaid Emad. - Malik Zuhlof - 6 - Anas Raed - Zaid Emad - Malik Zuhlof 1 P a g e - This lecture started with the excellent presentation of chronic fatigue syndrome and the role of Vitamin B12 in its treatment by our colleague Osama