Malria Drug

7/28/2019 Malria Drug

1/10

Mechanisms of Drug Action and Resistance(Focus on Antimalarials)

Chemotherapy is the primary means of treating protozoan

infections. Successful chemotherapy depends in a large part on theability to exploit metabolic differences between the pathogen andthe host. A problem confronting chemotherapy is the ability of thepathogen to mutate and become drug resistance. Specific examples,of mechanisms of drug action and resistance are discussed below.

Drug actiono Chloroquine and the food vacuoleo Antifolateso

Drugs involving redox mechanisms Drug resistance

o Chloroquine Other Links

Drug Action

Drugs act by specificallyinterferring with cellular orbiochemical processes, often called

'targets'. The classic example of adrug target is an enzyme which isinhibited by the drug. Effectivedrugs will exhibit a selectivetoxicity for the pathogen as compared to the host. Many factorscontribute to this selective toxicity (Box) and these factors are notmutually exclusive.Rational drug design seeks to exploit thesevarious factors to develop drugs which are highly toxic to thepathogen and at the same time exhibit minimal toxicity to the host.

Chloroquine and the Food Vacuole

The food vacuole is a lysosome-like organelle in which thebreakdown of hemoglobin and the detoxification of heme occurs(see a more detailed discussion of thefood vacuole). Chloroquineconcentrates up to several 1000-fold in the food vacuole of the

Selective Toxicity

unique target in parasite discrimination between host and

parasite targets

target more important to parasite thanhost

greater drug accumulation by parasite drug activation by parasite
http://www.tulane.edu/~wiser/protozoology/notes/drugs.html#actionhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#actionhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#folatehttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#folatehttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#redoxhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#redoxhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#resisthttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#resisthttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQRhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQRhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#bottomhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#bottomhttp://www.tulane.edu/~wiser/malaria/fv.htmlhttp://www.tulane.edu/~wiser/malaria/fv.htmlhttp://www.tulane.edu/~wiser/malaria/fv.htmlhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#bottomhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQRhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#resisthttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#redoxhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#folatehttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#action


2/10

parasite. Possible mechanisms for this selective accumulation ofchloroquine in the food vacuole are: 1) protonation and ion trappingof the chloroquine due to the low pH of the food vacuole; 2) activeuptake of chloroquine by a parasite transporter(s); and/or 3)

binding of chloroquineto a specific receptor inthe food vacuole.

The exact contributionsof these threepostulated mechanismsis not clear, but it isgenerally accepted thatchloroquine exerts it

toxic effect byinterferring with theconversion of freeheme to hemozoin.Large quantities ofheme are released as aresult of hemoglobindigestion in the foodvacuole. The free heme

can lyse membranes,lead to the generationof reactive oxygenintermediates, andinhibit many otherprocesses and thus isquite toxic. Heme isdetoxified in the foodvacuole via abiocrystallizationprocess in which theheme is sequestered into large insoluble crystals called hemozoin orthe malarial pigment. [See more detailed description ofhemozoinformation.] The exact mechanism by which chloroquine inhibitshemozoin formation is not known, but chloroquine can bind hemeand this binding may prevent the heme from being incorporated

Chloroquine (CQ) accumulates in the food vacuole of the

parasite. This accumulation may involve ion trappingfollowing protonation, specific transport, and/or binding to

a receptor (eg., heme). The major action of chloroquine is

to inhibit the formation of hemozoin (Hz) from the hemereleased by the digestion of hemoglobin (Hb). The free

heme then lyses membranes and leads to parasite death.

Chloroquine resistance is due to a decreased accumulationof chloroquine in the food vacuole. Two differenttransporters (CRT and MDR1) have been implicated in

resistance. The functions of these transporters and their

exact roles in chloroquine resistance are not known.
http://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoin


3/10

into the hemozin crystal. Parasite killing is therefore a result of theaccumulation of metabolic wastes (ie, heme) associated with thedigestion of hemoglobin.

Other quinoline containing anti-malarials, such as mefloquine andquinine, also appear to affect the food vacuole. However, it in notclear whether these drugs bind heme or affect the formation ofhemozoin. Furthermore, these drugs are weaker bases thanchoroquine and may not exhibit the same degree of ion trappingwithin the food vacuole.

The food vacuole provides many potential drug targets. In additionto the inhibition of hemozoin formation discussed above, specificinhibitors of the proteases involved in hemoglobin digestion are also

being investigated as potential antimalarials. [See more detaileddiscussion offood vacuole proteases.] The specialized functions ofhemoglobin digestion and hemozoin formation are unique to theparasite and not found within the host. Furthermore, both functions--generation of amino acids from hemoglobin and detoxification ofheme--are very important for the parasite.

Antifolates

Folate metabolism is the target of several antimalarials as well asdrugs used against other pathogens. Reduced folates serve a co-factors in a many one-carbon transfer reactions involved in thebiosynthesis of amino acids and nucleotides (see more onvitaminsand co-factors). Due to its high rate of replication the malariaparasite has a high demand for nucleotides as precursors for DNAsynthesis (see more onnucleotides and nucleic acids), and thus isparticularly sensitive to antifolates. The two primary targets ofantifolate metabolism are the de novo biosynthesis of folates anddihydrofolate reductase (DHFR).

The malaria parasite synthesizes folates de novo whereas thehuman host must obtain preformed folates and cannot synthesizefolate. The inability of the parasite to utilize exogenous folatesmakes folate biosynthesis a good drug target. Folate is synthesizedfrom 3 basic building blocks, GTP, p-aminobenzoic acid (pABA), andglutamate, in a pathway involving 5 enzymes. One of these
http://www.tulane.edu/~wiser/malaria/fv.html#proteasehttp://www.tulane.edu/~wiser/malaria/fv.html#proteasehttp://www.tulane.edu/~wiser/malaria/fv.html#proteasehttp://www.tulane.edu/~wiser/malaria/Summary.html#cohttp://www.tulane.edu/~wiser/malaria/Summary.html#cohttp://www.tulane.edu/~wiser/malaria/Summary.html#cohttp://www.tulane.edu/~wiser/malaria/Summary.html#nahttp://www.tulane.edu/~wiser/malaria/Summary.html#nahttp://www.tulane.edu/~wiser/malaria/Summary.html#nahttp://www.tulane.edu/~wiser/malaria/Summary.html#cohttp://www.tulane.edu/~wiser/malaria/Summary.html#cohttp://www.tulane.edu/~wiser/malaria/fv.html#protease


4/10

enzymes, dihydropteroate synthase (DHPS), is inhibited by sulpha-based drugs. Sulfadoxine and dapsone are two commonantimalarials that target DHPS. The sulfa drugs are structuralanlalogs of pABA and are converted into non-metabolizable adducts

by DHPS. This leads to a depletion of the folate pool and therebyreduces the amount of thymidylate available for DNA synthesis.

Simplified scheme of folate metabolism. The malaria parasite synthesizes folates de novo, but cannot

utilize preformed folates. Folates participate as co-factors in many biosynthetic processes. Of particular

note is the synthesis of thymidylate (dTMP) which is needed for DNA synthesis. The two primary targetsof antimalarial drugs which target folate metabolism are denoted with the boxed arrows.

DHFR is an ubiquitious enzyme that participates in the recycling offolates by reducing dihydrofolate to tetrahydofolate. The

tetrahydrofolate is then oxidized back to dihydrofolate as itparticipates in biosynthetic reactions (eg., thymidylate synthase).Inhibiting DHFR will prevent the formation of thymidylate and leadto an arrest in DNA synthesis and subsequent parasite death.Pyrimethamine and proguanil are the two most common DHFRinhibitors used as antimalarials.These drugs inhibit DHFR from theparasite to a greater degree than the host enzyme and thus show aselective toxicity towardsthe parasite.

Antifolate CombinationsDrugs Pathogens

pyrimethamine + sulfadoxine(or dapsone)

Plasmodium

trimethoprim (or

pyrimethamine)+ sulfadiazine

Toxoplasma


5/10

Most often inhibitors ofDHPS and DHFR are usedin combination (Table) fora synergistic effect and to slow the development of drug resistance.

Specific point mutations in these enzymes lead to a lower affinity forthe drugs. Resistance tends to develop rapidly in the presence ofdrug pressure in situations where a single mutation can lead to drugresistance. The use of drug combinations will slow the developmentof resistance since two independent mutations must occur forresistance to develop against both drugs. Fansidar, a combination ofsulfadoxine and pyrimethamine, is widely used for the treatment ofuncomplicated falciparum malaria.Trimethoprim, similar topyrimethamine, is commonly used in combination with other sulfadrugs for treatment of coccidia (Toxoplasma, Cyclospora,

and Isospora)and Pneumocystis.

Drugs Involving RedoxMechanisms

Several anti-protozoal drugsare believed to act viaoxidative stress (Table).

Metabolic processes willproduce reactive oxygen intermediates (ROI) which can damagecellular components such as lipids, proteins, and nucleic acids (seereview onoxidative stress). The high metabolic activity of mostprotozoan pathogens will result in production of even higher levelsof ROI. This is exemplified by the malaria parasite which producesROI as a consequence of hemoglobin digestion and the release offree heme. [See more detailed description ofheme and ROI.] Allcells have mechanisms by which the ROI can be detoxified(eg.,redox metabolism). Drugs which specifically increase the levelsof oxidative stress in the parasite may overwhelm these ROIdefense mechanisms and lead to parasite death. Levels of oxidativestress can be increased by drugs that are direct oxidants, as well asby drugs which participated in oxidation-reduction cycling,sometimes called futile redox cycling.

trimethoprim + sulfamethoxazole Cyclospora, Isospora,Pneumocystis

Possible Redox Agents

Drugs Pathogens

primaquine, artemisinin

derivatives

Plasmodium

metronidazole, tinidazole Giardia, Entamoeba,

Trichomonas

benznidizole, nifurtimox Trypanosoma cruzi
http://www.agronomy.psu.edu/Courses/AGRO518/Oxygen.htmhttp://www.agronomy.psu.edu/Courses/AGRO518/Oxygen.htmhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.tulane.edu/~wiser/malaria/Summary.html#redoxhttp://www.tulane.edu/~wiser/malaria/Summary.html#redoxhttp://www.tulane.edu/~wiser/malaria/Summary.html#redoxhttp://www.tulane.edu/~wiser/malaria/fv.html#hemozoinhttp://www.agronomy.psu.edu/Courses/AGRO518/Oxygen.htm


6/10

Many of the drugs participating in redox reactions need to beactivated before they are effective against their target(s). Forexample, metronidazole and other nitroimidazoles are broadspectrum drugs that affect a wide variety of anerobic bacteria and

protozoa. These drugs are activated by a reduction of the nitrogroup to an anion radical. The anion radical is highly reactive andwill form adjuncts with proteins and DNA leading to a loss offunction. In particular, the reactions with DNA result in strandbreakage and inhibition of replication and will lead to cell death.Reduction of nitroimidazoles requires strong reducing conditions andanaerobic organisms have more reduction potential than aerobicorganisms. This accounts for the selectivity of these compounds foranaerobic organisms. In other words, the drugs are preferentiallyactivated by the pathogens.

In the case of metronidazole, reduced ferredoxin appears to be theprimary electron donor responsible for its reduction (Figure). Thereis a good correlation between the presence of the pyruvate-ferredoxin oxidoreductase (PFOR) and sensitivity to metronidazole.All three of the protozoa affected by metronidazole (Table) lackmitochondria and have PFOR similar to that found in manyanaerobic bacteria. Aerobic organisms with mitochondria usepyruvate dehydrogenase instead of the PFOR for the production of

acetyl-coenzyme A.

Actions of nitroimidazoles. Nitroimidazoles (R-NO2) are activated by the parasite via a reduction


7/10

to an anion radical. This highly reactive anion radical will then damage DNA and proteinsresulting in parasite death. Metronidazole appears to be specifically reduced by ferredoxin

in Giaridia, Entamoeba, and Trichomonas. Aerobic organisms would utilize other electron donorsfor the reduction of nitroimidazoles and there would also be the possibility to establish futile

cycles leading to the generation of ROI in which oxygen is the final electron acceptor. NormallyNAD oxidoreductases carry out redox cycling with water being the final product.

Nitroimidazoles (eg, benznidizole) and related nitrofuran compounds(eg, nifurtimox) also are effective against Trypanosoma cruzi. Theelectron donors responsible for the initial reduction of these drugsae not known and the basis of specificity for the parasite is notclear. Both of these drugs are somewhat toxic and do not exhibitgood therapeutic indices. The mechanism of nifurtimox action isbelieved to involve futile redox cycling following its reduction,whereas benznidizole is speculated to inhibit specific reductases and

thereby decrease the ability of the parasite to remove ROI.

Oxidative Stress and G6PD Deficiency

Several human genetic diseases are known to confer some protection against malaria

(see innate resistance). Glucose-6-phosphate dehydrogenase (G6PD) deficient individuals

will have lower levels of reduced NADPH in their erythrocytes which is needed to maintainreduced glutathione. The lower levels of reduced glutathione will result in an increased

sensitivity to oxidative stress since glutathione peroxidase participates in the detoxification

of ROI. The increase levels of ROI due to the parasite's metabolism combined with the

decrease ability G6PD-deficient erythrocytes to remove ROI will result in a premature lysis

of the infected erythrocyte and therefore confer some protection against malaria. Theparasite not only needs to protect itselfs against ROI but it also needs to insure that the host

erythrocyte is not damaged before the parasite completes erythrocytic schizogony. In fact, ithas been suggested that the parasite may supply the host erythrocyte with glutathione to

increase its reducing capacity. Somewhat related, primaquine treatment is contraindicated in

G6PD-deficient patients since it can cause hemolytic anemia. This is likely related to the

ability of primaquine to generate ROI and the decreased reducing potential of G6PD-deficient erythrocytes.
http://www.tulane.edu/~wiser/protozoology/notes/malaria.html#IRhttp://www.tulane.edu/~wiser/protozoology/notes/malaria.html#IR


8/10

Drug Resistance

The emergence of drug resistanceseverely limits the arsenal of availabledrugs against protozoal pathogens.Parasites have evolved numerousways to overcome the toxicity of drugs(Box). Quite often drug resistance involves mutations in the drugtarget so that the drug does not bind or inhibit the target as well.Drug resistance can develop quickly in situations where a singlepoint mutation can confer resistance. Another mechanism of drugresistance involves expressing higher levels of the target. This canbe accomplished either through increased transcription andtranslation or gene amplification. This results in a requirement for

higher levels of drugs to achieve the same level of inhibition.Decreasing drug accumulation or metabolizing the drug to non-toxicproducts will result in less drug reaching the target and can alsocontribute to drug resistance. Drug resistance can also involve theaccumulation of mutations in the same or different targets whichwill have additive or synergistic effects. Parasites with mutations orgenetic polymorphisms which confer a decrease in drug sensitivitywill be selected under drug pressure.

Proteins and Mutations Associated with Drug ResistanceProtein Function Location Drugs Effected Major

Mutations

DHPS folate

metabolism

cytoplasm sulfadoxine,

dapsone

A437G (K540E,

A581G)

DHFR folatemetabolism

cytoplasm pyrimethamine,proguanil

S108N (N51I,C59R, I164L)

CRT transporter food vacuole chloroquine K76T

MDR1 transporter food vacuole mefloquine,quinine (?)

increased copy #,D86Y*

Cytochrome b electron

transport

mitochondria atovaquone Y268S/N/C

ATPase 6 calciumtransport

endoplasmicreticulum

artemisinins S769N

The proteins are: CRT = chloroquine resistance transporter; MDR1 = multi-drug resistance (P-

glycoprotein homologue); DHFR = dihydrofolate reductase; DHPS = dihydropterote sythetase;

ATPase6 = sarco/endoplasmic reticulum calcium-dependent ATPase orthologue. *Associated with

Mechanisms of Resistance

mutations in target gene increase production of target decrease drug accumulation

(including increase in efflux)

drug inactivation


9/10

an increased sensitivity to mefloquine and dihydroartemisinin and a decreased sensitivity to

chloroquine.

In some cases specific mutations have been associated with drugresistance (Table). Fansidar (SP) resistance is correlated with

specific mutations in the enzymes targeted by sulfadoxoine andpyrimethamine (dihydropterote sythetase and dihydrofolatereductase, respectively). Chloroquine resistance (discuss below inmore detail) has been correlated with mutations in a transporterfound on the food vacuole membrane (chloroquine resistancetransporter, CRT). Another food vacuole transporter, multi-drugresistance gene 1 (MDR1), has been implied to play an ancillary rolein resistance. The basis for resistance to mefloquine and quinine isnot clear, but the mdr1 gene has also been implicated.

Chloroquine. Chloroquine resistance is associated with a decrease inthe amount of chloroquine that accumulates in the food vacuole, thesite of action for chloroquine (see above). The mechanism for thisdecreased accumulation is controversial. Some studies have shownthat the decrease in drug accumulation is due to an increase in drugefflux. Whereas other studies suggest that diminished levels ofchloroquine accumulation is more important. The observation thatverapamil and related drugs can reverse the chloroquine resistantphenotype has led to speculation that an ATP dependent transporterplays a role in drug efflux and chloroquine resistance, similar to themultidrug resistance (MDR) in cancer. A MDR-like transporter,designated PfMDR1, has been identified on the food vacuolemembrane. However, no definitive correlations between PfMDR1and chloroquine resistance could be demonstrated. An ancillary rolefor PfMDR1 in chloroquine resistance cannot be ruled out though.

A genetic cross and mapping studies between a chloroquineresistant clone and a chloroquine sensitive clone resulted in the

identification of a 36 kb region on chromosome 7 associated withchloroquine resistance. One of the 10 genes in this 36 kb regionencodes a protein with 10 transmembrane domains and resembles atransporter protein similar to chloride channels. The gene has beendesignated aspfcrtand the protein is localized to the food vacuolemembrane. Several mutations in thepfcrtgene show correlationswith the chloroquine resistance phenotype and one mutation, a
http://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQhttp://www.tulane.edu/~wiser/protozoology/notes/drugs.html#CQ


10/10

substitution of a threonine (T) for a lysine (K) at residue 76 (K76T)shows a perfect correlation with chloroquine resistance. Presumablythese mutations affect the accumulation of chloroquine in the foodvacuole, but the exact mechanism of chloroquine resistance is not

known. Furthermore, the observation that chloroquine resistancehas arisen relatively few times and then subsequently spread haslead to speculation that multiple genes are involved in thedevelopment of resistance (see more discussion).

Additional Reading

Foley M and Tilley L (1998) Quinoline antimalarials.Mechanisms of action and resistance and prospects for newagents. Pharmacology & Therapeutics79:55

Hyde JE (2007) Drug-resistant malaria -- an insight.FEBSJournal 274, 4688-4698.

Ouellette M (2001) Biochemical and molecular mechanisms ofdrug resistance in parasites. Trop Med Int Health 6:874.

Rosenthal PJ and Goldsmith RS (2001) Antiprotozoal drugs. InBasic and Clinical Pharmacology, 8th edition. McGraw-HillCompanies Inc. (On-line edition available through Stat!RefBooks at theTulane Medical Library)

Samuelson J (1999) Why metronidazole is active against bothbacteria and parasites. Antimicrob Agents Chemother.43:1533.

Wellems TE and Plowe CV (2001) Chloroquine-resistantmalaria. J Inf Dis 184:770.
http://www.tulane.edu/~wiser/protozoology/notes/malaria.html#DRhttp://www3.interscience.wiley.com/journal/118546650/abstract?CRETRY=1&SRETRY=0http://www3.interscience.wiley.com/journal/118546650/abstract?CRETRY=1&SRETRY=0http://www3.interscience.wiley.com/journal/118546650/abstract?CRETRY=1&SRETRY=0http://www3.interscience.wiley.com/journal/118546650/abstract?CRETRY=1&SRETRY=0http://www.tulane.edu/~matas/http://www.tulane.edu/~matas/http://www.tulane.edu/~matas/http://www3.interscience.wiley.com/journal/118546650/abstract?CRETRY=1&SRETRY=0http://www3.interscience.wiley.com/journal/118546650/abstract?CRETRY=1&SRETRY=0http://www.tulane.edu/~wiser/protozoology/notes/malaria.html#DR

Malria Drug

Documents

Transcript of Malria Drug