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REVIEW

Antimalarial Drug Resistance and Novel Targets

for Antimalarial Drug Discovery
This article was published in the following Dove Press journal:
Infection and Drug Resistance

Melkamu Adigo Shibeshi Abstract: Malaria is among the most devastating and widespread tropical parasitic diseases
Zemene Demelash Kifle in which most prevalent in developing countries. Antimalarial drug resistance is the ability of
Seyfe Asrade Atnafie a parasite strain to survive and/or to multiply despite the administration and absorption of
medicine given in doses equal to or higher than those usually recommended. Among the
Department of Pharmacology, School of
Pharmacy, College of Medicine and factors which facilitate the emergence of resistance to existing antimalarial drugs: the
Health Sciences, University of Gondar, parasite mutation rate, the overall parasite load, the strength of drug selected, the treatment
Gondar, Ethiopia compliance, poor adherence to malaria treatment guideline, improper dosing, poor pharma­
cokinetic properties, fake drugs lead to inadequate drug exposure on parasites, and poor-
quality antimalarial may aid and abet resistance. Malaria vaccines can be categorized into
three categories: pre-erythrocytic, blood-stage, and transmission-blocking vaccines.
Molecular markers of antimalarial drug resistance are used to screen for the emergence of
resistance and assess its spread. It provides information about the parasite genetics associated
with resistance, either single nucleotide polymorphisms or gene copy number variations
which are associated with decreased susceptibility of parasites to antimalarial drugs. Glucose
transporter PfHT1, kinases (Plasmodium kinome), food vacuole, apicoplast, cysteine pro­
teases, and aminopeptidases are the novel targets for the development of new antimalarial
drugs. Therefore, this review summarizes the antimalarial drug resistance and novel targets
of antimalarial drugs.
Keywords: antimalarial, drug resistance, novel targets, vaccines

Introduction
Malaria is an infectious, hematologic disease causing death and illness in children
and adults, especially in tropical countries1 Malaria control requires an integrated
approach, including prevention, primarily vector control, and prompt treatment with
effective antimalarial drugs.2 Malaria is among the most devastating and wide­
spread tropical parasitic diseases in which most prevalent in developing countries.3
Malaria is caused by the Plasmodium parasite, which is transmitted by the bite of
a mosquito vector. Five species are known to infect humans: P. falciparum,
Plasmodium vivax, Plasmodium ovalae, Plasmodium malariae, and Plasmodium
knowelsi. The parasite P. falciparum causes the most dangerous, with the highest
rates of complications and mortality.3 Antimalarial drug resistance results in
a global resurgence of malaria making a major threat to malaria control.
Correspondence: Zemene Demelash Widespread and indiscriminate use of antimalarial drugs contributes to malaria
Kifle
Department of Pharmacology, School of parasites to evolve mechanisms of resistance.4,5
Pharmacy, University of Gondar, P.O. Box:
196, Gondar, Ethiopia
The malaria life cycle is very complex which requires two organisms as host,
Email [email protected] mosquito, and human being.6 The most common symptoms of malaria (chills, high

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fever, sweating, malaise, headache, and muscle aches) Ethiopia report between June 2016 and July 2017,
manifest usually one to four weeks after infection with 1,530,739 confirmed malaria illnesses (69.24%
the parasite; in relapsing Plasmodium parasites it ranges P. falciparum, 30.76% P. vivax) malaria illnesses from
from five to eight years, but these signs and symptoms these 356 deaths were reported.16
may also have seen in other diseases.7 Currently, available
malaria diagnostic tools for identification of plasmodium Drugs Used for the Treatment of
species in human blood samples include microscopy (light
or fluorescence)-gold standard method, immuno-
Malaria
Currently available antimalarial drugs are broadly categor­
chromatographic lateral flow assays (also called rapid
ized into three types. Aryl amino alcohol compounds
diagnostic tests, RDTs), serology, nucleic acid amplifica­
including quinine, quinidine, halofantrine, lumefantrine,
tion techniques (NATs) that include polymerase chain
chloroquine, amodiaquine, mefloquine, cycloquine, etc.
reaction (PCR) and isothermal amplification and others.8,9
Antifolate compounds: proguanil, pyrimethamine, tri­
According to the 2018 malaria report WHO estimated
methoprim, etc. Artemisinin compounds like artemisinin,
that approximately, 219 million cases of malaria from 90
dihydroartemisinin, artesunate, artemether, arteether,
countries, an increase of 2 million cases over 2016. Infants
etc.17,18
and young children in malaria-endemic countries of Africa
Most of the antimalarial drugs target the asexual ery­
typically experience several clinical episodes of malaria
throcytic stages of the parasite (blood schizonticidal
before they develop partial immunity. This protects against
drugs). Two types either fast-acting (Chloroquine, quinine,
severe disease and death from malaria.10,11 Malaria con­
and mefloquine) or slow-acting (Pyrimethamine, sulpho­
tinues to burden the overstretched health services in the
namides, and sulphone). Tissue schizonticidal drugs target
sub-Saharan region and is a serious public health problem.
the hypnozoites (dormant stage of the parasite) in the liver
The same report indicated that 3.1 billion US$ was
whereas gametocytocidal drugs destroy sexual erythrocy­
invested for malaria control and elimination program, of
tic forms of the parasite in the bloodstream preventing
which US$ 2.2 billion benefited the WHO African Region,
transmission of malaria to the mosquito. Sporontocides
followed by the WHO Southeast Asia Region US$
prevent or inhibit the formation of malarial oocysts and
0.3 billion. The WHO African region with 200 million
sporozoites in the infected mosquito.19
cases (92%) in 2017, followed by the WHO Southeast
Quinolines (affects polymerization of hemozoin), anti­
Asia region (5%), especially Sub-Saharan Africa suffers
folates (block dihydrofolate reductase and dihydropteroate
by far the greatest malaria burden worldwide and is cur­
synthetase enzymes of the parasite) and artemisinin (have
rently undergoing a profound demographic change.
various mechanisms), administered alone or in combina­
Almost 93% of all deaths due to malaria in 2017 were
tion to treat malaria18 Artemisinin combination therapy is
from Africa. Globally 266, 000 (61%) malaria deaths were
the cornerstone of malaria control in sub-Saharan Africa
estimated to be in children less than 5 years age.12 In most
such as artemether/lumefantrine and artesunate/amodia­
areas of Africa, P. vivax infection is essentially absent
quine. Because of the notorious capacities of
because of the inherited lack of the Duffy antigen receptor
Plasmodium falciparum to develop drug resistance, many
for chemokine on the surface of red blood cells that are
antimalarial programs have recently included dihydroarte­
involved in the parasite invasion of erythrocytes.13
misinin/piperaquine (DHA/PPQ) as a second-line antima­
However, in Brazilian Amazon, Madagascar, and Central
larial drug.20
Sudan implicated that individuals with negative Duffy
antigen receptor were infected with p. vivax. P.falciparum
species are dominant in Africa and the highest-burden of Resistance to Plasmodium vivax and
P. vivax infection is in Southeast Asia and South Plasmodium falciparum
America14 In Ethiopia, major malaria transmission seasons Before dealing with resistance to malaria it is better to
are from September to December and June to August.15 know the terminologies of recurrence, recrudescence,
According to the 2018 federal ministry of health (FMOH) relapse, and resistance (4R’s). Recurrence is the recurrence
of Ethiopia report many densely populated highland areas of asexual parasitemia following treatment (in P. vivax and
are malaria-free including the capital city of Addis Ababa. P. ovale infections only) or a new infection.
Health management information system (HMIS) of Recrudescence is the recurrence of asexual parasitemia

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Dovepress Shibeshi et al

after the treatment of the infection with the same infection combination therapy depending on chloroquine sensitiv­
that caused the original illness. Relapse is the recurrence ity in the area for radical cure of vivax malaria. Current
of asexual parasitemia in P. vivax and P. ovale malaria guidelines recommend a 14-day course of primaquine
deriving from persisting liver stages from persisting hyp­ administered either once or twice daily to reduce the
nozoites. Resistance is the ability of a parasite strain to risk of hemolysis and improve tolerability from gastro­
survive and/or multiply despite the proper administration intestinal disturbance.2
and absorption of an antimalarial medicine in the dose In Ethiopia first report of P. falciparum and P. vivax
normally recommended.21 Plasmodium vivax is continued chloroquine treatment failure in Debre Zeit, was in 1995.
to put a substantial burden on the malaria-endemic world The invasion of human red blood cells by the extracel­
with the morbidity and mortality due to its propensity to lular merozoite form of Plasmodium falciparum is
cause recurrent infections.22 Plasmodium vivax forms dor­ a process central to the pathogenesis of this devastating
mant liver stages (hypnozoites), which causes relapses of pathogen. In the present time control of multidrug-
infection weeks to months after the initial attack. resistant P. falciparum malaria has become a very diffi­
Recurrent infections can occur as often as every three cult task because endogenous allelic exchanges occurred
weeks, with relapses the main cause of vivax illness. in P. falciparum have increased the therapeutic failures
Even though chloroquine is the first-line treatment for P. and significantly increased the levels of resistance world­
vivax malaria in most endemic countries resistance is the wide. As evolution is an unending process how the
main problem facing chloroquine in different parts of the formation of drug-resistant mutant alleles stops is
world. In Africa and South America chloroquine resistance a very concerning question.31 Usually higher mean para­
to plasmodium falciparum first appeared in 1978 and 1996 sitemia index is seen in infected individuals with
respectively.23,24 P. falciparum but P. vivax infection generally exhibits
Chloroquine-resistant Plasmodium vivax was first low parasitemia index due to its preference to invade
reported in 1989 from Papua New Guinea. High-grade chlor­ reticulocytes rather than erythrocytes.32
oquine-resistant Plasmodium vivax is prevalent in areas such
as Indonesia and Oceania (regarded as epicenters of chlor­
oquine resistance).25 Both the acute illness and relapses from
Mechanism of Antimalarial Drug
hypnozoites can be effectively prevented by the administra­ Resistance
tion of a combination of chloroquine with primaquine (radi­ According to the World Health Organization (WHO), anti­
cal cure). Primaquine has activity against both blood and malarial drug resistance is defined as the ability of
liver stages, including against chloroquine-resistant strains. a parasite strain to survive and/or to multiply despite the
Severe P. vivax infections can cause cerebral malaria with administration and absorption of medicine given in doses
generalized convulsions and status epilepticus, severe ane­ equal to or higher than those usually recommended but
mia, hepatic dysfunction and jaundice, acute lung injury, within the tolerance of the subject, provided drug exposure
pulmonary edema, splenic rupture, acute renal failure, and at the site of action is adequate. Resistance to antimalarial
severe thrombocytopenia with or without bleeding from dif­ arises because of the selection of parasites with genetic
ferent parts of the body.26–28 mutations or gene amplifications that confer reduced
Primaquine has activity against both asexual and susceptibility.2
sexual blood stages of the parasite as well as against Resistance appears to be caused by a change in the
the liver stage schizonts and hypnozoites.29 Primaquine structure, function, or quantity of a protein. The change in
can result in significant hemolysis in people with glu­ the protein is mediated by genetic changes such as single
cose-6-phosphate dehydrogenase deficiency (G6PDd). nucleotide polymorphisms (SNP) or gene amplification.
G6PD deficiency is the most common heritable enzymo­ Because of antimalarial drug resistance is becoming the
pathy in the world, with a prevalence range of 2% to most difficult hurdle for the success of antimalarial ther­
40%.30 The WHO for radical cure of vivax malaria apy, so scientists are in continuous move researching to
currently recommends the use of a daily dose of overcome the problem. Resistant parasite strains will
0.25 mg/kg/day (3.5 mg/kg total dose) primaquine always emerge, requiring the continual generation of new
taken with food once daily, which can be either co- molecules. The novel drugs with a new mechanism of
administered with chloroquine or artemisinin action are entering into clinical trials.17

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Several factors facilitate the emergence of resistance to of drugs through induction of multidrug resistance (MDR)
existing antimalarial drugs. To mention some factors, the transporters. In malaria, MDR transporters are not the
parasite mutation rate, the overall parasite load, the primary mechanism of resistance (Table 1).37
strength of drug selected, the treatment compliance, and The antimalarial activity of Artemisinin is due to its
poor adherence to malaria treatment guidelines.33 unique trioxane structure with an endoperoxide bond.
Improper dosing, poor pharmacokinetic properties, fake Usually, semi-synthetic derivatives are used clinically
drugs lead to inadequate drug exposure on parasites.34 (artemether, artesunate, and dihydroartemisinin) because
Poor-quality antimalarial (falsified antimalarial without due to the low solubility of artemisinin.38 Artemisinin
active pharmaceutical ingredient (APIs)) may aid and combination therapy (ACT), currently recommended are
abet resistance by increasing the risk of hyperparasitaemia, artemether + lumefantrine, artesunate + amodiaquine, arte­
recrudescence, and hypergametocyopaenia, wrong APIs sunate + mefloquine, artesunate + sulphadoxine-
such as the use of halofantrine instead of artemisinin pyrimethamine, and dihydroartemisinin + piperaquine,
which without chemical analysis will be invisible to inves­ the current gold standard for malaria treatment but resis­
tigators but not to parasites.35,36 Frequently targeted bio­ tance is emerging in different areas. Resistance to
logical pathways by antimalarial drugs in parasites of Artemisinin and its derivatives are emerging and troubling
plasmodium are heme detoxification (in digestive vacuole) phenomena in malaria treatment.39 In the Greater Mekong
biosynthesis folate and pyrimidine and electron transport Subregion of Asia, the Artemisinin-based drug resistance
(in mitochondrion). Studies done during treatment with is emerging.40 As resistance to each new malaria drug
aryl amino alcohols quinine, lumefantrine (LMF), and arises, it becomes necessary to combine two or more
mefloquine (MFQ) from South East Asia showed that component drugs to slow the spread of resistance to reduce
copy-number changes in pfmdr1, as well as PfCRT and the chance of resistance combinations containing an arte­
PfMDR1 sequence variants, can affect the parasite’s misinin derivative that is currently in use.41 Within the
susceptibility.32 Unlike other diseases (eg Tuberculosis, malaria parasite-host hemoglobin is degraded by a series
AIDS), malaria drug resistance mechanisms are unique, of protease enzymes to release peptides and amino acids
as the parasite is capable of inducing resistance in the required for development and to create space within its
exact cellular target of the drug, drug resistance phenotype digestive vacuole in which buildup of hematin occurs
is mostly induced due to enhanced and non-specific efflux which is potentially toxic to the parasite.

Table 1 Summary of Some Antimalarial Drugs, Mechanism of Action, Site of Action, and Mechanism of Resistance
Antimalarial Drug Mechanism of Action Site of Mechanism of Resistance
Action

Antifolates ((pyrimethamine Inhibition of dihydrofolate reductase Cytosol Mutations in dihydrofolate reductase (DHFR)
(PYR) and cycloguanil (CYC)) (DHFR)

Antifolates (sulfadoxine (SDX)) Inhibition dihydropteroate synthetase Cytosol Dihydropteroate synthetase (DHPS)
(DHPS)

Naphthoquinones (Atovaquone Inhibits mitochondrial electron Mitochondria A single point mutation in the cytochrome
(ATQ)) transport b subunit (CYTb) of the bc1 complex

Antibiotics (Clindamycin (CLD) Inhibit protein translation inside the Inside the A point mutation in the apicoplast encoded 23S
and Doxycycline (DOX)) apicoplast apicoplast rRNA (CLD)

Artemisinin (ART) Alkylation of proteins and lipids ER, vesicular Mutation in K13
structures

4- aminoquinolines (CQ, AQ, They bind reactive heme and interfere Digestive Point mutations in the transporters PfCRT and
PPQ, Mannich base with its detoxification through vacuole PfMDR1, increased expression of the
pyronaridine (PND)) incorporation into chemically inert hemoglobinases plasmepsin 2 and 3 (PM2/PM3, in
hemozoin. the digestive vacuole), and might in some instances
involve mutant PfCRT

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Dovepress Shibeshi et al

Artemisinin and its derivatives have a fast onset of behind this low impact is the greater degree of acquired
action but are eliminated soon (half-life 0.5–1.4 h) from immunity there, resulting from repeated exposure to
humans for this reason it is essential to combine with slow P. falciparum, which builds host immunity to help control
clearing drugs to kill residual parasites.42 Artemisinin and drug-resistant infections.44 Mutant K13 results in lowered
its derivatives can be combined with other antimalarial ART interactions with P. falciparum phosphatidylinositol-
drugs at least for two reasons, first to prolong the half- 3-kinase (PfPI3K).54 In vitro and in vivo studies in many
life of Artemisinin and its derivatives, second, to prevent areas of Southeast Asia show that mutations in the K13
resistance.43 Recent reports in Equatorial Guinea showed propeller gene (PF3D7_1343700 or PF13_0238) are linked
that P. falciparum isolate was resistant to artemisinin.44 to artemisinin resistance.55

Molecular Mechanism of Molecular Markers of Antimalarial

Artemisinin Resistance Drug Resistance
Artemisinin possesses a long-acting effect against drug- Molecular markers of antimalarial drug resistance are used
resistant malaria parasites, and also able to reduce the to screen for the emergence of resistance and assess its
parasite burden in asymptomatic individuals who serve spread. It provides information about the parasite genetics
as reservoirs for malaria transmission.45 Artemisinin and associated with resistance, either single nucleotide poly­
its derivatives (artesunate, artemether, and arteether) are morphisms or gene copy number variations which are
potent and fast-acting drugs that cause a rapid decline in associated with decreased susceptibility of parasites to
parasitemia during the first days of treatment. Meshnick antimalarial drugs. Detection of molecular markers pro­
using mass spectroscopy observed that Artemisinin can vides a feasible means of tracking the emergence and/or
alkylate heme resulting in decomposition of the endoper­ spread of antimalarial drug resistance.56 P. falciparum
oxide bridge to produce carbon-centered free radicals chloroquine resistance transporter gene (PfCRT), chloro­
which are crucial for selectively toxic to malaria quine accumulates within the DV (digestive vacuole) of
parasites.46 Specific protein or enzyme is used as the parasites where there is mutant PfCRT, accumulation
a molecular target for Artemisinin. Invitro studies show of chloroquine in parasites is very less as compared to
that hemoproteins such as catalase, cytochrome c, and parasites expressing wild type PfCRT,57 as a result, chlor­
hemoglobin but not free globin, is alkylated by oquine-resistant parasites can export chloroquine via
Artemisinin.47 active transport,58 implying that mutant and wild type
Another molecular target is PfATP6; Artemisinin inhi­ PfCRT have different drug transporting properties.
bits of a parasite Ca2+ transporting ATPases (SERCA – P. falciparum multidrug resistance transporter 1
Sarco/endoplasmic reticulum membrane calcium ATPase). (PfMDR1) locates in the digestive vacuole of the parasite
SERCA reduces cytosolic free calcium concentrations by and function as a general importer sequestering toxic
actively concentrating Ca2+ into membrane-bound stores, metabolites and drugs into the digestive vacuole (DV).
an activity critical to cellular survival.48 In the parasite Pfmdr1 indirectly influences drug flux by affecting intra­
membrane, Artemisinin accumulates within neutral lipids cellular ions and PH.59 Studies show that wild type
and causes parasite membrane damage.49 Artemisinin has PfMDR1 transports quinine and chloroquine but not halo­
shown resistance in P. falciparum cultures50 and P. voelii fantrine while mutant PfMDR1 transports halofantrine but
mouse models51 and in vitro resistance in field isolates.52 not quinine or chloroquine.60
P. falciparum Kelch 13 (PfKelch13), the marker for The multidrug resistance-associated protein (PfMRP)
artemisinin resistance in P. falciparum malaria, is not an is a member of the ATP-binding cassette (ABC) proteins
enzyme or a pump but rather is predicted to be a substrate family and ABC transporter C subfamily. Genetic disrup­
adapter for a cullin E3 ligase, with a putative substrate of tion PfMRP leads to increased parasite susceptibility to
P. falciparum phosphatidylinositol 3-kinase (PfPI3K) and several antimalarial drugs like chloroquine, quinine, arte­
a redox sensor.53 Kelch-like protein K13 is a molecular misinin, piperaquine, and primaquine and accumulates
marker for Artemisinin resistance, but no detectable more glutathione (GSH), chloroquine, and quinine.61
impact in Africa (except one report with P. falciparum Cytochrome bc1 complex catalyzes the transfer of elec­
K13-variant infection from western Africa). The reason trons from ubiquinol to cytochrome c thereby maintaining

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the membrane potential of mitochondria used to produce efficacy is carried out at sentinel sites by national malaria
ATP by an ATP synthase. Mutations in the Cytochrome control programs using a standardized WHO protocol.
bc1 complex leads to atovaquone resistance this is because Treatment response is defined as the absence of parasite­
atovaquone binds to the ubiquinol binding site, thereby mia at follow-up, on day 28 or 42. WHO recommends
disrupting the electron transfer chain.62,63 a switch to another more effective first-line drug if a 10%
Imidazolopiperazine (IPZ) class of drug compounds treatment failure rate is reached.69,70
(saturated cyclic amines) has activity in both liver and
blood-stage parasites as an antimalarial drug for use in Novel Targets of Antimalarial Drugs
prophylaxis, treatment, and prevention of malaria disease The existing antimalarial drugs were identified based on
transmission. Whole-genome sequencing done by Prof the major metabolic pathway differences of the parasite
Paul and his co-workers of these drug-resistant with its host. The Key metabolic pathways of the
Plasmodium falciparum clones, genes associated with Plasmodium species, including oxidative stress, heme
drug resistance are identified, an acetyl-CoA transporter detoxification, fatty acid synthesis, and nucleic acid synth­
(PFACT) and a UDP-galactose transporter (PFUGT). esis are some of the novel targets for antimalarial drug
These mechanisms responsible for resistance are members discovery and development.71,72 Though most of the anti­
of a family of membrane transporter proteins (major facil­ malarial drugs used for many years, presently the use of
itator superfamily or MFS).MFS is the largest and most such drugs is limited as a result of drug resistance.
ubiquitous secondary transporter family responsible for the According to previous studies, there are no antimalarial
translocation of small molecules including metabolites, agents recognized to inhibit an identified antimalarial drug
nucleosides, oligosaccharides, amino acids, oxyanions, targets.73 In its place, the majority of the antimalarial
and drugs.64,65 agents were discovered in both in vitro model and animal
Imidazolopiperazine is promising drug candidates with models (in vivo). Thus, the exact mechanism of action of
the potential to aid in malaria elimination include KAF156 most antimalarial drugs is not known. In addition, the
and KAF179 (currently in Phase II clinical trials). They mechanism of antimalarial drug resistance was not well
possess low Nanomolar potency against P. falciparum known for most antimalarial drugs.72 In addition to
liver stages, asexual blood stages, and sexual stage increasing the need to develop new antimalarial drugs,
gametocytes.66 P. falciparum V-type H+ pyrophosphatase identifying countermeasures either to delay or minimize
(PFVP2) is located in the DV membrane and increased the development of resistance against new drugs is an
transcription of pfvp2 has been observed in-vitro when important phenomenon.
P. falciparum are exposed to chloroquine (10-fold up-
regulation) and lumefantrine (2-fold up-regulation). The Glucose Transporter PfHT1
up-regulation of pfvp2 implies that it could be involved Glucose is a source of energy for Intra erythrocytic malar­
in maintaining the H+ balance in the parasite DV and to ial parasites in which infected erythrocytes consume
compensate for H+ loss caused by the removal of proto­ higher energy than normal erythrocytes.74 P. falciparum
nated CQ.67,68 almost fully depend on glycolysis for energy production,
deprived of energy stores; depend on continuous uptake of
glucose as a source of energy. The Pyruvate is converted
Antimalarial Drug Resistance into lactate to yield ATP in the parasite, which necessitates
Surveillance for replicating in the intraerythrocytic site.75 Initially, via
Antimalarial drug resistance surveillance can be performed GLUT1 transporter Glucose is transported from the blood
through in vivo studies such as therapeutic efficacy stu­ into the parasitized erythrocyte, which is abundant in the
dies, in vitro/ex vivo studies of cultured malaria parasites, erythrocyte membrane.76 The Plasmodium glucose trans­
and molecular studies assessing known markers of anti­ porter P. Falciparium Hexose transporter (PFHT) is essen­
malarial drug resistance. As outcomes have direct clinical tial for parasite growth and survival,77 as well as, is the
relevance in therapeutic efficacy studies it is regarded as main transporter of glucose.78 GLUT1 transporter can only
the gold standard for informing antimalarial drug resis­ transport D-glucose, while P. Falciparum Hexose transpor­
tance and for drug regimen change as well. In malaria- ter non selectively transports both D-fructose and
endemic countries, routine monitoring of antimalarial drug D-glucose. Thus, the differences between PFHT and

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GLUT1 in their interaction with the substrates, proposed Table 2 Role of Kinase in the Plasmodium Parasite Life Cycle
that selective inhibition of P. Falciparum Hexose transpor­ Type Role in the Parasite Reference
ter is a potential novel target for the discovery of new Life Cycle
antimalarial agents.79 In the previous study, Compound
Serine/threonine- Crucial for asexual blood- [79]
3361 which is a long chain O-3-hexose derivative can protein kinase, casein stage parasites
hinder the uptake of fructose and glucose by kinase 2α (PfCK2α)
P. Falciparium Hexose transporter nevertheless, it cannot
Calcium-dependent Essential for parasite [79]
hinders hexose transport by mammalian transporters protein kinase 1 survival
(GLUT1 and 5). Similarly, Compound 3361 was reported (PfCDPK1)
to hinders the glucose uptake by P. vivax of P. Falciparum
Mitogen-activated Essential for completion of [80]
Hexose transporter.80 protein kinase 2 the asexual cycle
(PfMAP-2)
Targeting the Parasite Protein Kinases cGMP dependent Essential for parasite [81]
Kinases are involved in phosphorylation, transcriptional protein kinase (PfPKA) growth and survival
control, post-transcriptional control, and protein degrada­
Orphan protein kinase Significant for asexual [82]
tion in the plasmodium parasite life cycle. So, could be the
PfPK7 stage development in
strategic targets for the development of antimalarial drugs.
humans and oocyst
The most studied Cyclin-dependent kinases (CDKs) in production in mosquitoes
Plasmodium falciparum are P. falciparum protein kinase
5 (PfPK5), 6, and P. falciparum mitogen related kinase
(PfMRK). By in-vitro study two compounds, flavopiridol cell hemoglobin, which has a key role in the attainment of
and lomoucine have shown inhibition of PfPK5, by amino acid which is essential for parasite development and
decreasing DNA synthesis and changing total RNA synth­ growth. Investigation of this degradation pathway can be
esis and parasite growth.81 a promising method for the discovery and development of
The P. falciparum kinases play a significant role in the novel antimalarial agents. This pathway is started by
parasite differentiation and growth. Amongst numerous a series of protease enzymes that digest hemoglobin into
kinases, cyclin-dependent protein kinases (CDKs) are con­ small peptides. During proteolysis, heme is released from
spicuous targets for the development of drugs, numerous hemoglobin as a toxic byproduct which is detoxified by
cyclin-dependent protein kinases selective inhibitors were conversion into hemozoin. In the previous studies, hemo­
discovered for the management of different diseases such zoin comprises about 95% of the free iron synthesized
as neurological disorders, infectious diseases, and cancers. through hemoglobin digestion.86,87 In addition, two possi­
Presently, they become a potential novel target in the ble mechanisms responsible for the degradation of hemo­
discovery and development of new antimalarial globin were reported (degradation by hydrogen peroxide
drugs.82,83 The PfCDPK4 plays a key role in the formation inside the large digestive vacuole and glutathione-
of infectious sporozoites through the sexual phase of the dependent degradation within the cytoplasm).88–90
malarial life cycle. Compound 1294 which is PfCDPK4
inhibitor, revealed an antimalarial effect with a novel Electron Transport Chain (ETC)
mechanism of action through preventing the transmission The plasmodial mitochondrial electron transport chain is
of parasites from mosquitoes to humans.84 Likewise, produced from non-proton motive quinone reductases,
Imidazopyridine derivatives have shown significant such as malate quinone oxidoreductase (MQO),
PfCDPK1 inhibitory effect with nanomolar antimalarial (DHODH), (Alternative Complex I), type II NADH dehy­
activity in both in vitro and in vivo models (Table 2).85 drogenase (NDH2, glycerol 3-phosphate dehydrogenase
(G3PDH), dihydroorotate dehydrogenase, and succinate
Food Vacuole as a Drug Target dehydrogenase (SDH, Complex II), and proton motive
The blend of digestive vesicles provides a large digestive respiratory complexes, such as ATP synthase (Complex
vacuole/food vacuole through the growth of the malaria V), cytochrome c oxidase (Complex IV), and bc1 complex
parasite inside the human erythrocytes. Food vacuole is (Complex III). The electron transport chain needs cyto­
accountable for the degradation of 60–80% of the host red chrome c1 and ubiquinone (coenzyme Q) which serve as

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electron carriers between the complexes.91–93 The pool of effect in Kenyan102 and Indonesian103 adults when received
electron transport chain and carbon metabolism antimalar­ daily doses, even though the preventive activity was lower
ial targets that have been under the lamp post in recent than doxycycline in both trials (protective efficacy in Kenya
years, as well as suggest a promising new avenue for the was 93% for doxycycline vs 83% for azithromycin; in
validation of novel drug targets for the treatment of Indonesia 96% vs 72%, respectively). In Kenya, azithromy­
malaria. The interaction between the pathways vital for cin preventive activity was fairly deprived when adminis­
the parasite, such as aspartate metabolism, mitochondrial tered weekly (64%). Mass distribution of azithromycin for
tricarboxylic acid cycle, and pyrimidine biosynthesis, is the control of trachoma was linked with a decrease in malaria
described to create a road map of novel antimalarial parasitemia as compared to controls.104 Azithromycin +
agents.94 piperaquine was well tolerated in pregnant Papua New
Guinean women,105 even though preventive efficacy data
Apicoplast as Drug Targets are not obtainable.
Recently, blocking the P. falciparum ribosome and other
parts of the translational machinery accountable for pro­ Plasmodium Proteases
tein synthesis are becoming a promising target for the Plasmodium proteases are a regulatory and ubiquitous
discovery and development of novel antimalarial agents. catalytic enzyme that play a significant role in the survival
The plasmodium species have three genomes: apicoplast, of the plasmodium parasite and responsible for the hydro­
nuclear, and mitochondrial.95 The apicoplast is lysis of the peptide bond (Figure 1).106 The role of plas­
a chloroplast like organelle of apicomplexan parasites. modium proteases in the pathogenesis of malaria disease
The apicoplast resulted from endosymbiosis, leading to includes activation of inflammation, cell/tissue penetra­
an organelle that maintains certain specific functions, tion, invasion of erythrocyte, development of the parasite,
probably including fatty acid, heme, and amino acid immune evasion, autophagy, and hemoglobin and other
metabolism.96 The apicoplast genome of P. falciparum proteins breakdown.107 Plasmodium proteases such as
comprises a 35-kb DNA which is small in size.97 The aspartate, serine, cysteine, metallo, threonine, and gluta­
apicoplast is a non-photosynthetic plastid that is vital for mate are auspicious drug targets for the treatment of
the malarial parasite since it covers a large number of malaria since the disruption of the plasmodium proteases
important metabolic biochemical pathways (biosynthesis gene inhibits the degradation of hemoglobin and the
of fatty acid, isoprenoid precursors, and heme synthesis) growth of the parasite in the erythrocyte stages.108
for the Plasmodium falciparum survival. Human beings do Proteases are generally used for the rupture and subsequent
not have these metabolic biochemical pathways which are reinvasion of erythrocytes by merozoite-stage parasites and the
important for ideal drug targeting.98,99 degradation of hemoglobin by intraerythrocytic trophozoites.
Even though the majority of proteins of this organelle are For instance, drugs that inhibit Plasmodium cysteine proteases
encoded in the nuclear genome and are subsequently trans­ are the potential targets for malarial treatment and shown
ported to the apicoplast, it also encodes a full set of tRNAs, potential effects.109 Cysteine proteases have different roles in
some ribosomal proteins, three genes for the subunits of an Plasmodium parasites including hemoglobin hydrolysis (pro­
oligomeric RNA polymerase, a gene for the elongation factor vide amino acids for parasite protein synthesis, maintain the
PfTu and a gene contributing to the Fe–S pathway.100 Since osmotic stability of malaria parasites),110 erythrocytic ruptur­
the apicoplast possess unique metabolic pathways such as ing, helps merozoites to be released,111 erythrocyte invasion
isoprenoid, heme synthesis, and fatty acid, which are not also it has a role on non-erythrocytic parasitic stages (Table 3).
found in the human,101 it could be a potential drug target
for the management of malaria. As reported in the previous Aminopeptidases
study, protein syntheses inhibitors play a key role in the Amino peptidases catalyze the cleavage of amino acids from
clinical success of potent antibiotics. Azithromycin, the amino terminus of peptides and proteins and are distributed
Clindamycin, and Doxycycline revealed antimalarial activity widely in prokaryotes and eukaryotes as either integral mem­
since they can inhibit the ribosomes within the apicoplast and brane or cytosolic proteins (Figure 1). They play a role in
Plasmodium species mitochondria, resulting in loss of the protein and peptide metabolism, activation/inactivation of bio­
normal function of these organelles.95 For prevention of logically active peptides, removal of the N-terminal methio­
malaria, azithromycin has shown a noticeable protective nine from newly synthesized proteins, and the trimming of

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Dovepress Shibeshi et al

Hemoglobin (Hb)
PDT PDT
Proteases
Amino acids

Small Peptides Aminopeptidas

Glutathione dependent e
degradation
PDT
PDT

Toxic Heme Degradation

Detoxification
Oxidation

Hematin

PDT

Hemazoin

Figure 1 Targets of proteases and amino peptidase, malaria parasite detoxification mechanism.
Abbreviation: PDT, possible drug targets.

antigens for presentation by the major histocompatibility com­ inhibit Na+-TPase 4 ion channel,116 KAF156/GNF156/
plex-1 system.112 Bestatin inhibits the growth of P. falciparum (Cyclic amine resistance unknown mechanism of locus
in vitro and in vivo and is active against the intraerythrocytic (PfCARL) inhibitor),117 Albitiazolium/SAR9727/(Inhibit
stages. Bestatin appears to inhibit both leucine aminopeptidase the transport of choline into the parasite),118 DSM265
(PfLAP) and membrane alanine aminopeptidase, (PfA-M1) by (Inhibit dihydroorotate dehydrogenase enzyme),119
chelating the active metal ions in their metal-binding centers. Methylene Blue (Prevents haem polymerisation by inhibit­
Inhibitors capable of binding compactly within the active site ing P. falciparum glutathione reductase),120 Sevuparin/
and chelating the tightly bound metal ions of both PfA-M1 and DF02/(Anti-adhesive polysaccharide derived Blocks mer­
PfLAP. It has two metal-binding sites, a readily exchangeable ozoite invasion and sequestration),121 MMV048
site, and a tight binding site may prove more potent and show (Inhibiting the parasite enzyme phosphoinositol 4-kinase
greater anti-malarial activity.113–115 enzyme),122 MMV390048 (Phosphatidylinositol 4-kinase
(PfPI4K) inhibitor),123 Fosmidomycin + piperaquine
A Recent Achievement in the (DOXP pathway), Artefenomel (oz439) + Piperaquine
Discovery and Development of (Synthetic endoperoxide),124 OZ277+ Piperaquine
Antimalarial Agents (Inhibit Pf-encoded sarcoplasmic endoplasmic reticulum
Drugs currently in Phase I, II and III trials for blood-stage calcium ATPase), P218 (PfDHFR inhibitor),125 M5717/
treatments of malaria includes KAE609 (cipargamin) DDD498/(Protein-making machinery of the malaria

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Table 3 Mechanism of Action of Protease Inhibitors therapeutic agents targeting old targets is both imperative
Name of Drug Mechanism of Action on Cultured aspects in fighting drug-resistant malaria. The future anti­
Parasite malarial drug development will better target medicines
with a distinctive mechanism of action.
Leupeptin Causes the food vacuole to swell and fill
with dark-staining material, blocks the
processing of hemoglobin, Inhibits lysis of Abbreviations
erythrocyte membranes ABC, ATP binding cassette; ACT, artemisinin combina­
Anti-pain Inhibits lysis of erythrocyte membranes tion therapy; ART, artemisinin; ATQ, atovaquone; CDKs,
cyclic dependent kinases; CLD, clindamycin; CQ, chlor­
E-64 Causes the food vacuole to swell and fill
oquine; CYC, cycloguanil; Cytb1, cytochrome b subunit 1;
with dark-staining material, inhibits lysis of
parasitophorous vacuole, Blocks the
DHA/PPQ, dihydroartemisinin/piperaquine; DHFR, dihy­
processing of hemoglobin drofoliate reductase; DHPS, dihydropteroate synthase; DV,
digestive Vacuole; G6PDd, glucose-6-phosphate dehydro­
Chymostatin (serine Inhibits erythrocyte invasion
protease inhibitor)
genase deficiency; GSH, glutathione; IPZ, imizolopipera­
zine; LMF, lumefantrine’ MFQ, mefloquine; MFS, major
facilitator superfamily; NATs, nucleic acid amplification;
parasite, liver- stage P. falciparum),126 SJ733 (The P-type PAM, pregnancy associated malaria; PCR, polymerase
Na+–ATPase transporter),116 and Spiroindolone (ciparga­ chain reaction; Pfact, Plasmodium falciparum acetyl CoA
min) inhibits PfATP4, a parasite plasma membrane Na+- transport; PfA-M1, Pf membrane alanine aminopeptidase;
ATPase that regulates sodium (maintains low-level Na+ in PfCRT, Pf chloroquine-resistant transporter; PfKelch13, Pf
the cytosol) and osmotic homeostasis. Cipargamin is used kelch like protein 13; PfLAP, Pf leuci ne aminopeptidase;
in the treatment of falciparum and vivax malaria. PfMDR1, pf multidrug resistance 1; Pfmrk, Pf mitogen
Inhibition of PfATP4 increases a Na+ in the cytosol as related kinases; pfMRP, Pf multidrug resistance-associated
Na+ moves into the cell, down its electrochemical gradient protein; PfPI3K, Pf phosphatidylinositol-3-kinase; Pfpk5,
leading to a concomitant increase in cytosolic pH Plasmodium falciparum protein kinase 5; SERCA, sarco/
(PfATP4-mediated acid load). Mutation in PfATP4 results endoplasmic reticulum Ca2+ ATPase.
in cipargamin.127 Artefenomel is a new synthetic antima­
larial peroxide that clears parasitemia rapidly in both Ethical Approval
P falciparum and P vivax malaria. It has a good safety Not applicable.
profile and long half-life (for a single dose malaria
cure).128 Acknowledgment
We would like to acknowledge the University of Gondar
Conclusion for providing materials.
In conclusion, present and future therapeutic targets for the
discovery and development of novel antimalarial agents Funding
were reviewed. The frequently emerging antimalarial drug There is no funding to report.
resistance including combination therapies globally forces
the scientists to search and develop antimalarial drugs with Disclosure
novel mechanisms of action. Resistance to two highly The authors declare that they have no competing interests.
dominant species Plasmodium falciparum and
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