Novel molecular target in malaria parasite chemotherapy; limitations and future

prospects.

A seminar paper submitted

By

unekwe queensly ifunanya

reg no; 2020312002

the department of pharmacology and therapeutics

faculty of basic clinical sciences

chukwuemeka odumegwu ojukwu university awka,campus.

In partial fulfillment of the requirement for the award of a masters degree in

pharmacology and therapeutics

Supervisors; dr azikiwe

Date;

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 SUMMARY
BACKGROUND:Malaria is a mosquito-borne disease that infects the human liver and RBCs caused by the parasite Plasmodium.
It is transmitted through the bite of infected anopheles mosquito causing symptoms that typically include fever,
fatigue,vomiting and headaches . The world health organisation’s (WHO) global initiative aims to reduce the burden of malaria
disease but has been massively challenged by the emergence of parasitic strains resistant to traditional and emerging
antimalarial drugs.
AIM: This review aimed to highlight the urgent need to develop new and effective anti-malarial molecules;which could be
achieved either by the identification of the new drugs for the validated targets, or by further refining the existing anti-malarials,
or by identifying novel targetsS for the malaria chemotherapy.
MATERIALS:Electronic databases (Pubmed connect, R Discovery, Google scholar) were searched for papers, published from
2005-2023) that reported modern advancement in the biology of the parasite and availiability of the different genomic
techniques , providing a wide range of novel targets in the development of new therapy.
RESULTS:Based on the evolving understanding of the physiology of Plasmodium, identification of potential targets for drug
intervention has been made in recent years, resulting in more than 10 unique potential anti-malarial drugs added to the
pipeline for clinical development.
CONCLUSION :The present and future therapeutic targets for the discovery and development of novel antimalarial agents were
reviewed. The frequently emerging antimalarial drug resistance including combination therapies globally forces the scientists to

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search and develop antimalarial drugs with novel mechanisms of action. This review will focus on novel targets and
therapeutics against malarial, new drugs in clinical development, challenges with antimalarials

Keywords: malaria; plasmodium; novel; anti-malarial drug

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 Chapter 1

INTRODUCTION

Malaria is a protozoal infection caused by certain species of Plasmodium . It is

transmitted from person to person through the bite of infected female anopheles
mosquito which carries the plasmodium parasite.

Malaria infection begins when an infected female anopheles mosquito bites a person,
injecting Plasmodium parasite, in form of sporozoites,into the bloodstream. The
sporozoites pass quickly into the human liver.the sporozoites multiply asexually in the
liver cells over the next 7-10days, causing no symptoms( Baer et al .,2007). In an animal
model, the parasites, in the form of merozoites, are released from the liver cells in
vesicles,journey through the heart, and arrive in the lungs, where they settle within lung
capillaries. The vesicles eventually disintegrate, freeing the merozoites to enter the
blood phase of their development. In the bloodstream, the merozoites invade red blood
cell(erythrocytes) and multiply again until the cell burst. Then ,they invade more
erythrocytes. This cycle is repeated, causing fever each time parasites break free and
invade blood cells. Some of the infected blood cells leave the cycle of asexual
multiplication. Instead of replicating, the merozoites in these cells develop into sexual
forms of the parasite, called gametocytes, that circulate in the blood stream. When a
mosquito bites an infected human, it ingests the gametocytes, which develop further into
mature sex cells called gametes. The fertilized female gametes develop into actively
moving ookinetes that burrow through the mosquito’s midgut wall and form oocysts on
the exterior surface. Inside the oocyst, thousands of active sporozoites develop. The
oocyst eventually bursts, releasing sporozoites into the body cavity that travel to the
mosquito’s salivary glands. The cycle of human infection begins again when the mosquito
bites another person(Baer et al ., 2007)

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 The six species of plasmodium known to cause malaria in human are; Plasmodium
falciparum , Plasmodium vivax , Plasmodium ovale curtis , Plasmodium ovale wallikeri ,
Plasmodium malariae, and Plasmodium knowlesi , and are transmitted by the bite of
infected female Anopheles mosquitoes which are widely distributed throughout Africa ,
Asia , and Latin America ( Ezeokoil et al 2023). However , P falciparum which,
incidentally , is the main cause of severe malaria , is also the most prevalent of the
parasites on the African continent and responsible for most malaria deaths globally
( Ezeokoil et al 2023).

In the absence of an effective vaccine, the therapeutic use of antimalarial agents remains
the only method for management and prophylaxis of malarial disease. Several studies
showed that the efficacies of most antimalarial agents compromised by the emergency of
drug-resistant Plasmodiums( Patel et al 2017). Resistance reported for nearly all
available antimalarial agents that reinforced the urgent need to develop new
antimalarial agents against existing validated targets, as well as search for novel
targets(Menard et al., 2017). The development of a novel antimalarial agent that cts
toward both transmissible gametocytes stages and the intraerythrocytic proliferative
asexual, in particular those of resistant parasite species, is urgently required(Mishra et
al., 2017). Several enzymes ,ion channels, transporters ,interacting molecules in red
blood cells (RBC) invasion, and molecules responsible for oxidative stress in the parasite
, lipid metabolism , and degradation of hemoglobin are promising novel targets to
develop new antimalarial drugs against rapidly mutating malarial parasites(Sahu et al.,
2008).

The potential of new antimalarial agents judged by several requirements: novel mode of
action with no cross-resistance to the current antimalarial agent, single-dose cures,
effective against both the asexual blood stages and the gametocytes responsible for
transmission. Besides , the new antimalarial agent should have the efficacy to prevent
infection (chemoprotective agents), and clear P. vivax hypnozoites from the liver(anti-
relapse agents)( Diagana ., 2015).

The purpose of this review is to discuss new targets in the chemotherapy of malaria
parasite which are under investigation by different organizations and researchers , and
their roles in overcoming anti-malaria drug resistance in the near future.

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CHAPTER 2

ANTI-MALARIAL DRUGS

2.1 Antimalarial drugs

Available antimalarial drugs are many , including quinolones, antifolates ,artemisinin

derivatives , naphthoquinones, trioxolanes, and antimicrobials such as lincosamide,
tetracycline, and macrolides. Unfortunately , a single antimalarial agent effective against
all species and all sexual and asexual stages of plasmodium is yet to be developed. Most
antimalarial drugs exhibit considerable stage and species selectivity(figure 1),
mandating combined antimalarial regimens for proper efficacy and to prevent the
development of resistance.

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2.1.2 current treatments

Periodic fever has been recognized since antiquity and was empirically treated by the
Quechua of Peru with ground bark of Cinchona trees, from which quinine was extracted
in 1820. Formal therapeutic testing started only in the late 19th century after Charles
Laveran discovered Plasmodium as the cause of malaria. Quinolones became the
standard therapy for malaria between the 1920s and 1940s(Gachelin et al., 2017).
Although the exact mechanism of action of these drugs is unclear, interference with
hemoglobin metabolism leading to toxic accumulation of heme and the inhibition of
glycolytic enzymes have been proposed to explain antimalarial activity in infected
erythrocytes(Foley &Tilley 1997). Quinine was the first widely used alkaloid to treat
malaria; however, its significant toxicity and the emergence of P. falciparum resistance
limited its use, promoting introduction of other quinolones including primaquine,
mepacrine, and chloroquine.

Chloroquine was developed during World war II and became the antimalarial drug of
choice due to its safety , cost and efficacy. Development of resistance by P. falciparum,
first reported in 1957(Ippolito et al., 2021) and later spreading throughout the world ,
has limited its use to selected areas. Primaquine phosphate and recently approved
single-dose tafenoquine (8-aminoquinolines) are medications with activity against
P.vivax and P. ovale hypnozoites and prevent relapse. Pyronaridine is another quinoline
derivative used in combination with artesunate with good efficacy against
uncomplicated P. falciparum malaria(Pryce et al., 2019).

Antifolates are the second class of medication to combat malaria and are considered
shizonticidals. There are three commonly used drugs in this group: sulfadoxine ,
pyrimethamine , and proguanil. They are usually combined with other antimalarial
drugs such as artesunate + sulfadoxine – pyrimethamine for treatment or atovaquone +
proguanil for chemoprevention(WHO guideline for malaria, 2022). They act mainly by
inhibiting enzymes necessary to produce active folates, which in turn serve as essential
co-factors in nucleic acid biosynthesis and cell division. Sulfadoxine inhibits
dihydropteroate synthase, while pyrimethamine and proguanil inhibit dihyrofolate
reductase.

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Artemisinin , a sesquiterpene lactone endoperoxide, and its derivatives including
artemether, dihydroartemisinin, arteether , and artesunate are first –line therapies for
uncomplicated and complicated (severe) malaria. Artemisinin was extracted in 1972
from Artemisia annua. Full understanding of its mechanism of action is evolving, but it
involves interaction with the heme group inside RBCs, generating free radical that
mediate protein alkylation and DNA damage ( Gopalakrishnan & Kumar, 2015) ,
resulting in inhibition of parasitic molecules ( calcium ATPase and proteosomes)
(Bridgford et al., 2018). However , downsides of artemisinin and its derivatives include
their short half-life , poor bioavailability, lack of activity against latent phases of the
parasiste(hypnozoites), and emergence of resistance especially in parts of Southeast
Asia and Africa. Therefore , it is recommended that artemisinin derivatives be combined
with other antimalarial agents to prevent treatment failure and recrudescence(WHO
guideline for malaria, 2022). The World Health Organisation recommends the following
artemisinin-based combination therapies (ACTs) : artemether- lumefantrine,
artesunate-amodiaquine ,artesunate-mefloquine, artesunate-pyronaridine, artesunate-
sulfadoxine-pyrimethamine, and dihydroartemisinin- piperaquine.

2.2 NOVEL TARGETS OF ANTIMALARIAL DRUGS AND THEIR LIMITATIONS

The current available antimalarial agents identified based on the major metabolic
pathway differences of the Plasmodium species with its host. The major metabolic
pathways of the parasite , including heme detoxification, fatty acid synthesis, nucleic acid
synthesis, and oxidative stress are some of the novel sites for drug design (Oyelade J ,
Isewon I , et al 2019). Though most of the antimalarial drugs used for many years ,
presently the use of such drugs is limited as a result of drug resistance. According to past
studies , there are no antimalarial agents recognized to inhibit an identified antimalarial
drug targets( Comer E, Beaudoin J.A et al 2014). In its place, the majority of the
antimalarial agents were discovered in both in vitro model and animal models(in vivo).
Thus , the exact mechanism of action of most antimalarial drugs is not known (Fidock
D.A , Nwaka .S. et al 2004). In addition , the mechanisms of emergence of resistance are
poorly understood for most of the drugs. Genetic , molecular and pharmacological
approaches have shown that different targets of older drugs are resistant due to
mutations on their key enzymes or transporters ( Rosenthal , 2005).

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2.3 THE NEED OF NEW TARGETS FOR ANTIMALARIAL DRUGS

Malaria elimination needs an integrated strategy, including new and old drugs, vaccines ,
vector control , and public health measures (Schellenberg et al 2018). Considering the
high mortality, morbidity, the emergence and spred of resistance to existing drugs, there
is no question that new drugs are required. To achieve this goal, antimalarial drug
research should focus on validated targets in order to generate new drug candidates. On
the other hand, there is a need to identify new targets for the future by studying the basic
metabolic and biochemical processes of the malaria parasite. The need for new
metabolic targets stem from two main reasons; firstly , apart from artemisinin –type
compounds and atovaquone there is an overall lack of chemical diversity in the
antimalarial drugs in use, which has led to considerable cross reistance between drugs
(Antony H.A , Parija S.C , 2016). Secondly , because of the confusing array of putative
chemotherapeutic targets, so many have not been validated.

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 CHAPTER 3

NEW MOLECULAR TARGETS FOR MALARIA PARASITE CHEMOTHERAPY.

Identification of novel drug targets and the design of new compounds acting on new
targets is nowadays widely used approach all over the world to combat issues raised by
emergency of resistance to existing drugs( Aide et al , 2019).During the last years(2019-
2022), several studies (Stanway et al 2019 ; Mbaba et al 2020; Jaromin et al 2020; Ali et
al 2021 ; Diallo et al 2021 ; Guleria et al 2021; Somsri et al 2021; Tehlan et al 2022) were
published discussing new approaches to identify novel drug targets for Plasmodium spp.
Identifying genes encoding the high metabolic activities behind rapid development of
single sporozoite(invading the liver) into thousand of daughter –merozoites(invading
RBCs) should reveal several potential drug targets.

Therefore, investigating inhibitors specific to the new target proteins of the malaria
parasite has been exploited for drug target identification. Since the unveiling of the
P.falciparum genome, several novel targets for drug intervention have emerged. These
potential antimalarial drug targets the crucial metabolite biosynthesis, membrane
transport, and signaling system , and the hemoglobin degradation processes(Deu E. ,
2017)

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1) Glucose transporter( PfHT1) as a drug target

Glucose is a source of energy for intra-erythrocytic malaria parasites in which infected

erythrocytes consume higher energy than normal erythrocytes(Mehta M , Sonawat H.M
et al 2006). P. falciparum almost fully depend on glycolycis for energy production,
deprived of energy stores; depend on continuous uptake of glucose as a source of energy.
The pyruvate is converted into lactate to yield ATP in the parasite, which necessitates for
replicating in the intra-erythrocytic site(Tilley L, Dixon M.W et al 2011). Initially , via
GLUT1 transporter, glucose is transported from the blood into the parasitized
erythrocyte, which is abundant in the erythrocyte membrane(Dickerman B.K et al
2016). The plasmodium glucose transporter P. falciparum Hexose transporter (PFHT) is
essential for parasite growth and survival( Kraft T.E ,Armstrong C et al 2015), as well as
is the main transporter of glucose(Kirk k, 2001). GLUT1 transporter can only transport
D-glucose, while P.falciparum Hexose transporter non-selectively transports both D-
fructose and D-glucose.

Thus ,the differences between PFHT and GLUT1 in their interaction with the substrates,
proposed that selective inhibition of P. falciparum Hexose transporter is a potential
novel target for the discovery of new antimalarial agents(Heitmeier M.R , et al 2019). In
the previous study , compound 3361 which is a long chain 0-3 hexose derivatives can
hinder the uptake of fructose and glucose by P. falciparium Hexose transporter
nevertheless, it cannot hinder hexose transport by mammalian transporters (GLUT1 and
5). Similarly , compound 3361 was reported to hinder the glucose uptake by P. vivax of P.
falciparium Hexose transporter.

2) Mitochondrial respiratory chain as a drug target

Although several drugs are used targeting Plasmodium mitochondrial respiratory chain
machinery, none escapes drug resistance due to gene mutations. This is because almost
all exhibit a mode of action targeting single active site of cytochrome bc1 complex. A
study designed a selective inhibitor of P.falciparum nicotinamide adenine
dinuleotide(PfNADH)with simultaneous allosteric inhibitory potency on both active sites
of PfNDH2. The investigator claimed that RYL-581 was the first report of developing
multi-targeting allosteric inhibitory drug as a novel antimalarial drug(Yang et al 2021).

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3) TARGETING THE PARASITE PROTEIN KINASES

Kinases are involved in phosphorylation transcriptional control, post- transcriptional

control and protein degradation in the plasmodium parasite life cycle. So ,could be the
strategic targets for development of antimalarial drugs. The most studied cyclin
dependent kinases (CDKs) in Plasmodium falciparum are P.falciparum protein kinase 5
(PfPK5), 6 and P. falciparum mitogen related kinase(PfMRK). By in-vitro study , two
compounds , flavopiridol and lomoucine have shown inhibition of PfPk5, by decreasing
DNA synthesis and changing total RNA synthesis and parasite growth(Jirage et al 2010).

Table 1

TYPE ROLE IN THE PARASITE LIFE CYCLE

Serine/ threonine protein kinase, casein kinase 2- Crucial for asexual blood-stage parasites
alpha(PfCK2alpha)

Calcium dependent protein kinase Essential for parasite survival

1(PfCDPK1)

Mitogen – activated protein kinase Essential for completion of asexual cycle

2(PfMAP-2)

cGMP dependent protein kinases(PfPKA) Essential for parasite growth and survival

Orphan protein kinase(PfPK7) Significant for asexual stage development

in humans and oocyst production in
mosquitoes.

Role of kinase in the plasmodium parasite life cycle.

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4) FOOD VACUOLE AS A DRUG TARGET

Food vacuole is accountable for the degradation of 60-80% of the host red cell
hemoglobin, which has a key role in the attainment of amino acid which is essential for
parasite development and growth ( Melkamu et al 2022). Investigation of this
degradation pathway can be a promising method for the discovery and development of
novel antimalarial agents. This pathway is started by a series of protease enzymes that
digest hemoglobin into small peptides. During proteolysis, heme is released from
hemoglobin as a toxic byproduct which is detoxified by conversion into hemozoin
(Melkamu et al 2022).

In the previous studies, hemozoin comprises about 95% of the free iron synthesized
through hemoglobin digestion(Banerjee et al 2002). In addition ,two possible
mechanism responsible for the degradation of hemoglobin were reported; degradation
by hydrogen peroxide inside the large digestive vacuole and glutathione- dependent
degradation within the cytoplasm(Eggleson et al 1999).

5) APICOPLAST AS DRUG TARGETS

Recently ,blocking the P.falciparum ribosome and other parts of the translational
machinery accountable for protein synthesis are becoming a promising target for the
discovery and development of novel antimalarial agents; apicoplast, nuclear , and
mitochondrial(Sheridan et al 2018). The apicoplast is a chloroplast like organelle of
apicomplexan parasites. The apicoplast resulted from endosymbiosis, leading to an
organelle that maintains certain specific functions, probably including fatty acid, heme,
and amino acid metabolism(Kohler et al 1997; Melkamu et al 2022). The apicoplast is a
non- photosynthetic plastid that is vital for the malarial parasit since it covers a large
number of important metabolic biochemical pathways(biosynthesis of fatty
acid,isoprenoid precursors, and heme synthesis) for the Plasmodium falciparum
survival. Human beings do not have these metabolic biochemical pathways which are
important for ideal drug targeting(Lim and Fadden , 2010 ; Limenitakis and Soldati ,
2011)(Melkamu et al 2022).

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6) PLASMODIUM PROTEASES AS DRUG TARGETS

Plasmodium proteases are a regulatory and ubiquitous catalytic enzymes that play a
significant role in the survival of the plasmodium parasite and responsible for the
hydrolysis of the peptide bond(Teixeira et al 2011)(Melkamu et al 2022). These
enzymes catalyze the degradation of proteins into smaller peptides and amino acids in a
process called proteolysis. Most proteins are activated or deactivated through
proteolysis for the survival of the organism(James Abugri et al 2022). A review
published in 2019, proposed 12 proteases as potential drug targets(Mishra et al 2019).
The reviewers proposed five cysteine ( falcipains/vivapains 1 and 2, serine repeat
antigens 5 and 6, dipeptidyl aminopeptidase I, three metallo( DPAP III, alanyl and leucyl
aminopeptidases), two aspartyl (plasmepsins II and V), a threonine(HsIV), and a
subtilisin- like protease. Three crucial biological processes are assigned to proteases;
hemoglobin(Hb) degradation, synthesis of protein required for growth, survival, and
differentiation, and RBCs invasion and egress(Mishra et al , 2019).

1)Aspartylproteases : Plasmodium genome showed 10 plasmepsins(PLM), however ,

only four (II, V, IX, and X) were suggested promising targets. While II and V contribute in
Hb degradation, IX and X are involved in RBCs invasion and egress cascade(Mishra et al
2019 ) . The crystal structure of PLMs II, and V showed conserved features with relative
evolutionary divergence from host aspartyl proteases that increased their potentiality as
drug targets(Favuzza et al 2020) . Their inhibition by statine and allophenylnorstatine-
based inhibitors that block Hb degradation was suggested(Belete , 2020).

II) metalloproteases (MPs): alanyl and leucine aminopeptidases(M1 and M17,

respectively) were identified contributing in Hb digestion. All PfM17 and most of PfM1
were strictly localized in the cytosol. Besides , PfM1 was expressed in three isoforms, one
of which was minimally expressed in the PV( Matthew et al 2021). Because both MPs
possess tightly bound zinc ions in their active site, the reviewer recommended
investigating zinc ion chelating agents that inactivate MPs proteolytic functions( Mishra
et al 2019).

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 In an attempt to develop inhibitors with dual activity against both MPs, a report studied
their binding sites and substrates preferences in three species, P. falciparum, P.vivax
and P.berghe. Although , the results revealed largely conserved substrate specificity
profiles, the investigators succeeded to recognize an approach that changed the
substrate length and its intra-peptide sequence, i.e high possibility to develop selective
inhibitors(Malcolm et al 2021). In the same year, the same Australian investigators
identified the metal active site of PfM17 and PvM17 that played a structural role
regulating M17 catalytic activity. The investigators demonstrated that binding of metal
ions to M17 active site regulated the dynamic equilibrium between inactive oligomers to
active hexamers. This means that conversion of inactive oligomers(mono, di and tetra)
to active hexamer is a dynamic process directionally controlled by the metal active site.
Accordingly , the study suggested M17 oligomerization a novel approach for developing
new antimalarial drug instead of using MP inhibitors that blocks M17 active site(Malcom
et al 2021).

III) Threonine proteases: Plasmodium HsIV showed high similarity and conservancy in
all species with highly different similarity to human orthologues. In addition to PLMs II
and V, PLM III previously known as histoaspartyl proteases and expressed in merozoites
digestive vacuole was recently proposed a potential drug target(Sojka et al 2021).

IV) Subtilisin- like protease 1(SUB1): it has an additional role in processing proteases
required for RBCs invasion and egress cascade. The crystal structure of Plasmodium
SUB1 showed scissile bond allowing unusual interaction of its active site with substrate
residues on prime and non –prime sides. Mature SUB1 was strongly suggested a novel
drug target(Mishra et al 2019).

7) Heat shock protein (HSP)as a molecular drug target

Since it belongs to HSP70 family, glucose- regulated protein(GRP78), also known as Bip,
was investigated as novel drug target(Chen et al 2018). It is a luminal molecule in ER to
maintain hemostasis and has essential role in protein folding and membrane
modifications. Plasmodium GRP78 proved its essentiality for schizonts and gametocytes
survival and growth. During ,stress conditions, accumulation of misfolded proteins
triggers the unfolded protein response(UPR) to restore normal ER hemostasis(Sherif,
2022).

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Recently , an American study identified P.falciparum HSP40 chaperone with a C-terminal
thioredoxin(Trx) domain, termed PfJ2 localized in ER. Besides, four protein disulfide
isomerases(PDIs), members of the Trx superfamily , with a redox regulatory role of PfJ2
were identified. Knocking down of the gene encoding PDIs revealed that PfPD18 was
essentially required for growth and survival of asexual erythrocytic stages. The
investigators suggested that PfJ2- PfPD18 interaction acted with GRP78(HSP70-BiP)to
accelerate ER protein folding. The investigators demonstrated that interaction between
ER-Pfj2 and PfPD18 could be blocked by covalent inhibitors. Therefore ,oxidative folding
process in P. falciparum EC was proposed a drug target and using these inhibitors in a
combined multi-target therapy(Cobb et al 2021).

OTHER TARGETS INCLUDE

A)The Hb trafficking pathway: phosphoinositide lipids (PPIs) play a key roles in cell
motility, and cytoskeletal reorganization. Hence , they are distributed throughout
cellular membrane, and concentrated in Golgi apparatus, and endosomes. In
Plasmodium spp, they have a crucial role in Hb trafficking pathway from infected RBCs
cytosol to the digestive vacuoles of intra- erythrocytic stages. Knocking down the gene
encoding the specific putative phosphoinositide – binding protein(PfPX1) led to
significant growth alteration. The investigators demonstrated its binding with
phophatidylinositol-3-phosphate(P13P) localized in the digestive vacuole membrane to
facilitate Hb trafficking. Therefore , Plasmodium PX1 was proposed a potential drug
target, and inhibitors of phosphoinositide metabolism through inhibition of P13K, and
P14K are currently in development as a novel antimalarial drugs(Mukherjee et al 2022).

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B) VITAMINS : possible use of the enzymatic routes of vitamins biosynthesis for the
discovery of new generation of antibiotics as novel antimalarial drugs was investigated.
Since vitamins B1(thiamine) and B6(pyridoxal) are involved in crucial metabolic process
of carbohydrates and amino acids, molecules involved in their biosynthesis were
proposed potential drug targets. Using proteomic and genomic data available for
bacteria, a study(Barra et al 2020) demonstrated that thiamin required three enzymes
(ThiM, ThiM and ThiE), while pyridoxal required only two (Pdx1 and Pdx2) . Absence of
these enzymes in humans renders them potential drug targets(Barra et al 2020).

c) HOST –DIRECTED ANTI-MALARIAL DRUGS

i) Eryptosis enchancement: similar to all intracellular protozoa, Plasmodium spp

manipulate host cell signaling pathway, eg, programmed cell death pathway. Since RBCs
life span is 115 days, they undergo apoptotic features such as nuclear apoptosis, cell
shrinkage and membrane ruffling i.e. gradual degradation(eryptosis). For rapid
establishment of P.falciparium in host RBCs, erythrocytic stages induce significant stress
on host cell signaling to delay eryptosis( Sherif, 2022 ). To avoid drug resistance for anti-
malarial drugs, a review(114) was published discussing host apoptotic signaling
pathways for future perspectives using host-directed therapies to enhance eryptosis. In
addition to eryptosis enhancement, the reviewers discussed Plasmodium molecules
expressed to manipulate host apoptotic signaling pathway through buffering
intracellular calcium levels, exporting amino acids and blocking host cellular
kinases(Boulet et al 2018)

ii) Host TK : interestingly, a new strategy was proposed utilizing the absence of TK in
Plasmodium spp. Human RBCs are known to express TK involved in phosphorylation of
the cytoplasmic domain of band 3(cdb3), a RBC’ transmembrane protein.
Phosphorylated cdb3 within RBC membrane cytoskeleton accelerates separation of
Ankyrin and spectin- based membrane cytoskeleton from the lipid bilayer, resulting in
membrane breakdown.

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 A recent study observed significant increase of cdb3 phosphorylation on maturation of
Plasmodium merozoites. Therefore , the investigators hypothesized that merozoites
might trigger host TK to increase cdb3 phosphorylation facilitating their egress, i.e
inhibition of host TK would prevent merozoites egress. Library online search for kinase
inhibitors with potential antimalarial activity revealed only one subset, Syk inhibitors.
Although , the investigators observed insignificant results, they suggested that selective
Syk inhibitors might be potential novel anti-malarial drugs without future drug
resistance. In 2020, the hypothesis utilizing host target for development of novel
antimalarial drug was achieved in a clinical trial in Vietnam and Laos(Kesely 2020) .

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 CHAPTER 4

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The role of plasmodium proteases in the pathogenesis of malaria disease includes ;
Activation of inflammation, Cell/Tissue penetration, Invasion of erythrocyte,
Development of the parasite, Immune evasion, Autophagy, and Hemoglobin and other
protein breakdown( Roy ,2017).

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Plasmodium proteases such as aspartate, serine, cysteine, metallo, threonine, and
glutamate are auspicious drug targets for the treatment of malaria since the disruption
of the plasmodium proteases gene inhibits the degradation of hemoglobin and the
growth of the parasite in the erythrocyte stages(Verma et al , 2016).

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