**2. Molecular markers of resistance to Quinoline-based drugs**

#### **2.1** *P. falciparum* **chloroquine resistance transporter (***pfcrt***)**

The *P. falciparum* Chloroquine Resistance Transporter (*pfcrt*) gene is a putative transporter, has a weight of 49 kDa, is a member of the drug transporter superfamily, and localized to the parasite digestive vacuole [1, 2]. Mutations within the *pfcrt* are the primary responsible for resistance to chloroquine. This was identified after a genetic cross experiment between CQ-sensitive HB3 and CQ-resistant Dd2 clones. Genetic analysis of the CQ-resistant progeny identified mutation in a single genetic locus on chromosome 7.A quantitative trait loci (QTL) analysis mapped a mutation on the 13-exon of the *pfcrt* gene [1, 3]. Studies by [4] have confirmed that mutation in the *pfcrt* gene is associated with chloroquine resistance in a genome-wide association study. A single mutation, resulting in the change in amino acid from K76T confers resistance to CQ in both labs adapted and field isolated *P. falciparum* strains. Removal of this mutation in CQ-resistant strains (Dd2 from Southeast Asia and 7G8 from South Africa) resulted in the total loss of resistance to chloroquine in these strains [5].

The mechanism of CQ resistance after the replacement of a positively charged lysine (K) with a neutral threonine (T) results in the expulsion of deprotonated CQ out of the digestive vacuole. The expulsion is achieved through either active transport or facilitated diffusion. This results in decreasing access of the CQ to heme, which is its target [6].

There are other mutations in the *pfcrt* gene which introduce different amino acids in the wild-type amino acids CVMNK, which compensates for the altered PfCRT function due to *pfcrt* K76T mutation and may subsequently modulate drug susceptibility in the parasite. These mutations occur in the surroundings of K76T (position 72–76). These mutations that occur at positions 72–76 may be unique to a particular geographic location. For example, the CVIET mutations at positions 72–76 are mostly found in parasites from Africa and Southeast Asia, while the SVMNT mutations at position 72–76 are found in South America, the Philippines, and Papua New Guinea [7].

#### P. falciparum *and Its Molecular Markers of Resistance to Antimalarial Drugs DOI: http://dx.doi.org/10.5772/intechopen.98372*

The use of *pfcrt* K76T mutations in epidemiology surveillance does not only apply to chloroquine resistance but also some partner drugs used in artemisininbased combination therapy. For example, the introduction of mutant *pfcrt* into CQ-sensitive GC03 strain resulted in reduced susceptibility to both amodiaquine and its primary metabolite desethylamodiaquine (DEAQ ) [8]. Studies conducted by [9] using field isolates showed the selection of *pfcrt* K76T in AQ recrudescence treatment outcome. Parasite resistance to AQ or DEAQ is not solely dependent on *pfcrt* mutation, but rather a combination of mutation(s) in both the *pfcrt* and *pfmdr1* gene [10].

The *pfcrt* K76T mutation does not only results in resistance to CQ and AQ but also results in increased susceptibility to lumefantrine [11], quinine, halofantrine, mefloquine, artemisinin and its derivatives [8, 12].

## **2.2** *P. falciparum* **multidrug resistance protein 1**

The *P. falciparum* multidrug resistance protein 1 (*pfmdr1*) is a member of the ATP-binding cassette (ABC). The *pfmdr1* is also known as the P-glycoproteins homolog 1 (Pgh-1) [6]. The PfMDR1 is localized in the membrane of the DV. The PfMDR1 is a transporter and functions by regulating drug accumulation in the parasite's DV [6].

The *pfmdr1* plays a very important role in the parasite response to different antimalarial drugs. The two mechanisms used by the *pfmdr1* gene to regulate antimalarial drug response are through increased *pfmdr1* copy number or by introducing mutations in the gene. Increased copy number of *pfmdr1* has been associated with reduced *in vitro* susceptibility to halofantrine, quinine, mefloquine, dihydroartemisinin, and artesunate [13]. Most importantly, increased *pfmdr1* copy number in clinical isolates is the cause of mefloquine monotherapy [14] or artesunate-mefloquine combination treatment failures [15]. The validation of increased *pfmdr1* copy number and its involvement in mefloquine, lumefantrine, halofantrine, quinine, and artemisinin resistance was proven in an experiment that involved the knockout of one of the two copies of drug-resistant FCB strains, resulting in the reversal of its resistance to make it susceptible to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin [16].

The polymorphisms which occur in different haplotypes of *pfmdr1* result in resistance to different antimalarial drugs. These mutations alter the substrate specificity of *pfmdr1* [17]. The *pfmdr1* N86Y mutation has been associated with CQ and AQ treatment failure, although the association to CQ is weak [18]. The *pfmdr1* D1246Y have been reported to be involved in resistance to AQ/DEAQ. In East Africa, the *pfmdr1* 86Y-184Y-1246Y haplotype was selected for an AQ recrudescence treatment outcome [19]. In other studies using field isolates from Columbia observed high AQ IC50 for parasites with *pfmdr1* D1246Y [20]. The *pfmdr1* N86-F184-D1246 haplotype is associated with resistance to lumefantrine in Africa [21, 22] whiles the *pfmdr1* N1042D was associated with increased in vitro lumefantrine IC50 values in isolates from the Thai-Myanmar border [23]. The *pfmdr1* S1034C/N1042D/D1246Y mutations are associated with reduced susceptibility to quinine [24]. The *pfmdr1* and *pfcrt* alleles may interact to confer higher resistance to AQ and DEAQ [10].

#### **2.3** *P. falciparum* **multidrug resistance-associated protein (PfMRP)**

The *P. falciparum* Multidrug Resistance-Associated Protein (*pfmrp*) belongs to the ABC transporter family [25]. The *pfmrp* acts as a transport regulatory protein. Mutations in the *pfmrp* have been associated with resistance to some antimalarial

drugs such as quinine and chloroquine [25]. The Y191H and A437S have been shown to have a weak association to CQ-resistance in Asia and the Americas respectfully, while Y191H and A437S are associated with quinine resistance in the Americas. Recent studies have also reported the selection of *pfmrp* 856I alleles following the use of artemether-lumefantrine for the treatment of malaria [26]. The *pfmrp* 1466 K has been reported in sulfadoxine-pyrimethamine recrudescence treatment outcome [26].

The validation of the contribution of *pfmrp* to quinine and CQ resistance was reported by [27] after showing that knock out of PfMRP in CQ-resistant strain W2 rendered the parasite to be susceptible to CQ and quinine. Parasite with disrupted PfMRP also showed reduced IC50 values for primaquine, piperaquine, and artemisinin. The reduced IC50 for these drugs was modest, showing a reduced IC50 ranging from 38–57%. These may suggest that *pfmrp* might act as a secondary determinant in the modulation of parasite resistance to these antimalarial drugs [28].

#### **2.4** *P. falciparum* **Na+/H + exchanger 1 (***Pfnhe-1***)**

The *P. falciparum* Na+ /H+ exchanger 1 (*Pfnhe-1*) gene is a putative Na+ /H+ exchanger found on chromosome 13 in the parasite genome. Some polymorphisms in the *pfnhe-1* are involved in resistance to some antimalarial drugs whiles other polymorphisms result in increased susceptibility to other antimalarial drugs [3]. Parasites with the D-and N-rich polymorphism (microsatellite ms4760–1) have been reported to be resistant to quinine in clinical isolates from Asia, Southeast Asia, and Central and South America [3]. Resistance to quinine by this locus is ambiguous, with some scientists reporting increased quinine IC50 values in one study [29], and decreased quinine IC50 values in another study [30].

The destruction of *pfnhe-1* in CQ and quinine resistant parasite strains 1BB5 and 3BA6 lead to an approximately 30% decrease in quinine mean IC50 values, but the knockdown of *pfnhe-1* in CQ-sensitive GC03 strain did not lead to the reduction in quinine mean IC50 values [31]. These results suggest that *pfnhe-1* contributes to quinine resistance in a strain-specific manner, and also other parasite genetic background factors are required for quinine resistance in parasites [28].

#### **2.5 Plasmepsin II &III (***pfmp2* **and** *pfmp3***)**

The plasmepsins are aspartic proteases in *P. falciparum* that are involved in the degradation of hemoglobin. They are approximately 38-kDa in weight. The *pfmp2* and *pfmp3*cleave hemoglobin in the parasite's digestive vacuole [32]. Piperaquine, an aminoquinoline drug targets the *pfmp2* and *pfmp3* to inhibit them as its mode of action. An increase in *pfmp2* and *pfmp3* copy numbers have been associated with piperaquine resistance [33].

#### **3. Molecular markers for resistance to antifolates**

The antifolates used in malaria treatment are pyrimethamine, sulfadoxine, and proguanil. The proguanil is a cycloguanil metabolite that functions by interfering with folate metabolism [34]. The mode of action of pyrimethamine and cycloguanil is by inhibiting the dihydrofolate reductase (DHFR) enzyme, whiles sulfadoxine acts by inhibiting the dihydropteroate synthase (DHPS) enzyme, all involved in the folate metabolism pathway [34]. The sulfadoxine–pyrimethamine is used in a combination therapy to treat CQ-resistant parasites mostly in pregnant women in most malaria-endemic countries in Africa [34, 35].

#### P. falciparum *and Its Molecular Markers of Resistance to Antimalarial Drugs DOI: http://dx.doi.org/10.5772/intechopen.98372*

Mutations in the DHFR are associated with resistance to pyrimethamine and cycloguanil, while mutations in DHPS are associated with sulfadoxine [36]. The *pfdhfr* S108N, N51I, C59R, and I164L are associated with pyrimethamine resistance, while *pfdhfr* A16V/S108T confers greater resistance to cycloguanil compared to pyrimethamine [37]. The quadruple mutant (S108N/N51I/C59R/ I164L combination), which is mostly found in Asia but rare in Africa confers high levels of resistance to sulfadoxine–pyrimethamine [38]. The *pfdhps* S436A/F, A437G, K540E, A581G, and A613S/T mutations have been associated with resistance to sulfadoxine, with the *pfdhps* A437G mutation observed either alone or in combination with other mutations in field isolates [39]. The amplification of GTP-cyclohydrolase I, a gene involved in the upstream biosynthesis of folate is mostly seen with the *pfdhfr* I164L mutation in *P. falciparum* clinical isolates, and this is taught to compensate for the reduced efficiency of the *pfdhfr* I164L mutation in the parasite [40].
