In order to counter the malarial parasite’s striking ability to rapidly develop drug resistance, a constant supply of novel antimalarial drugs and potential drug targets must be available. The so-called Harlow-Knapp effect, or “searching under the lamp post,” in which scientists tend to further explore only the areas that are already well illuminated, significantly limits the availability of novel drugs and drug targets. This chapter summarizes the pool of electron transport chain (ETC) and carbon metabolism antimalarial targets that have been “under the lamp post” in recent years, as well as suggest a promising new avenue for the validation of novel drug targets. The interplay between the pathways crucial for the parasite, such as pyrimidine biosynthesis, aspartate metabolism, and mitochondrial tricarboxylic acid (TCA) cycle, is described in order to create a “road map” of novel antimalarial avenues.
- Plasmodium falciparum
- drug design
- drug target validation
- protein interference
- metabolic map
“Portrait of a serial killer,” a commentary published in 2002 in Nature Journal states: “Malaria may have killed half of all the people that ever lived” . Despite the effort and funds spent on malaria eradication, it continues to infect approximately 200 million people worldwide every year and kill one in every four infected . While effective in the past, current antimalarials are becoming less and less reliable as the parasite rapidly develops drug resistance . There have been a number of extensive reviews covering the recent status of antimalarial research and parasite’s resistance [3–11]. The shared message highlighted in these articles is that a constant supply of novel antimalarials is urgently required. Similarly to the Harlow-Knapp effect described for human kinase research , the majority of the antimalarial research is currently aimed at optimization of existing drugs targeting the known and validated pathways.
The currently used antimalarial drugs can be classified into few classes based on the mode of action [3, 7]. Briefly, the groups that receive the most attention of the researchers include the artemisinins and chloroquine-like compounds, which target the food vacuole and heme processing and detoxification [13, 14], antifolates targeting the mitochondrial dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), such as proguanil [15, 16], and mitochondrial inhibitors targeting the electron transport chain and consequently the pyrimidine biosynthesis. Unfortunately, resistance has been reported for nearly all available treatments [3, 7]. Unsurprisingly, compounds such as artemisinin and quinolines that target a broad range of essential pathways within the parasite have successfully been used for nearly 40 years before the widespread of resistance had been reported. In contrast, single-target drugs, such as antifolates and atovaquone, have lost their efficacy within few years of clinical use [11, 17]. A number of promising approaches to counter the fast emerging drug resistance suggested by Verlinden et al. include extension of combination therapy to three or more orthogonal drugs, development and use of multitargeting compounds interfering with unrelated targets, and deeper look into the unexplored alternative targets . In all three cases, in order to successfully overcome the parasite’s remarkable ability to develop resistance to nearly all drugs used against it, by far, a number of novel validated drug targets must be significantly expanded.
This chapter summarizes the pool of the mitochondrial and carbon metabolism targets that have been “under the spotlight” in recent years, as well as suggest a promising new avenue for the validation of novel drug targets. We will focus on the interplay between the pathways crucial for the parasite, such as pyrimidine biosynthesis, aspartate metabolism, and mitochondrial TCA cycle, in order to create a “road map” for further antimalarial drug development.
2. The Harlow-Knapp effect
A scientific analogue of biblical “The rich get richer and the poor get poorer” can be rephrased as “the propensity of the biomedical and pharmaceutical research communities to focus their activities, as quantified by the number of publications and patents, on a small fraction of the proteome”  or the “Harlow-Knapp effect.” It was first noted by Harlow and colleagues  and further expanded by Knapp group , based on the analysis of the amount of publications and patents featuring human protein kinases. Kinases are known to regulate the majority of the cellular pathways including those involved in cancer and other diseases. It was observed that despite the availability of human kinome  more than three quarters of protein research was still focused on just 10 per cent of the kinases that were already known before the kinome publication . Edwards and co-workers have also noticed that “the availability of research tools influences a protein’s popularity.” In other words, scientists tend to further explore the well-known systems, ignoring the less studied biomolecules where the probing tools are yet unavailable.
The availability of such tools for each system greatly limits the research opportunities and the attention to said system. Antimalarial research is not an exception to Harlow-Knapp effect: a limited opportunity for genetic manipulation  and complex life cycle of the parasite makes novel drug target validation highly challenging. Similarly to the human kinase research scientists tend to “keep looking under the spot light” among the few already validated targets, such as mitochondrial bc1 complex in malaria (target of the widely used Atovaquone), trying to optimize the existing compounds. Since first mentioned in the literature, there have been published more than 40 articles featuring plasmodial bc1 complex  and to the date it remains one of the most cited plasmodial enzyme.
Dihydroorotate dehydrogenase from
This divergent approach should be further exploited for other targets in order to yield novel and more potent scaffolds and support the antimalarial research.
3. Combinational therapy
The compound artemisinin and its derivatives have long been considered the most active and potent antimalarials for their efficacy against nearly all parasite stages [9, 14]. Artemisinins are believed to cause alkylation of proteins and heme and lead to oxidative damage within the parasite as well as affect the heme-related detoxification, although the exact mode of action is still a subject of debate [9, 14, 28]. Artemisinin-based combination therapy (ACT) is still recommended by World Health Organisation (WHO) for the treatment of uncomplicated falciparum and non-falciparum malaria in nearly all areas . ACT implies the use of the fast acting artemisinin component, responsible for the rapid parasitemia clearance, in combination with another long-acting drug partner to eliminate the remaining parasites and suppress the selection of artemisinin resistance . Despite the recent widespread of artemisinin-resistant falciparum malaria in Southeast Asia , the proven efficacy of combination therapy suggests that there is a pressing need for greater variety of highly effective antimalarial compounds. Combination of two or more drugs with different mode of action and resistance mechanisms significantly lowers the chances of the parasites to develop resistance to such treatment . Thus, the research focus should be extended from optimization of existing compounds to development of novel research tools in order to explore and dissect other potentially druggable pathways of the parasite and thus bypass of the Harlow-Knapp effect. As stated by Verlinden et al.: “History has clearly indicated that new antimalarials must be continually developed in the ensuing event of resistance development to the current antimalarial arsenal.” The occurrence of drug resistance in malaria is significantly faster than the development of antimalarials . Thus, a constant supply of novel unrelated antimalarial compounds with orthogonal modes of action is urgently required.
4. The mitochondria as drug target for
P. falciparum malaria
Mitochondria are organelles that act as the power plants of the cell, as they produce energy for all cellular activities. There are several molecular and functional differences between the mitochondria of
5. Electron transport chain (ETC)
The plasmodial mitochondrial electron transport chain (ETC) is composed of non-proton motive quinone reductases, such as dihydroorotate dehydrogenase (DHODH), malate-quinone oxidoreductase (MQO), glycerol 3-phosphate dehydrogenase (G3PDH), type II NADH dehydrogenase (NDH2, Alternative Complex I), and succinate dehydrogenase (SDH, Complex II), and proton motive respiratory complexes, including bc1 complex (Complex III), cytochrome
5.1. Dihydroorotate dehydrogenase (DHODH)
Given the essential role of the
5.2. Cytochrome bc1 (complex III)
The cytochrome bc1, also known as ubiquinol:cytochrome c oxidoreductase or complex III, is the only enzyme complex common to almost all respiratory ETCs . This complex is composed of 11 different polypeptides, and its catalytic core is composed of three subunits, namely cytochrome b, cytochrome c1, and Rieske protein, also known as iron-sulfur protein (ISP) [66–68]. Cytochrome bc1 is found in the inner mitochondrial membrane and functions as a transporter of protons into the intermembrane space through the oxidation and reduction of ubiquinone in the Q cycle [67–70]. This enzymatic complex contains two distinct binding sites for the reduction and oxidation of ubiquinol and ubiquinone, both located within cytochrome b. The Qo site acts to oxidize ubiquinol near the intermembrane space, whereas the Qi site binds and reduces ubiquinone near the mitochondrial matrix [71, 72].
Although the crystal structure of plasmodial bc1 complex has not been solved, the high degree of sequence homology with other organisms of which the X-ray crystal structure is known (e.g.
Aside of atovaquone, other bc1 complex inhibitors were described, as acridones , quinolones [79–81], pyridones [82, 83], and benzene sulfonamides . Although many compounds have presented inhibitory potential against bc1 complex, this target might be considered underexploited, since the majority of these compounds target the Qo site . The Qi site of cytochrome bc1 has been far less explored and only the binding of a few compounds has been reported [86–89].
5.3. Type II NADH dehydrogenase (NDH2)
Instead of the canonical multimeric complex I, or NADH:dehydrogenase, found in mammalian mitochondria, the
So far, no crystal structure of the
In more recent efforts, Biagini et al.  undertook a high-throughput screen (HTS) against
Although the recent efforts to inhibit NDH2 with antimalarial purposes have been a good improvement in the knowledge of its potential as a drug target, the report of
5.4. Mitochondrial glycerol-3-phosphate dehydrogenase (mG3DH)
Mitochondrial glycerol 3-phosphate dehydrogenase (mG3DH) is a ubiquinone-linked flavoprotein embedded in the mitochondrial inner membrane that transfers reducing equivalents directly from glycerol 3-phosphate into the electron transport chain [108, 109]. The
5.5. Succinate dehydrogenase (SDH)
The succinate dehydrogenase (SDH), also known as succinate: ubiquinone oxidoreductase (SQO) or complex II, is an enzymatic complex involved in both TCA cycle, functioning as a primary dehydrogenase, and in mitochondrial ETC, functioning as electron donator . This dual role makes SDH a direct connection between major systems in aerobic energy metabolism. The enzyme has been isolated and characterized from prokaryotic [114–117] and eukaryotic organisms [118–121], including
5.6. Malate-quinone oxyreductase (MQO)
The malate-quinone oxidoreductase (MQO) is a peripheral membrane-bound flavoprotein, which catalyzes the oxidation of malate to oxaloacetate, reducing ubiquinone .
Although malaria parasites generate most of their ATP through aerobic glycolysis during the blood stage of their life cycle, they appear to possess a complete ATP synthase complex .
6. Tricarboxylic acid (TCA) cycle
Further metabolomic studies suggest that
Recently, Ke et al.  reported significant flexibility in TCA cycle metabolism of
Although the fully functional TCA cycle appears to be dispensable for parasite survival in asexual blood stages , the interplay of some TCA enzymes with other essential pathways still represents an interesting target for antimalarial drug development. Below, we describe the role of three enzymes (aspartate aminotransferase, malate dehydrogenase, and fumarate hydratase) in
6.1. Aspartate aminotransferase
The enzyme aspartate aminotransferase (AspAT) catalyzes the reversible reaction of l-aspartate and α-ketoglutarate into oxaloacetate and l-glutamate. The AspAT from
6.2. Malate dehydrogenase
The enzyme malate dehydrogenase (MDH) catalyzes the reversible NAD(P)+-dependent oxidation of oxaloacetate to malate. Like other members of the NAD+-dependent dehydrogenase family, the MDHs possess two functional domains, the catalytic domain and the NAD+-binding domain. Protozoan MDHs are differentiated into two subdivisions: mitochondrial and cytosolic MDHs, the first being part of the TCA cycle, providing oxaloacetate for the generation of citrate and NADH to fuel the mitochondrial electron-transport chain. The mitochondrial MDH is absent in
The crystal structure of
6.3. Fumarate hydratase
Fumarate hydratase (FH) is an enzyme that catalyzes the reversible conversion of fumarate to malate. Although
Fumarate is a side product of the purine salvage pathway and acts as metabolic intermediate of the TCA cycle. As previously mentioned,
7. Pyrimidine biosynthetic pathway
A key-step for spreading of malaria parasites in the human host is the extensive and rapid replication of parasite DNA, which depends on the availability of essential metabolites, such as pyrimidines [154, 155]. In the
7.1. Carbamoyl phosphate synthetase II
Carbamoyl phosphate synthetase II (CPSII) is responsible for the first step of the
7.2. Aspartate transcarbamoylase (ATC)
Aspartate transcarbamoylase (ATC, EC 220.127.116.11) catalyzes the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate. Previous studies with human tumor tissues showed significantly elevated levels of ATC nearly in all samples . In
Similarly to CPSII,
7.4. Orotate phosphoribosyl transferase and orotidine 5′-monophosphate decarboxylase
The last two steps of the pyrimidine biosynthesis in
The enzyme orotate phosphoribosyl transferase (OPRT) catalyzes the formation of orotidine 5′-monophosphate (OMP) from α-D-phosphoribosyl pyrophosphate (PRPP) and orotate, the fifth step of the pyrimidine biosynthesis . The OPRT inhibitors reported so far includes the compound 5′-Fluoroorotate, an alternative substrate for this enzyme that was shown to inhibit the
A recent study of the transition state analogues of
Recently, the crystal structure of
Orotidine 5′-monophosphate decarboxylase (OPDC) catalyzes the final step of
8. Protein interference assay (PIA) as drug validation tool
We have recently proposed a novel promising drug-target validation approach that relies on common feature of all biological systems—oligomerization . Oligomerization is a self-assembly of two or more copies of one protein molecule (or different molecules) into one object. Recent analysis shows that majority (60%) of non-redundant protein structures available in the Protein Data Bank (PDB) represent dimerization or higher oligomerization order (Hashimoto
Another important aspect of oligomerization is remarkable selectivity and binding affinity. Large surface area of the intraoligomeric interfaces and evolutional diversity allow oligomeric partners selectively bind to each other with no cross-reactivity in the system. In majority of cases, purification of oligomeric proteins from both native and recombinant sources can be performed without any foreign protein incorporations in the assembly. Unlike the active sites and cofactor binding sites where evolutionary constraints restrict the sequence diversity to retain the function, oligomeric interfaces are significantly less conserved among homologous proteins [195, 196]. Thus, small molecule compounds reacting with the conserved active site of target enzyme of the parasite will likely interact with the host’s homologous enzyme.
Direct interference with protein self-assembly would provide an opportunity for a highly selective modulation of protein activity or function both
9. Making (breaking) bad proteins
The recently proposed protein interference assay (PIA)  involves the utilization of structural knowledge (data) and mutagenic modification of one (or more) partner proteins in the assembly. These modifications may affect the binding site for a cofactor, catalytic activity, or disrupt the oligomeric interface of the target protein. Thus, recombinant and, most importantly, controlled co-expression of both wild type and its inactive (hyperactive) mutant would allow the formation of the complex with modified activity
Previously mentioned homodimeric
Despite the obvious limitation of PIA approach to oligomeric proteins, this assay would still allow partial assessment of the system of interest, as many of the studied pathways are likely to involve at least one oligomeric assembly. We suggest that PIA would also allow re-evaluation of the previously studied promising targets where conventional validation approaches have failed.
In order to assess a gene’s product role, one must possess a set of tools, such as genetic manipulations (e.g. knockout, silencing etc.), to modulate the target function
In addition, the use of small molecule inhibitor approaches
Insufficient amount of effective target validation tools significantly limits the understanding of human pathogenic systems and hinders the rate of novel drug development. A constant supply of robust and effective techniques is needed in order to successfully dissect yet unexplored parasitic pathways, provide the basis for rational drug design, and counter-balance the ability of many human pathogens to rapidly develop drug resistance. We believe that protein interference assay (PIA) will enrich the currently available research toolset.
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