Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

future science group 10.4155/fmc-2017-0250

Review

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Plasmodium falciparum dihydroorotate

dehydrogenase: a drug target against
malaria
Lucas VB Hoelz2 , Felipe A Calil1 , Maria C Nonato, Luiz CS Pinheiro2 & Nubia Boechat*,2
1
Laboratório de Cristalografia de Proteı́nas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
Avenida do Café s/n Monte Alegre, 14040-903 Ribeirão Preto, SP, Brazil
2
Departamento de Sı́ntese de Fármacos, Instituto de Tecnologia em Fármacos, Farmanguinhos – FIOCRUZ, Fundação Oswaldo
Cruz. Rua Sizenando Nabuco 100, Manguinhos, Rio de Janeiro, RJ 21041-250, Brazil
*Author for correspondence: [email protected]

Malaria remains one of the most lethal infectious diseases worldwide, and the most severe form is caused
by Plasmodium falciparum. In recent decades, the major challenge to treatment of this disease has been
the ability of the protozoan parasite to develop resistance to the drugs that are currently in use. Among P.
falciparum enzymes, P. falciparum dihydroorotate dehydrogenase has been identified as an important tar-
get in drug discovery. Interference with the activity of this enzyme inhibits de novo pyrimidine biosynthesis
and consequently prevents malarial infection. Organic synthesis, x-ray crystallography, high-throughput
screening and molecular modeling methods such as molecular docking, quantitative structure–activity
relationships, structure-based pharmacophore mapping and molecular dynamics simulations have been
applied to the discovery of new inhibitors of P. falciparum dihydroorotate dehydrogenase.

First draft submitted: 20 October 2018; Accepted for publication: 17 May 2018; Published online:
18 July 2018

Keywords: antimalarial • dihydroorotate dehydrogenase • malaria • PfDHODH • Plasmodium falciparum

According to the WHO, 212 million cases of malaria occurred worldwide in 2015; these led to 429,000 deaths,
mainly among children under 5 years of age in African countries. Malaria is found in both tropical and subtropical
regions of the planet and in 91 countries. The increasing number of malaria cases that have occurred in first-world
countries due to globalization have drawn attention to this disease, which, together with AIDS and tuberculosis,
represents one of the most serious global public health problems [1].
Malaria is caused by a protozoan of the genus Plasmodium, of which five species are infectious to humans:
Plasmodium falciparum, which produces the most severe form of malaria; Plasmodium vivax, Plasmodium malariae,
Plasmodium knowlesi and Plasmodium ovale, which are divided into P. ovale wallikeri and P. ovale curtisi. These
species are primarily transmitted by the bite of infected female mosquitoes of the genus Anopheles [2–4].
Vector control, chemoprophylaxis and chemotherapy with antimalarial drugs are the primary methods used to
eliminate or reduce the number of cases of malaria [5]. Most antimalarials operate via mechanisms that target one
or two phases of the parasite’s life cycle. Several drugs are available, each of which acts at a different phase of the
parasite’s life cycle to prevent development of the parasite in the host [6]. However, the ability of Plasmodium species
to evade the action of current drugs by developing resistance has become a great challenge to malaria treatment in
recent decades, requiring the discovery of more available and effective drugs [7–9].
The antimalarial drugs that are currently in use fall into three main classes: quinoline derivatives, antifolates and
artemisinin derivatives (Supplementary Figure 1) [9–12].

• Quinoline derivatives. Quinine, an alkaloid isolated from Cinchona bark, was the first compound used to
treat malaria. Its use led to the development of synthetic derivatives such as chloroquine (CQ), amodiaquine,
primaquine, mefloquine, pyronaridine and piperaquine (Supplementary Figure 1). Cases of resistance to these
drugs have also been reported [9,13]. The quinolines are active against the erythrocytic forms of P. falciparum and
P. vivax. CQ was originally the most effective drug and has been the first choice for antimalarial treatment for
 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

a long time, but abusive use has led to the emergence of CQ-resistant parasites, rendering this drug ineffective
in many regions of the world [13]. If used against P. vivax and P. ovale hypnozoites, primaquine, which inhibits
the formation of gametocytes, acts against the slowly developing hepatic forms of P. vivax infection that are
responsible for relapses [9].
• Antifolates. These compounds constitute a class of antimalarials that act as schizonticides in the blood and are
divided into classes I and II (Supplementary Figure 1):
Class I. Sulfadoxine, which belongs to the type I class of antifolate drugs, has a structure similar to that of
p-aminobenzoic acid. Sulfadoxine interrupts the formation of dihydrofolic acid by inhibiting dihydropteroate
synthase, which is necessary for the synthesis of nucleic acids [11].
Class II. Cycloguanil and pyrimethamine belong to the type II class of antifolate drugs; they inhibit dihydrofolate
(DHF) reductase in the parasite, thereby preventing the reduction of DHF to tetrahydrofolate, which is important
in the synthesis of nucleic acids and amino acids. DHF reductase inhibitors are potent schizonticidal agents that
act on asexual forms of the parasite. The use of this class of drugs has been reduced due to the capacity of the
parasites to develop resistance [10,11].
• Artemisinin and its derivatives. Dihydroartemisinin, artemether, arteether and artesunate are known for their
ability to rapidly reduce the number of parasites present (Supplementary Figure 1). These drugs are poorly
effective as monotherapies for treatment of malaria due to their low bioavailability and short half-life, and due to
cases of resistance, their use is primarily indicated as part of artemisinin-based combination therapy [14,15]. The
endoperoxide bridge of these compounds can undergo reductive cleavage in the presence of ferrous ions from the
heme group of hemoglobin, thus generating free radicals that alkylate or modify the proteins of the parasite and
lead to its death [6]. These drugs are blood schizonticides and act on the gametocytes, thus limiting transmission
to other hosts and reducing the spread of resistant forms (Supplementary Figure 1) [16].
• An antimalarial drug that is used in combination with proguanil for the treatment of malaria is atovaquone
(Supplementary Figure 1). This hydroxyl-1,4-naphthoquinone derivative inhibits oocyst development in the
mosquito and pre-erythrocytic development in the liver and interferes with cytochrome electron transport.

Nucleotide biosynthetic pathways

Nucleotide biosynthesis can occur in two distinct ways in nature: via the de novo pathway or via salvage pathways.
Amino acids, ribose-5 -phosphate, CO2 and NH3 are the metabolic precursors with which de novo synthesis is
initiated. It is notable that the free nitrogen bases do not act as intermediates in the de novo pathways. The purine
ring is constructed while it is bound to the ribose; during this process, one or a few atoms are added at a time.
In contrast, the pyrimidine ring is synthesized as orotate bound to the ribose and is subsequently converted to
the pyrimidine nucleotides required by the cell. In contrast, the salvage pathways recycle the free bases and the
nucleosides released during the breakdown of nucleic acids [17].
Although many parasites are unable to synthesize purine nucleotides through de novo biosynthesis and instead use
the salvage pathways for this purpose, most nonparasitic organisms possess the de novo pathway for production of
pyrimidine nucleotides. In addition, many organisms that affect human health, such as the bacterium Helicobacter
pylori (responsible for stomach ulcers) and the parasite P. falciparum (responsible for malaria), do not possess the
salvage pathway for pyrimidine nucleotides [18,19].
Inhibitors of the de novo synthesis of pyrimidine nucleotides form an important group of chemotherapeutic
agents, and cells that depend on this metabolic pathway are susceptible to such inhibitors. T-lymphocytes and
cancer cells, for example, depend on this pathway to support DNA synthesis, glycosylation of proteins and
synthesis of membrane lipids. Inhibitors of this metabolic process have been used in the treatment of cancer and to
prevent rejection of transplanted organs [20].
Although the primary structures of the enzymes that make up this metabolic pathway differ considerably among
prokaryotes, protozoa, fungi and animals, the de novo biosynthesis of pyrimidines represents one of the earliest
metabolic processes, and all six of its steps have remained intact during evolution [21].
De novo synthesis of pyrimidine nucleotides begins with formation of the carbamoyl phosphate molecule by
the enzyme carbamoyl phosphate synthetase. In the second step, which is catalyzed by aspartate transcarbamylase,
carbamoyl phosphate reacts with aspartate to release N-carbamyl aspartate. This step is tightly regulated in bacteria,
and aspartate transcarbamylase is one of the best-studied allosteric enzymes. In the third step of pyrimidine
nucleotide synthesis, the pyrimidine ring is closed to form dihydroorotate by the removal of water from N-
carbamyl aspartate in a reaction that is catalyzed by dihydroorotase. In the fourth step, dihydroorotate is oxidized

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Review Hoelz, Calil, Nonato, Pinheiro & Boechat

to release orotate, a pyrimidine derivative. This reaction is catalyzed by dihydroorotate dehydrogenase (DHODH).
Once orotate is formed, the ribose-5-phosphate side chain supplied by 5-phosphoribosyl-1-pyrophosphate is bound
to it to release the orotidylate in a reaction mediated by orotate-phosphoribosyl transferase. Finally, orotidylate-
phosphate decarboxylase catalyzes the decarboxylation of orotidylate to generate uridylate, which leads to the
formation of all other pyrimidine nucleotides (Supplementary Figure 2) [22].

Dihydroorotate dehydrogenases
The fourth enzyme that acts in the de novo biosynthesis of uridylate, the precursor of all pyrimidine nucleotides,
is DHODH (EC 1.3.1.14, 1.3.1.15, 1.3.5.2 or 1.3.98.1, depending on the type); it catalyzes the oxidation of
dihydroorotate to orotate according to a ping-pong-type enzymatic mechanism [23–25].
DHODH was first detected in 1953 by Lieberman and Kornberg in extracts of the anaerobic bacterium Zymobac-
terium oroticum (now known as Clostridium oroticum) [26]. Over the past 30 years, DHODH has been identified as
the pharmacological target of a number of chemical and natural compounds such as Arava R
(leflunomide, which is
approved for the treatment of rheumatoid arthritis in humans), isoxazole, triazine, bicinchoninic acid and quinone
derivatives [27–29]. These compounds interfere in uncontrolled reactions of the immune system, assist in fighting
parasitic infections such as malaria and boost antiviral therapies by decreasing the intracellular concentration of
pyrimidine nucleotides [30]. Currently, interest has arisen in exploiting DHODH inhibition as a strategy to combat
a broad range of diseases [31–38], including for malaria where the triazolopyrimidine DSM265 has been advanced
to clinical development [35–38].
In the performance of its biological functions, DHODH uses flavin mononucleotide (FMN) as a cofactor. In
the initial phase of the enzymatic reaction, FMN is reduced and the dihydroorotate substrate is oxidized. In the
second half of the reaction, FMN is reoxidized (FMNH2 is converted to FMN) with the aid of a third molecule
that acts as an electron acceptor (Figure 1) [39].
According to their primary structures and cellular locations, the DHODH enzymes of various organisms can
be divided into two classes, Class 1 and Class 2 [40]. Class 1 enzymes can be subdivided into Classes 1A, 1B
and a new class (1S) that was found in Sulfolobus solfataricus [41]. The Class 1 enzymes are found in the cytosol,
whereas enzymes belonging to Class 2 are associated with cytosolic or mitochondrial membranes [42,43]. Due to their
association with membranes, all members of Class 2 possess an extension in the N-terminal region known as the
membrane domain; this extension allows interaction of the enzymes with the membrane [44,45]. Enzymes belonging
to Class 1 are found in Gram-positive bacteria, anaerobic fungi and in lower eukaryotes such as trypanosomatids. In
contrast, enzymes belonging to Class 2 are found in eukaryotes and in certain prokaryotes such as Gram-negative
bacteria. The division of DHODHs into two classes also correlates with the preferences of the enzymes for different
electron acceptors and with their oligomeric states. The enzymes of Class 1A are homodimeric; as oxidizing agents,
they use oxygen molecules or molecules that are soluble in water, such as fumarate, which oxidizes FMNH2 for
the regeneration of FMN. DHODHs of Class 1B are heterotetrameric enzymes that use NAD+ as an oxidizing
agent and contain not only FMN but also a flavin adenine dinucleotide molecule and a [2Fe-2S] cluster [46,47].
Class 1B enzymes appear to prevail in Gram-positive bacteria, some of which express forms 1A and 1B. In contrast,
the 1A form appears as a single form in selected eukaryotes, for example, in species of the genera Leishmania
and Trypanosoma. Class 2 enzymes are monomeric proteins that use hydrophobic molecules (e.g., ubiquinone) as
oxidizing agents [22,43].
P. falciparum DHODH (PfDHODH) is a Class 2 enzyme that contains 569 amino acids (Figure 2) [48]. Figure 2
also shows the sequences of DHODHs from a variety of organisms, including P. berghei, which has high importance
as a laboratory model. The DHODHs of S. mansoni and H. sapiens, two other Class 2 DHODHs, display the
N-terminal helices that are used to anchor the enzyme to the membrane and are also unique to Class 2 DHODHs.
The residues shown in red in the P. falciparum sequence are exclusive to certain Plasmodium species and do not
occur in Schistosoma species or in humans. The pink triangle shows the catalytic residue conserved in all Class 2
DHODHs, in other words, a serine that is conserved in all Class 2 DHODHs; in Class 1 DHODHs, the catalytic
residue is a cysteine.

X-ray structures
Crystallization
DHODHs from 14 different organisms have been successfully crystallized. Currently, approximately 160 structures
of DHODH, all determined by x-ray crystallography, have been deposited in the Protein Data Bank (PDB). Among

10.4155/fmc-2017-0250 Future Med. Chem. (Epub ahead of print) future science group
 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

Dihydroorotate (DHO) Orotate (ORO)

FMN (Oxidized) FMNH2 (reduced)

DHODH

CoQH2 (reduced) CoQ (Oxidized)

Figure 1. Enzymatic reaction catalyzed by dihydroorotate dehydrogenase. For the ribbon representation of
Plasmodium falciparum DHODH, the PDB code 5TBO was used as a model.
DHODH: Dihydroorotate dehydrogenase; PDB: Protein data bank.

these, the first PfDHODH structure was determined (2.4 Å) by Hurt et al. in 2006 [50]. The PfDHODH crystals
are complexed with A771726 (1) (teriflunomide, an active metabolite of leflunomide; Supplementary Figure 4)
and orotate; they were obtained by removal of the signal peptide and the transmembrane region and grown using
the sitting-drop vapor-diffusion technique at 277 K with sulfate salt as the precipitant, ammonium acetate as a
buffer and the detergent pentaethylene glycol monooctyl ether (C8E5) in the crystallization solution. In fact, the
use of a detergent in both the purification and crystallization steps is considered obligatory for stabilization of the
Class 2 DHODH N-terminal membrane-associated domain.
All of the other DHODHs whose structures were determined later were crystallized using the hanging-drop
vapor-diffusion technique at 293 K also in the presence of sulfate salt and ammonium acetate. The only ex-
ception was described by Ross et al. in 2014; those authors used the buffers lithium chloride and/or 2-(N-
morpholino)ethanesulfonic acid together with polyethylene glycol (PEG) 3350 [51]. Other components used
include PEG 4000, glycerol, d, l-dithiothreitol (DTT) and the detergent N,N-dimethyldodecylamine N-oxide,
which was used in the purification protocol and/or during crystallization [38,51–58].
The first crystal structure of PfDHODH described by Hurt et al. was found to contain a missing or disordered
region (residues 375–414) that is not present in Class 2 enzymes such as those of humans or Schistosoma spp.
(Figure 2) [50]. In fact, removal of a 30-residue-long loop (residues 384–413, shown in red in Figure 2) was
found to be necessary to obtain reproducible diffraction-quality crystals [54]. Steady-state kinetic analysis of the

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P. falciparum 158 F E S Y N PE F F L Y D I F L K F C L K Y I D G E I C H D L F L L L GK Y N I L P Y D T S N D S I Y A C T N I K H L D F I N P F
P. malariae 199 F E S Y N PE F F MY D V F L K F C L K Y I D S E L C H D L F L L L GK F R L L P Y D T S N D S I Y A L S N I K D L N F I N P F
P. berghei 132 F E S Y N PE F F MY D V F L D F C L N Y V D S E V C H D L F L L L GK Y G L L P Y D T S N D S V Y A T S D I K N L N F I N P F
P. ovale 158 F E S Y N PE F F L Y D I F L N L C L K Y V D C E V C H D L F L H L GK Y N L L P Y D T S N D S A Y A T S D V K Y I H F L N P F
P. vivax 160 F E S Y D PE F F L Y D V F L KM L L K Y V D G E T C H E L F L L MGK Y K L L P Y D T G K D N I Y S C S E I KG L N F I N P F
P. knowlesi 170 F E S Y D PE F F L Y D I F L K F L L K Y V D G EMC H D L F L L MGK Y N ML P Y D T S K D S I Y S C T G I GG L N F I N P F
S. mansoni 24 Y S G · N EH F Y K DW F L P T A R L L V R D G E T A H N L S V Y L A S Y G F I P H K Q R N S F P Q L K C K V F G L E F D H P I
H. sapiens 30 A T G · D ER F Y A E H L MP T L Q G L L · D P E S A H R L A V R F T S L G L L P R A R F QD S DML E V R V L G H K F R N P V

P. falciparum 222 G V A A G F D K N GV C I D S I L K L G F S F I E I G T I T P R GQ T G N A K P R I F R D V E S R S I I N S C G F N NMGC D K

P. malariae 263 G V A A G F D K N GV S I D S I L K L G F S F I E I G TM T P K P Q K G N E K P R I F R D N E T K S I I N S C G F N N I GC D K
P. berghei 196 G V A A G F D K N G I C I D S I L K L G F S F I E I G T I T P K P Q K G N N K P R I F R D V E N K S I I N A C G F N N I GC D K
P. ovale 222 G V A A G F D K N GV C I D S I L K L G F S F I E I G T V T P R A Q K G N A K P R I F R D I E T K S V I N A C G F N N I GC D K
P. vivax 224 G V A A G F D K N GV C I D G I L K L G F S F I E I G T I T P K A Q K G N E R P R I F R D L E T R S I I N S C G F N NMGC D E
P. knowlesi 234 G V A A G F D K N GV C I D G I L K L G F S F I E V G T I T P K P Q K G N D K P R I F R D V E T R S I I N C C G F N N I GC D E
S. mansoni 87 G L A A G F D K D GK A FM G L L N A G F S H I E V G T V T P N P Q L G N A R P R I F RW T E K E A V V N R C G F N S D GH D A
H. sapiens 92 G I A A G F D K H GE A VD G L Y K M G F G F V E I G S V T P K P Q E GN P R P R V F R L P E D Q A V I N R Y G F N S H G L S V

P. falciparum 286 V T E N L I L F R K RQ E E D K L L S KH I VG V S I G K N K D T V N I V DD L K Y C I N K I G R Y A D Y I A I N V S S P N T P
P. malariae 327 I T T N L I E F R K RQ E K D K I L S RH I VG V S I G K N K N T I N I I DD L S Y C I K K I A R Y A D Y I A I N V S S P N T P
P. berghei 260 V T E N L I N F R K KQ E E D K L L S KH I VG V S I G K N K H T E N I V DD L K Y S I Y K I A R Y A D Y I A I N V S S P N T P
P. ovale 286 V C E N L I S F R K KQ E N D K L L S RH I VG V S I G K N K D T DN I T DD L S Y C I E K I A R Y A D Y I A I N V S S P N T P
P. vivax 288 V C K N L K R F R E RQ K T D K L L Q RH L VG V S L G K N K D S P D I L QD L S Y C I G K I G R Y A D Y I A I N V S S P N T P
P. knowlesi 298 V T E N L K R F R E KQ K T D K L L H RH F VG V S L G K N K D S A D I L E D L S Y C I S R I G K Y A D Y I A I N V S S P N T P
S. mansoni 151 V Y E R L · K D R PWE GR G V · · · · · · I G V N L G C N K T S A D P T A D Y V A G V R K F G E V A D Y L V I N V S S P N T P
H. sapiens 156 V E H R L · R A R QQK QA K L T E D G L P L G V N L G K N K T S V D A A E D Y A E G V R V L G P L A D Y L V V N V S S P N T A

P. falciparum 350 G L R D N Q E A G K L K N I I L S V K E E I D N L E K N N I M N D E S T Y N E D N K I V E K K N N F N K N N S HM M K D A K D N
P. malariae 391 G L R D N Q E S A K L K N I I L S V QG E I D K L E RG D K N S E V D T A L C I T N S I S N HN D E N K · · · · · · · · · · · ·
P. berghei 324 G L R D N Q E S N K L K N I I L F V KQ E I N K I E Q I G H N · · · G E · T · · · · · · · · · · · · · · · · · · · · · · · · · ·
P. ovale 350 G L R D N Q E S T K L K N I I L H V QR E V T R L E E S H T N R L A G E R T · · · · · · · · · · · · · · · · · · · · · · · · · ·
P. vivax 352 G L R D H Q K G E R L H G I I QR V K E E V A K L D GG G A P L G G A T T G G A A MGG A T TG E A V VG K A P P D E A A T G G
P. knowlesi 362 G L R D N Q Q S E R L Q R I I L R V K E E V D K L E E S N V · · · · · · · · · · · · · · · · VG D D V V R · · · · · · · · · · G
S. mansoni 208 GL R S L Q T K E K L R D L L SK V L A AR N Q L S K · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
H. sapiens 219 G L R S L QGK A E L R R L L T K V L Q E R D G L R R V H · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

P. falciparum 414 F L WF N T T K K K P L V F V K L A P D L NQ E Q K K E I A D V L L · · E T N I D GM I I S NT T T Q I · · · · · · · · · ND I
P. malariae 443 · L W I NT T K R R P L V F V K L S P D L E E N D R K K I A Q V L L · · E T D I D GM I I S NT T I K N · · · · · · · · · F N I
P. berghei 358 · F WM N T I K K K P L V F V K L A P D L E N S E K K K I A Q V L L · · D T G I D GM I I S N T T I N K · · · · · · · · · MD I
P. ovale 388 · T WV N T T Q R K P L I F V K L A P D L E N S E K K K I A Q V L L · · E T G V D GM I I S NT T T N K · · · · · · · · · F D I
P. vivax 416 E PWA N T T K R R P L I F V K L A P D L E E G E R K S I A N V L L · · N A E V D GM I I C NT T T Q K · · · · · · · · · F N I
P. knowlesi 400 D T WV N T S K R K P L V F V K L A P D L E E G E K K K I A D V L L · · K T K V D GM I I C NT T T E K · · · · · · · · · F N I
S. mansoni 235 · · · · · · · · · K T P I L L K I S P D E N DQ N L K D I V E V A L D S K T R I D GM I I S N T T L T T Y E E A V A CG A A P I
H. sapiens 248 · · · · · · · · · R P A V L V K I A P D L T SQ D K E D I A S V V · · K E L G I DG L I V T NT T V S R · · · · · · · · P AG L

P. falciparum 467 K S F E N K K · · · GG V S G A K L K D I S T K F I C E MY N Y T N KQ I P I I A S GG I F SG L D A L E K I E A G A S V C Q L
P. malariae 495 K S F E N K K · · · GG V S G Q K L K D I S T N L I A E MY N Y T N K K I P I I A S GG I F S A E D A L E K I E A G A S V C Q L
P. berghei 410 K S F E D K K · · · GG V S G K K L K D L S T N L I S D MY I Y T N KQ I P I I A S GG I L T G A D A L E K I E A G A S V C Q L
P. ovale 440 K S F E N K K · · · GG V S G E K L K V I S T N F I S E MY L Y T E K K I P I I A S GG I F SG A D A L E K I E A G A S V C Q L
P. vivax 469 K S F E D K K · · · GG V S G E K L K G V S T HM I SQ MY N Y T N GK I P I I A S GG I F T G E D A L E K I E A G A S V C Q L
P. knowlesi 453 K NF QD K K · · · GG V S G E K L K D V S T K F I SQ MY N Y T N K K I P I I A S GG I F T G K D A L E K I E A G A S V C Q L
S. mansoni 290 P GN N K QN V V Y GG L S G R P L F E K S T D C L R K V S A L T K GA I P L I G V GG I S CG E D A L S K L N A G A S L V Q L
H. sapiens 293 Q GA L R S E T · · GG L S G K P L R D L S T Q T I R E MY A L T Q GR V P I I G V GG V S SG Q D A L E K I R A G A S L V Q L

P. falciparum 528 Y S C L V F N G M K S A V Q I K R E L NH L L Y Q RG Y Y N L K E A I G R K H S K S
P. malariae 556 Y S C L V F N G V K T A V K I K R E L NN L L Y Q RG Y Y N L E E A I G K R H K RG
P. berghei 471 Y S C L V F N G V K S A I Q I K R E F NN A L Y Q KG Y Y N L R E A I G K K H S NA
P. ovale 501 Y S C L V F N G V K S A I K I K R E F NN L L Y Q RG Y Y N L R E A I G K K H E R D
P. vivax 530 Y S C L V F N G M K A A V R I K R E L DH L L Y Q RG Y Y K L G D A V G R A H R R A
P. knowlesi 514 Y S C L V F N G M K A A V R I K R E L DH L L Y Q RG Y Y K L E D A I G K A H R RG
S. mansoni 354 Y T S F V Y Q G P P V A H K V A R E I NK LKMT S · · · · · · · · · · · · · · · ·
H. sapiens 355 Y T A L T F W G P P V VG K V K R E L E A L L K E QG F GG V T D A I G A D H R R ·

Figure 2. Primary sequence alignment of Class 2 dihydroorotate dehydrogenases. The species shown in the alignment were selected
from Plasmodium spp. linked to human malaria. Plasmodium berghei was selected based on its importance as a laboratory model, and S.
mansoni and H. sapiens were chosen for comparison. The sequences are shown only from the N-terminal helices (first two helices in the
sequence), which are a singular characteristic of Class 2 DHODHs. The pink triangle shows the conserved catalytic residue. Identical
(shaded blue) and conserved (light blue) residues are boxed. All identified residues that interact with the ligands in the quinone-binding
tunnel are indicated by stars (blue stars indicate more important residues). The portion of the sequence shown in red forms a loop with a
length of 30 residues (residues 384–413) that was found to be necessary to obtain reproducible diffraction-quality crystals (figure
assembled in ESPript 3.0, [49]).
DHODH: Dihydroorotate dehydrogenase.
 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

Table 1. Plasmodium falciparum dihydroorotate dehydrogenase complexes that have been crystal-
lized and structurally determined to date.
PDB ID Deposit year Resolution Space group Ligand Ref.
1TV5 2004 2.4 ˚A H32 A771726 [50]
3I6R 2009 2.5 ˚A P 64 DSM74 [54]
3I65 2009 2.0 ˚A P 64 DSM1 [54]
3I68 2009 2.4 ˚A P 64 DSM2 [54]
3O8A 2010 2.3 ˚A P 64 Genz-667348 [52]
3SFK 2011 2.95 ˚A P 64 DSM267 [53]
4CQ8 2014 1.98 ˚A C121 Genz-669178 [51]
4CQ9 2014 2.72 ˚A P 1 21 1 IDI-6253 [51]
4CQA 2014 2.82 ˚A P 1 21 1 IDI-6273 [51]
4ORM 2014 2.07 ˚A P 64 DSM338 [55]
4RX0 2014 2.25 ˚A P 64 DSM265 [38]
5BOO 2015 2.8 ˚A P 65 DSM265 [38]
5DEL 2015 2.2 ˚A P 64 DSM59 [56]
5FI8 2015 2.32 ˚A P 64 DSM422 [57]
5TBO 2016 2.15 ˚A P 64 DSM421 [58]
PDB: Protein data bank.

construct lacking amino acid residues 384–413 (PfDHODH384–413 ) demonstrated that the catalytic efficiency
and inhibitor-binding properties of the loop enzyme were similar to those of the wild-type enzyme [54].
It is worth mentioning that all PfDHODH crystal structures available in the PDB have been solved in the
presence of both orotate and potent Class 2 DHODH inhibitors (Table 1).

Description of the structures

In general, DHODHs show a typical α/β-barrel fold with a central barrel composed of eight parallel β-strands
(β1–β8) wrapped by eight α-helices (α1–α8). Class 2 DHODHs present an additional membrane-association motif
arranged as a pair of α-helices, αA and αB. These two helices are connected by a short loop and form a small
hydrophobic N-terminal domain. This small N-terminal region is directly linked to the larger C-terminal domain
of the human enzyme, where the binding sites for the substrate and FMN are located [32]. A tunnel that consists
almost entirely of hydrophobic amino acids is formed between the N-terminal helices and the α/β-barrel domain.
The tunnel begins at the surface of the protein, ends adjacent to FMN and is the site at which Class 2 DHODH
inhibitors bind (Figure 3) [59].
The first reported crystal structure of PfDHODH was obtained in the presence of A771726, a human DHODH
(HsDHODH) inhibitor and the primary metabolite of leflunomide, which is used as a drug to treat rheumatoid
arthritis. A771726 displays reduced activity against the parasite enzyme as a consequence of charge and size
variations within the DHODH-binding pockets [50]. In particular, the residues that are important for the ligand
target site (Leu172, Cys184, Phe188 and Val532) present in the P. falciparum enzyme are substituted by Phe, Ala,
Ala and Thr, respectively, in the human enzyme [50].
The inhibitor-binding site of Class 2 DHODHs can be divided into two main portions: a hydrogen-bonding
site consisting of Arg256 and His185 and a hydrophobic cavity formed by the helices αA and αB. Reduced
conformational flexibility is observed near the FMN-binding site where both Arg256 and His185 are located. On
contrary, αA and αB display a high level of structural variability that allows alternative modes of binding between
protein and ligands. For instance, Deng et al. showed that in addition to the hydrogen-bond interaction with His185
and hydrophobic interactions, the N-phenylbenzamide inhibitor (DSM59, 2, Supplementary Figure 4) is able to
undergo an additional stacking interaction of its nitro group with the protein amide between Gly181 and Glu182,
to form a salt bridge with Arg265 and to form a halogen bond between one of the dichloraniline chlorines and the
carbonyl of Gly535. Flexibility associated with sequence variability at the hydrophobic N-terminal domain can also
modulate affinity and species selectivity of Class 2 DHODH inhibitors. For example, triazolopyrimidine-based
inhibitors were shown to bind to the same hydrophobic patch in both Plasmodium and mammalian DHODHs by
exploiting distinct binding mechanisms [55].

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αE

αB

ORO FMN
78Z
αA

α3

α4
α8

βB
C
αD βA

αG

Figure 3. Cartoon representation of the monomeric structure of Class 2 Plasmodium falciparum dihydroorotate
dehydrogenase (protein data bank ID: 5TBO). The N-terminal α-helices αA and αB (shown in dark violet) are present
only in Class 2 DHODHs and supply the target site for inhibitors (e.g., 78Z [DSM421], shown in cyan). The cofactor
FMN (shown in pale yellow) and the oxidized substrate ORO (shown in orange) are also present.
DHODH: Dihydroorotate dehydrogenase; FMN: Flavin mononucleotide; ORO: Orotate.

Mapping of the interactions between PfDHODH and ligands (Supplementary Figure 3 & Supplementary Table
1) highlighted the importance of hydrogen-bond interactions between the ligands and His185. Arg265, Val532,
Phe227 and Gly181 are also described as interacting with ligands through hydrophobic interactions. Other residues
such as Leu172, Cys184, Phe188, Ile263 and Leu531 also contribute to the inhibitor-binding pocket and show
hydrophobic interactions with the ligands in the great majority of structures (11 or more). The ligand Genz-
669178 (5-(4-cyano-2-methyl-1H-benzimidazol-1-yl)-N-cyclopropylthiophene-2-carboxamide, 3; Supplementary
Figure 4) is the ligand with the highest number of interactions among all structures, in other words, 17 in total
(hydrophobic and hydrogen bonds). Most of these residues (indicated by blue and yellow stars in Figure 2) appear
to be important and are conserved among Plasmodium species, especially considering the structural basis approach
to the development of new inhibitors of malarial DHODH. the structural basis approach in the development of
new compounds against malarial DHODH.

PfDHODH kinetics & activity assays

The bireactant PfDHODH presents a kinetic mechanism that appears similar to that of other Class 2 DHODH
enzymes when steady-state analysis is performed [23,24,60,61]. The initial velocity of PfDHODH catalysis over a
range of dihydroorotate (DHO) and decylubiquinone (CoQD ) cosubstrate concentrations displays parallel lines
that are consistent with a ping-pong kinetic model (Supplementary Figure 5) [62,63].
DHODH activity has traditionally been measured using a standard colorimetric assay that monitors 2,6-
dichloroindophenol (DCIP) reduction as a decrease in absorbance that can be monitored at 600 nm. This assay
couples the reduction of DCIP with the reoxidation of ubiquinone coenzyme (CoQ), which is stoichiometrically

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 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

coupled to orotate formation. Experiments to determine Km (the Michaelis constant, defined as the substrate
concentration at half of the maximum velocity) and kcat (the turnover number, the number of substrate molecule
each enzyme site converts to product per unit time, when the enzyme is fully saturated with substrate) for both
substrates were conducted by varying the concentrations of CoQ and DHO until saturating concentrations were
reached [55,62,63]. For PfDHODH, kcat is approximately 18 s-1 , and Km is approximately 16 and 71 μM for DHO
and CoQD , respectively. Similar kinetic constants were found for PfDHODH384–413 , for which kcat is 18 s-1 and
Km is 21 and 105 μM for DHO and CoQD , respectively [54].
Although CoQD is broadly used in activity and inhibition assays of Class 2 DHODHs from numerous organisms,
the use of this substrate is not the only option [64]. In previous studies of PfDHODH, CoQ0 , CoQ4 , CoQ6 , CoQ10
and Vitamin K3 were used, and the catalytic efficiency of the enzyme was evaluated. CoQ6 , CoQD and CoQ4 , in
that order, presented lower Km , whereas CoQ0 and Vitamin K3 (which lack an isoprenoid tail) displayed Km values
up to seven-times higher [39]. CoQ10 has also been tested, but due to solubility problems, assays using this substrate
are often unfeasible. Although CoQD is used due to its high efficiency and availability, it is expensive, which makes
it impractical for use in countries that lack financial support. In this sense, CoQ0 provides a suitable alternative
because of its price and the fact that, like CoQD , it is sensitive to ligands/inhibitors.
Alternative enzymatic assays are always needed to ensure accurate measurement of activity for the tested com-
pounds and to improve the quality and/or reduce the cost of assays. A new fluorescence intensity assay similar to
the DCIP assay was developed for monitoring DHODH activity. In this alternative assay, CoQD is substituted
by resazurin, a fluorogenic dye that when reduced to resorufin changes from a blue nonfluorescent state to a pink
fluorescent state [65].
Another justification for the development of this promising new assay, especially for PfDHODH inhibition
studies, is the fact that DCIP has been reported to act as an alternative substrate for CoQD and the observed activity
is not only due to the reduction of the quinone but also to the dye [54]. One should be aware that under typical assay
conditions and depending on the type and concentration of quinone substrate, 10–30% of the observed activity
is directly related to the reduction of DCIP by DHODH; this can seriously interfere with the interpretation of
inhibition experiments [66,67].
Therefore, the type of substrate used and the accuracy of the assay can influence inhibition studies. It is
widely known that many parameters can affect enzymatic activity, inhibition potency and mechanism studies. The
incubation protocol applied prior to inhibition assays is also an important parameter that can greatly influence the
kinetic parameters and can compromise appropriate comparisons among different experiments.

Medicinal chemistry of Plasmodium DHODH inhibitors

Application of high-throughput screening & enzyme structural information to the discovery of
Plasmodium DHODH inhibitors
High-throughput screening (HTS) is a robust method that is used in drug design; it can, for example, provide
an effective means of identifying novel chemical scaffolds with inhibitory activity against a specific therapeutic
target [68,69]. Due to the utilization of robots, detectors and software, this technique enables a series of analyses of
chemical compounds to be conducted in a short time, and it has been used in the discovery of new Plasmodium
DHODH inhibitors [69].
In 2005, Baldwin et al. applied HTS using 220,000 drug-like compounds to select effective inhibitors against
Plasmodium DHODH. The most active compound, a phenyl benzamide derivative (4; Supplementary Figure 4),
presented an IC50 value against malarial DHODH of 16 nM and was 12,500-fold less active against HsDHODH [69].
A series of potent and selective PfDHODH inhibitors of the triazolopyrimidine class was also identified using
HTS [70]. In 2008, the first selective inhibitor, DSM1 (5; Supplementary Figure 4) was shown to be active against
both PfDHODH and P. falciparum-infected cells with an IC50 of approximately 50 nM. Nevertheless, this inhibitor
was almost inactive in a mouse model of malaria infection, probably due to its activity as a metabolic inducer and its
rapid metabolism [70,71]. A subsequent study identified a phenyl-substituted triazolopyrimidine analog (DSM74,
6; Supplementary Figure 4) with low predicted metabolism in human liver microsomes, also showing prolonged
exposure in mice or rats. This compound was also able to inhibit P. berghei when administered at a dose of
50 mg/kg for 4 days. These studies provided the first proof that DHODH could be used as a therapeutic target to
reduce in vivo infection with Plasmodium species. Nevertheless, although DSM74 presented higher bioavailability
than DSM1, it was 6-fold less potent [71]. Two years later (2011), using structural information on Plasmodium
DHODH, Coteron et al. provided a lead optimization of PfDHODH inhibitors of the triazolopyrimidine class,

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adding substituents in the C2 position of the triazolopyrimidine ring. This study yielded a potent compound,
DSM265 (7; Supplementary Figure 4), with good pharmacokinetic profile and in vivo efficacy in mouse models;
it is currently in clinical trials [35–38,53].
Studies using HTS with subsequent lead optimization also identified thiophenecarboxamide derivatives (Genz-
669178, 3; Genz-667348, 8; and Genz-668857, 9; Supplementary Figure 4) that inhibit PfDHODH with IC50s
ranging from 20 to 50 nM and presented high potency against P. falciparum-infected 3D7 cells (half maximal
effective concentration, EC50 , equal to 7–20 nM). Moreover, after twice daily oral administration, these derivatives
showed good plasma bioavailability, suppressing parasitaemia in the malaria mouse model (effect of concentration on
median effective dose, ED50 , equal to 13–21 mg/kg). Among these compounds, Genz-667348 provided cure after
administration of 100 mg/kg daily. Interestingly, all derivatives presented drug-like physicochemical properties, not
showing activity in cytochrome P450 (CYP) enzymatic inhibition or human ether-a-go-go-related gene (hERG)
analysis and thereby qualifying as candidates for preclinical research [52,72].
In 2016, Phillips et al. investigated substitution of the pentafluorosulfanyl(SF5 )-aniline group of DSM265
with a series of trifluoromethyl (CF3 )-pyridinyls while maintaining the core triazolopyrimidine scaffold. This
study identified the compound DSM421 (10), which presented improved solubility, lower intrinsic clearance,
increased plasma exposure after oral administration and a long-predicted human half-life compared with DSM265.
DSM421 showed excellent efficacy in the severe combined immunodeficiency (SCID) mouse model of P. falciparum,
supporting the prediction of a low human effective dose (<200 mg). Importantly, DSM421 showed equal activity
against both P. falciparum and P. vivax field isolates, whereas DSM265 was more active against P. falciparum.
DSM421 has the potential to be developed as a single-dose cure or as a once weekly chemopreventive for both P.
falciparum and P. vivax malaria, leading to its advancement as a preclinical development candidate [58].
Recently, Ross et al. performed a HTS work to select PfDHODH inhibitors against mutants forms related to
drug resistance. Hence, the authors screened 23 (11–33, Supplementary Figure 6) active compounds tested in
PfDHODH mutant cell lines, which presents cross-resistance against triazolopyrimidine derivatives. In addition,
some combination of those inhibitors in pairs were active in the suppression of resistance, representing, with further
investigation, potential new antimalarials [73].

Discovery & development of PfDHODH inhibitors using computational approaches

As described previously, 15 x-ray crystallographic structures of PfDHODH [38,51–55,57,58,74,75] have been reported
since 2006; the most recent of these was published in 2016 [58]. Consequently, the number of articles reporting
the discovery of new PfDHODH inhibitors has increased; these primarily include papers in which molecular
modeling methods such as molecular docking, quantitative structure–activity relationships (QSAR), structure-
based pharmacophore mapping and molecular dynamics simulations were used [74–79].
In 2006, Heikkilä et al. applied the SPROUT computational package, to the x-ray crystal structures of Plasmodium
(PDB code: 1TV5) and HsDHODH (PDB code: 1D3H) complexed with A771726 inhibitor (Supplementary
Figure 4) to obtain novel PfDHODH inhibitors. The resulting small-molecule templates were used to design and
prepare two amide derivatives of anthranilic acid (34 and 35; Figure 4A); these probably bind to the hydrophobic
ubiquinone channel of PfDHODH, thus facilitating interactions with Arg65 and His185. These inhibitors (34
and 35) showed selectivity for Plasmodium DHODH with IC50 values of 42 and 93.4 μM, respectively [80].
A set of compounds possessing a common hydrophilic moiety linked to a variety of hydrophobic aromatic or
heteroaromatic rings were also synthesized by Heikkilä et al. Inhibitors with nanomolar affinity for PfDHODH
(36, IC50 = 0.16 ± 0.05 μM; 37, IC50 = 0.44 ± 0.06 μM; Figure 4B) were identified. Moreover, two types of
molecular docking softwares, Autodock 3.0 [81] and eHITS [82,83], were used to model the binding of the inhibitors
within the ubiquinone channel; the modeling predicted hydrogen bonds with His185, Arg265 and Tyr528, similar
to those observed for the cocrystallized A77–1726 inhibitor (Figure 4A; PDB code: 1TV5). These results were
also supported by mutagenesis data. Moreover, these two potent PfDHODH inhibitors (36 and 37) inhibited the
growth of cultured parasites at low micromolar levels of 1.8 and 4.0 μM, respectively (Figure 4B) [84].
Furthermore, Davies et al. used the SPROUT package to design potent inhibitors of PfDHODH (PDB code:
1TV5) and HsDHODH (PDB code: 1D3H), also based on the structure of A771726 (Supplementary Figure 4).
The compounds presented higher activity against both enzymes than the activity observed for A771726, for which
the majority of the inhibitors were selective for HsDHODH. Therefore, the authors proposed that the variations
at the hydrophobic moiety (highlighted in red in Figure 4C) only result in small selectivity for PfDHODH over
HsDHODH (38 and 39, Figure 4C). Additionally, several PfDHODH-selective inhibitors make strong hydrogen

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 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

HO2C
CO2Et
F3C H
O N
HN CO2Et
CN
N X
H R O
HO
(34) R = 1,1'-biphenyl, IC50 = 42.6 ± 4.6 µM (36) X = S, IC50 = 0.16 ± 0.05 µM
A771726 (1) (35) R = 2-bromo-naphtyl, IC50 = 93.4 ± 6.4 µM (37) X = NH, IC50 = 0.44 ± 0.06 µM

O
R3
O
R2 CO2Et
Tail or
H O
N Head R1
O R2 O
R4 CN Malonate Derivatives Meldrum’s Acid Derivatives
N R3
H
R1
HO

(38) R1 = Cl, R2 = Cl, R3 = H, R4 = Cl; Inhibition (%) Inhibition (%)

IC50 (PfDHODH) = 10.2 µM Compound Structure PfDHODH HsDHODH
IC50 (HsDHODH) = 16.5 µM (50 µM) (50 µM)

(39) R1 = CF3, R2 = H, R3 = Cl, R4 = H; Malonate Derivatives

IC50 (PfDHODH) = 57.2 µM R1 = CN
IC50 (HsDHODH) = 86.6 µM 40 R2 = m-CO2Et 23 ± 3 2±2
R1 = CO2Et
41 R2 = p-CO2Et 60 ± 6 8±4

F3C R1 = CN
O 42 R2 = p-CO2Et 66 ± 5 12 ± 4
R3 = m-Cl
N N
H Meldrum’s Acid Derivatives
O
R2 = m-CO2H
Leflunomide 43 R3 = p-Cl 50 ± 4 21 ± 2
R2 = m-CO2Et
44 0±2 1±6
R3 = H

Figure 4 PfDHODH inhibitors: compounds reported by Heikkilä et al. (A & B), Davies et al. (C) and Cowen et al. (D).
PfDHODH: Plasmodium falciparum dihydroorotate dehydrogenase.

bond with His185; this is not possible in HsDHODH, probably because the equivalent His56 (in the human
enzyme) forms a hydrogen bond with Tyr147 [85].
In 2010, Cowen et al. synthesized a series of mono- and di-substituted N-arylaminomethylene malonate deriva-
tives with inhibitory activity against PfDHODH and HsDHODH [86] and predicted their modes of binding to
both enzymes, also using the molecular docking softwares, Autodock 3.0 [81,87] and eHITS [82,83]. Their results
showed that compounds containing an ester group at either the meta or the para positions of the aryl moiety (40
and 41; Figure 4D) generally bind more tightly to PfDHODH, likely due to hydrogen-bond interactions with
Arg135 (nonspecific binding among DHODH species). In addition, based on the molecular docking simulations,
the authors suggested that ester inhibitors with a carboxyl group at the R1 position participate in a salt bridge
interaction with Arg265 in PfDHODH and with Arg136 in HsDHODH, thus interacting more effectively with
the hydrophobic cavities of the enzymes (43 and 44; Figure 4D) [88]. Interestingly, increased enzyme affinity were
also observed for inhibitors with a chlorine group in the aryl (42; Figure 4D). Nevertheless, the enamine inhibitors
derived from Meldrum’s acid, compounds with rigid cyclic moiety at the head position, are less potent in both
enzymes (40, 41, 43 and 44; Figure 4D) [86].
In the same year, Ojha et al. explored molecular docking and QSAR methods using triazolopyrimidine-based
PfDHODH inhibitors [88]. Molecular shape analysis using molecular shape descriptors, together with electronic,

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R2 (6) R = H, R1 = CF3, R2 = H,
R1 IC50 (PfDHODH) = 0.28 µM
IC50 (HsDHODH) = > 100 µM
HN (57) R = CF2CH3, R1 = CF3, R2 = H,
IC50 (PfDHODH) = 0.038 µM
N N
R IC50 (HsDHODH) = > 100 µM
N N
(9) R = CF2CH3, R1 = SF5, R2 = H,
IC50 (PfDHODH) = 0.033 µM
IC50 (HsDHODH) = > 100 µM

Steric bulks
are favored
Electron-rich
Ar substituents
HN Figure 5. Triazolopyrimidine-based Plasmodium
enhance
7 8 1 falciparum dihydroorotate dehydrogenase inhibitor
6 N N the activity
2 studies. (A) Lead optimization presenting the IC50
5
N 3a N selectivity for the Plasmodium enzyme over the human
3 (58) AR = 3-F, 4-CF3-PH,
4 enzyme as reported by Coteron et al. (B) 3D-QSAR
IC50 (PfDHODH) = 77 µM (Comparative Molecular Field Analysis, Comparative
Hydrogen bond (59) AR = 2-anthracenyl, Molecular Similarity Index Analysis) molecular docking
acceptor IC50 (PfDHODH) = 56 µM and dynamics performed by Shah et al. showing two of
the most active compounds and the respective IC50
values.

spatial, physicochemical and structural descriptors, was used in the QSAR studies in which a combined set of
descriptors was attempted for model development. Among the different models developed, the classical QSAR
model with only physicochemical descriptors, which was obtained from the genetic function approximation coupled
with partial least squares technique [88], was the best model according to the r2 (overall) (0.733) and R2 p (0.767) criteria.
From the QSAR models, the authors concluded that: the phenyl should be unsubstituted at the ortho position; at
the meta position, groups with moderate hydrophobicity and volume might increase PfDHODH inhibition; and
at the para position, the presence of groups with volume and hydrophobicity high but restricted is necessary for
DHODH inhibition (Supplementary Figure 7). Interestingly, these conclusions were also supported by the results of
the molecular docking analyses using Discovery Studio 2.1 (Accelrys, Inc., CA, USA). Moreover, molecular docking
has suggested that the 2-methyltriazolopyrimidine ring interacts with certain hydrophilic (Arg265 and His185) and
selected hydrophobic amino acids (Ileu263, Gly181, Leu176, Cys175, Cys184, Val532 and Leu172), whereas the
substituted phenyl ring binds to a hydrophobic pocket of the enzyme (Gly535, Leu531, Phe227, Phe188, Leu189,
Met536, Ileu237, Leu240 and Leu197; Supplementary Figure 7). Therefore, the new proposed compounds (45–56,
Supplementary Figure 7) showing binding interactions similar to those of previously synthesized inhibitors, are
likely to be active PfDHODH inhibitors [88].
One year later, Coteron et al. conducted lead optimization around the previously identified triazolopyrimidine-
based series of PfDHODH inhibitors [53]. Thus, the x-ray structure of PfDHODH complexed to compound
DSM267 (57; PDB code: 3SFK; Figure 5A) [53] was aligned, analyzed and compared with PfDHODH bound
to compound JZ5 (DSM74, 6; PDB code: 3I6R; Supplementary Figures 3B & 5A) [54] using the PYMOL
program [89,90]. This structural analysis led to the identification of a potent and selective inhibitor (DSM265, 9;
Supplementary Figures 4 & 5A) that killed both drug-sensitive and drug-resistant strains of Plasmodium. This
inhibitor presented potency comparable to CQ in a P. falciparum-humanized mouse model, demonstrating high
oral bioavailability, low clearance and a long half-life. Thus, these studies identified the first triazolopyrimidine
inhibitor that could, with further development, represent a drug candidate [38].
Shah et al. conducted a QSAR study using CoMFA and 35 triazolopyrimidine analogs reported as PfDHODH
inhibitors [91], providing simpler and better 3D-QSAR models in less time than those obtained by conventional
CoMFA modeling [92]. Thus, from the CoMFA and CoMSIA contour map analysis [93], the authors showed that the
presence of bulky groups around the aryl ring is more favored. The electrostatic contours of CoMFA also indicated
that the presence of electron-rich substituents could increase the activity of PfDHODH inhibitors (Figure 5B).

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 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

These high points were also reported by Ojha and Roy [88], who showed that compounds in this class with
hydrophobic substituents at the meta and para positions show better potency. Hence, the data obtained from
the CoMSIA contour maps are similar to those obtained from the CoMFA contour maps with respect to steric
and electrostatic effects, except for a steric favorable region around the aryl ring in the case of CoMSIA. The
hydrogen-bond contour map also proposes a favorable acceptor area around nitrogen 4 of the triazolopyrimidine
ring (Figure 5B). Furthermore, molecular docking and molecular dynamics simulations were used to study the
binding mode of the two most active compounds of the series (58 and 59; Figure 5B); where the results of these
simulations agreed well with those obtained using CoMFA–CoMSIA contour maps [93].
In 2013, structure-based pharmacophore mapping, molecular docking, binding energy calculations and binding
affinity predictions were presented by Wadood and Ulhaq as part of a virtual screening strategy for the design of new
and potent PfDHODH inhibitors [94]. Thus, a structure-based pharmacophore model was constructed considering
significant interactions as observed in the cocrystal of PfDHODH. Consequently, the best model was used to select
87 compounds from the ChemBridge database (http://www.chembridge.com/search/search.php). Additionally, the
molecular docking method, using the GOLD program [95] and the MOE software package (www.chemcomp.com),
was applied to further screen the selected compounds. Based on these results, 26 compounds (60–85; Supplementary
Figure 8) were predicted as new PfDHODH inhibitors [94].
In addition, Tseng et al. reported a computational study using PfDHODH inhibitors. QSAR pharmacophore
generation and molecular docking-based pharmacophore (DBP) development methods were used with a dataset
consisting of 38 compounds in the training set and 29 compounds in the test set [91]. The Hypo1 model, the
resulting 3D-QSAR pharmacophore model, showed a high correlation coefficient (r) of 0.935, high prediction
accuracies for the training and test sets of 89.4 and 72.4%, respectively, and a low root-mean-square deviation value
(RMSD) of 2.15. Additionally, two DBP models were constructed considering the conformations of all 255 docked
poses (DBP-All255) and the conformation of the Top1 scoring pose (DBPTop1). The DBP-All255 model showed
correlation coefficient (r = 0.908) and RMSD (2.54) values that were comparable to those obtained with the Hypo1
model. However, the DBPTop1 model presented poor statistical significance (r = 0.302) and a high RMSD value
(5.15). Thus, the authors showed that the pharmacophore features of Hypo1 are complementary to the functional
residues (Phe171, His185, Phe188, Phe227, Tyr258 and Arg265) within the active site of PfDHODH, making
this model attractive for use in the virtual screening of databases [96].
QSAR pharmacophore models were also constructed using the structures of PfDHODH inhibitors. The best
resulting model was used for virtual screening of the National Cancer Institute database (https://www.cancer.gov).
Additionally, molecular docking and molecular mechanics/generalized born surface area-binding energy calculations
were used as a basis for further selection of the hit compounds. This virtual screening resulted in the identification of
nine new compounds (86–94; highlighted in red, Supplementary Figure 9) that presented PfDHODH inhibition
values higher than 25% at a concentration of 10 μM; three of these compounds exhibited IC50 values in the
range of 0.38–20.00 μM (86–88; Supplementary Figure 9). The most active inhibitor, 86 (NSC336047), showed
much greater selectivity against PfDHODH (IC50 = 0.38 μM) than did HsDHODH (IC50 ≥ 100 μM) and also
inhibited parasite growth (PfNF54) with an IC50 of 26 μM (Supplementary Figure 9). Moreover, 13 compounds
were active against the growth of the parasite with IC50 values of ≤50 μM; four of these displayed PfNF54 IC50
values in the range of 5.07–12.03 μM (95–98; highlighted in blue in Supplementary Figure 9) [97].
Hou et al. constructed four 3D-QSAR models based on multilinear regression and support vector machine
methods using a dataset of 255 inhibitors of PfDHODH. The QSAR models displayed good prediction quality,
on the training sets and test sets, with a leave-one-out values (q2 ) and correlation coefficients (r) greater than 0.66
and 0.85, respectively. In addition, the mean square errors on the training and test sets were lesser than 0.32 and
0.37, respectively. The results showed that hydrogen-bonding capability, atom polarizability and ring complexity
are the main features necessary for active against PfDHODH. Therefore, the authors proposed four (99–102) new
putative PfDHODH inhibitors with predicted average IC50 values ranging from 2.92 to 6.45 nM (Figure 6A) [98].
Recently, Azeredo et al. applied molecular docking simulations to 15 designed and synthesized 7-arylpyrazolo[1,5-
a]pyrimidine compounds with activity against PfDHODH. The docking results using Autodock 4.2 [99] showed
that the best inhibitors (103–105; Figure 6B), which also presented low toxicity, were docked at the same binding
pocket to the inhibitor JZ8 (DSM1, 5, binding energy = -11.9 kcal/mol; Figure 6B & Supplementary Figure 4).
Moreover, these compounds displayed similar values of binding energy (-11.2 to -11.6 kcal/mol) in interactions
with the same residues in the cavity of PfDHODH, which is composed of hydrophilic (His185, Arg265 and
Tyr528) and hydrophobic (Leu172, Phe188 and Phe227) pockets and the cocrystallized water molecule HOH15

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Review Hoelz, Calil, Nonato, Pinheiro & Boechat

O O
N S N S
Cl N N Cl N N
H H
Cl
Cl (99) F (100)
Hydrogen bond
Predicted average IC50 = 6.45 nm Predicted average IC50 = 2.92 nm with His 185

O
N H NO2
S NH
Cl N N N
O
H Cl H H N N
O
R1
F N
Cl N R2
F (101) Cl (102)
Predicted average IC50 = 4.98 nm Predicted average IC50 = 5.98 nm

Hydrogen bond Hydrogen bond

with Tyr 528 with Arg 245

(103) R1 = CF3, R2 = CH3

Hydrogen bond with IC50 = 0.016 ± 0.01 µM
His 185 and Tyr 528 O2 N Binding energy = -11.6 kcal/mol
O
(104) R1 = CH3, R2 = CF3
O Hydrogen bond
with Arg 245 IC50 = 6.00 ± 1 µM
Binding energy = -11.2 kcal/mol
X
(105) R1 = CH3, R2 = CH3
(106) X = NCH2CH3; IC50 = 4.00 ± 1 µM
IC50 (PfDHODH) = 0.094 ± 0.031 µM Binding energy = -11.6 kcal/mol
IC50 (PfDHODH) = > 100 µM
(5) JZ8 (DSM1);
(107) X = S;
Binding energy = -11.9 kcal/mol
IC50 (PfDHODH) = 0.38 ± 0.06 µM
IC50 (PfDHODH) = > 100 µM

Figure 6. Molecular modeling methods applied to the design and the development of pfDHODH inhibitors: (A) Potential PfDHODH
inhibitors proposed by Hou et al. using 3D-QSAR models based on a dataset of PfDHODH inhibitors showing the respective calculated IC50
values. (B) Molecular docking study of triazolopyrimidine-based PfDHODH inhibitors, as reported by Azeredo et al., showing the main
interactions between the inhibitors and enzyme, the binding energy and the IC50 values. (C) New tricyclic β-aminoacrylate derivatives, as
reported by McConkey et al., that act as PfDHODH inhibitors showing the respective interactions predicted by molecular docking and the
IC50 values.
PfDHODH: Plasmodium falciparum dihydroorotate dehydrogenase; QSAR: Quantitative structure–activity relationship.

(Figure 5B), as described previously [88,91]. These computational results correlated quite well with the experimental
values obtained from enzymatic assays, in which it was shown that the presence of a CF3 group at the 2- or
5-position of the pyrazolo[1,5-a]pyrimidine ring led to increased inhibitory activity [99].
In 2017, molecular docking simulations were used in a work published by McConkey et al. In this work, two new
tricyclic β-aminoacrylate derivatives (106 and 107, Figure 6C) were described as selective PfDHODH inhibitors
of HsDHODH with IC50 values of 0.094 ± 0.03 and 0.38 ± 0.06 μM, respectively (Figure 6C). Although 1 H
and 13 C Nuclear Magnetic Resonance (NMR) spectroscopic data showed that these compounds undergo cis–trans
isomerization, molecular docking studies using the eHITS [82,83] and Autodock 4.2 [100] programs indicated no
preferential conformation or double-bond configuration in the binding of either compounds to PfDHODH via
interactions with His185, Arg265 and Tyr528 residues (Figure 6C) [101].

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 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

F
CF3 SF5 SF5
CF3

HN HN HN
HN HN F
N N N N N N F F
N N N N
N N N N N
N N N N N
DSM1 (5) DSM74 (6) DSM161 (111) DSM190 (112) DSM265 (9)
IC50 = 0.047 µM IC50 = 0.28 µM IC50 = 0.13 µM IC50 = 0.19 µM IC50 = 0.008 µM
CF3

CF3 CF3

N
HN HN HN HN O HN HN

N N N N N N N N N N F F

N N N N N N N N N N

DSM71 (113) DSM151 (114) DSM68 (115) DSM155 (116) DSM96 (117) DSM421 (10)
IC50 = 0.15 µM IC50 = 0.077 µM IC50 = 0.4 µM IC50 = > 100 µM IC50 = 0.047 µM IC50 = 0.053 µM

HN Cl HN

N N F F N N

N N N CF3

DSM426 (118)
IC50 = 0.0063 µM IC50 = 0.16 µM

S O
O S O
O O O
N N N N
H N N
O H H H O
O O O
O O O
(122) (123) (124) (125)
IC50 = 0.092 µM IC50 = 0.02 µM IC50 = 0.018 µM IC50 = 0.006 µM

O
CN
H
N N
S H Cl
N O OH
N Cl

(133) Cl (137)
NC IC50 = 0.04 µM IC50 = 10.2 µM

Figure 7. Structure of synthesized Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with their
respective values of IC50 .

Methodologies for the synthesis of PfDHODH inhibitors

The DSM1 inhibitor (5, IC50 = 0.047 μM) was obtained using a three-step synthetic method. Condensa-
tion of 3-amino-[1,2,4]triazole (108) with substituted ethyl acetoacetate in acetic acid produced substituted
7-hydroxy-[1,2,4]triazolo[1,5-a]pyrimidine (109). Chlorination with POCl3 yielded the corresponding 7-chloro-
[1,2,4]triazolo[1,5-a]pyrimidine (110), which, upon treatment with substituted amines, resulted in the desired
products (Figure 7 & Supplementary Figure 10) [54,69,70,102]. Continuing the work described by Phillips et al.,

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Review Hoelz, Calil, Nonato, Pinheiro & Boechat

Gujjar et al. prepared DSM74 (IC50 = 0.28 μM) using the same methodology (Figure 7) [71,102].
Other studies were performed to optimize the aromatic functionality with the aim of improving the potency
and the in vivo properties of triazolopyrimidine derivatives, which identified two new compounds DSM161 (111;
IC50 = 0.13 μM) and DSM190 (112; IC50 = 0.19 μM) that showed good plasma exposure and better efficacy in
the P. berghei mouse model (Figure 7) [102].
The compound DSM265 (7; Figure 7) was identified as a potent and selective PfDHODH inhibitor
(IC50 = 0.008 μM) that displays a potency against P. falciparum similar to that of CQ in a humanized mouse
model. It displays an excellent oral bioavailability, a long half-life and low clearance. Therefore, DSM265 was the
first candidate in the triazolopyrimidine series to be selected as a preclinical drug development candidate [38,53].
To discover new PfDHODH antagonists, modifications of nitrogen atoms of the [1,2,4]triazolo[1,5-a]pyrimidine
ring have been made giving rise to other heterocyclic systems. The imidazo[1,2-a]pyrimidine derivatives DMS71
(113; IC50 = 0.15 μM) and DMS151 (114; IC50 = 0.077 μM) and increased or retained the potency, respectively,
compared with a [1,2,4]triazolo[1,5-a]pyrimidine analog DSM74 (6; IC50 = 0.28 μM). However, the pharmacoki-
netics and metabolic stability of the triazolo[1,5-a]pyrimidine derivative remain more favorable. The pyrazolo[1,5-
a]pyrimidine derivative DSM68 (115; IC50 = 0.4 μM) displays a moderate loss of activity. The NH group of
the arylamine moiety is also essential and cannot be replaced by an amide DMS155 (116; IC50 > 100 μM);
such replacement leads to complete loss of activity. Purine, pyrazolo[3,4-d]pyrimidine and [1,2,4]triazolo[4,3-
b]pyridazine derivatives were inactive or showed low potency. The potency of the [1,2,4]triazolo[1,5-a]pyridine
derivative DSM96 (117; IC50 = 0.4 μM) was reduced compared with that of DMS1 (5; IC50 = 0.047 μM) proving
the importance of the pyrimidine ring (Figure 7) [103]. Although DSM151, an imidazo[1,2-a]pyrimidine deriva-
tive, displayed more inhibitory potency than DSM74, its in vivo efficacy was reduced owing to its poorer plasma
exposure. The N4 substitution forms the derivative pyrazolo[1,5-a]pyrimidine DSM68 (IC50 = 0.4 μM), leading
to a moderate loss of activity. N1 is also essential and cannot be replaced by an amide (DMS155 IC50 > 100 μM),
as such replacement leads to complete loss of activity [103].
The replacement of the SF5 -aniline moiety, present in the DSM265 inhibitor (9; Supplementary Figures 4 & 7),
by CF3 -pyridinyl group led to the discovery of DSM421, a potent and selective inhibitor of PfDHODH. DSM426
showed an IC50 value of 0.053 μM against PfDHODH enzyme and displayed efficacy in the P. falciparum mouse
malaria model. This compound represents a potential preclinical drug for use against both P. falciparum and P.
vivax as a chemopreventative or a single-dose cure (Figure 7) [58]. These data suggest that this derivative has the
potential for further development in the treatment of malaria [57].
Boechat et al. reported the synthesis and activity of 2-methyl-7-(naphthalen-2-ylamine)-5-
(trifluoromethyl)pyrazolo[1,5-a]pyrimidines (118) that display inhibitory activity against PfDHODH
(IC50 = 0.16 μM; Figure 7). One of these compounds was synthesized in a reaction between 3-aminopyrazole
(119) and ethyl 4,4,4-trifluoroacetoacetate producing the 2-methyl-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-
7(4H)-one (120) intermediary. Consequently, the reaction of compound 120 with POCl3 gave the derivative 121.
Finally, compound 121 reacted with β-naphthylamine, via an aromatic nucleophilic substitution, to yield the
inhibitor 118 (Figure 7 & Supplementary Figure 11) [99].
A novel series of dihydrothiophenone and dihydrofuranone derivatives was also shown to include PfDHODH
inhibitors. The most potent and selective inhibitors 122–125 (Figure 7 & Supplementary Figure 12) showed
inhibitory activity against PfDHODH with IC50 values of 0.02, 0.092 and 0.018 μM, respectively [104]. The
dihydrothiophenone (122, 123) derivatives were synthesized in a three-step procedure (Supplementary Figure 12).
From the appropriate arylamines (126, 127), the arylthiocyanates (128, 129) were obtained. When these are treated
with triphosgene followed by condensation with ethyl 4-chloroacetoacetate, the target derivatives were produced
122, 123. Moreover, the reaction of diethyl malonate (130) with chloroacetyl chloride (131) produced the 4,5-
dihydrofurane derivative (132), which reacts with arylamines (126, 127) to yield the target derivatives 124, 125
(Figure 7 & Supplementary Figure 12) [104].
The derivative 5-(4-cyano-2-methyl-1H-benzo[d]imidazol-1-yl)-N-cyclopropylthiophene-2-carboxamide (133)
(Figure 7 & Supplementary Figure 13) was identified as a potential drug development candidate. This com-
pound is a potent and selective inhibitor of PfDHODH (IC50 = 0.04 μM) compared with HsDHODH. The
reaction of 3-fluoro-2-nitrobenzonitrile (134) with 5-aminothiophene (135) using BnEt3 NCl as a catalyst pro-
duced the aminothiophene 136. The compound 133 was formed via a peptide-coupling methodology using
O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and cyclopropy-
lamine (Figure 7 & Supplementary Figure 13) [52,105].

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 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

In addition, several compounds were screened for activity against PfDHODH and HsDHODH, compound
137, which is two-times more selective for the Plasmodium enzyme (IC50 = 10.2 μM) than for the human
enzyme (IC50 = 16.5 μM; Figure 7 & Supplementary Figure 14), was selected. Suzuki coupling of (2,6-
dichlorophenyl)boronic acid (138) with 4-bromo-2-chloroaniline (139) was performed to obtain the substituted
biphenylaniline (140). The 5-methylisoxazole-4-carbonyl chloride (141) was obtained by treatment of the cor-
responding acid with thionyl chloride. Thus, 140 was treated with 141 to yield the corresponding isoxazole-4-
carboxamide 142. The O–N bond of the isoxazole ring was cleaved via treatment with NaOH to produce compound
137 in 100% yields (Figure 7 & Supplementary Figure 14) [85].

Conclusion & future perspective

Malaria is considered the deadliest disease in human history. Despite all efforts aimed at prevention and treatment
of the disease, malaria is still considered one of the most serious public health problems in the world and accounts
for almost than half a million deaths yearly. Moreover, the ability of Plasmodium spp. to develop resistance to current
therapies represents a new and threatening challenge to malarial treatment in the next generations. However, a num-
ber of new antimalarial compounds at different stages of preclinical and clinical development have been described.
Some fixed-dose combinations have been prequalified and marketed; these include ASMQ, ASAQ Winthrop,
Coartem-Dispersible, Synriam and Eurartesim. New experimental antimalarial molecules are currently undergoing
clinical trials (Phase I: MMV390048, ELQ300, CDRI97/78, SJ557733, P218, PA21A092 and ACT481840;
Phase II: OZ439, ferroquine, AQ13, methylene blue, KAE609, DSM265 and 21A092, KAF156/GNF156, fos-
midomycin; and Phase III: tafenoquine and trimethoprim) [12,15,106–109].
One of the current strategies used to fight against malaria exploits the inability of Plasmodium spp. to synthesize
pyrimidine nucleotides through the salvage pathway, relying exclusively on the de novo biosynthesis of the pyrimidine
pathway. Our review detailed efforts to develop potent and selective inhibitors against the enzyme DHODH, the
fourth and only redox reaction in the de novo biosynthesis of the pyrimidine metabolic pathway.
X-ray crystallography has allowed mapping of the 3D structure of DHODH from P. falciparum and has provided
insight into how known ligands interact with their macromolecular targets, thus contributing to the design of future
generations of inhibitors. The latter has been beautifully performed with the aid of molecular modeling methods
such as molecular docking, QSAR, structure-based pharmacophore mapping and molecular dynamics simulation.
Finally, state-of-the art organic synthesis has been successfully applied to create several DHODH inhibitors,
including compounds that are currently in clinical trials. The achievements described in this work represent an
important starting point in the fight against malaria.

Acknowledgements
The authors thank the Coordination of Improvement of Higher Education (CAPES), the São Paulo Research Foundation (FAPESP),
and the National Council of R&D of Brazil (CNPq) for the fellowships granted to the authors. The authors also thank M Maetani
and S Schreiber of the Broad Institute of MIT and Harvard, for providing DSM265 for the incubated inhibition assays.

Financial & competing interests disclosure

The authors also thank the Foundation for Research of the State of Rio de Janeiro (FAPERJ), for financial support. The authors have
no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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Review Hoelz, Calil, Nonato, Pinheiro & Boechat

Executive summary
Background
• Malaria is one of the most serious global public health problems.
• Vector control, chemoprophylaxis and chemotherapy using antimalarial drugs are the main actions used to
eliminate or reduce the number of cases of malaria.
• The antimalarials fall into three main classes: quinoline derivatives, antifolates and artemisinin derivatives.
Nucleotide biosynthetic pathways
• Inhibitors of the de novo synthesis of pyrimidine nucleotides make up an important group of chemotherapeutic
agents.
Dihydroorotate dehydrogenases
• The enzyme dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate by a
ping-pong-type enzymatic mechanism.
• The DHODH enzymes of different organisms can be divided into two classes (Class 1 and Class 2).
• Plasmodium falciparum DHODH (PfDHODH) is a Class 2 enzyme.
X-ray Structures
• DHODHs from 14 different organisms have been successfully crystallized.
• The PfDHODH crystals are complexed with A771726 (teriflunomide, a leflunomide active metabolite) and orotate.
Description of the structures
• DHODHs show the typical α/β-barrel fold consisting of a central barrel composed of eight parallel β-strands
(β1–β8) wrapped by eight α-helices (α1–α8).
• Mapping of the interactions between PfDHODH and ligands highlighted the importance of hydrogen-bond
interactions between the ligands and His185.
• Other residues appear to be important and are conserved among Plasmodium species.
PfDHODH kinetics & activity assays
• The bireactant PfDHODH presents a kinetic mechanism that is similar to that of other Class 2 DHODH enzymes.
• DHODH activity has been measured using the standard colorimetric assay.
• A new fluorescence intensity assay was developed for monitoring DHODH activity.
Medicinal chemistry of PfDHODH inhibitors
Application of high-throughput screening & enzyme structural information to the discovery of Plasmodium
DHODH inhibitors
• A series of potent and selective PfDHODH inhibitors of the triazolopyrimidine class was also identified using
high-throughput screening (HTS).
• Studies using HTS with subsequent lead optimization also identified thiophenecarboxamide derivatives.
• HTS method select PfDHODH inhibitors against mutant forms related to drug resistance.
Discovery & development of PfDHODH inhibitors using computational approaches
• SPROUT computational package, a suite of program modules for de novo structure-based molecular design.
• N-arylaminomethylene malonate derivatives with inhibitory activity against PfDHODH and human DHODH.
• Triazolopyrimidine-based PfDHODH inhibitors studies using molecular docking and quantitative structure–activity
relationship methods.
• x-ray structure of PfDHODH complexed with triazolopyrimidines derivatives.
• Virtual screening strategy for designing new and potent PfDHODH inhibitors.
• A virtual selection method using CoMFA and CoMSIA reported triazolopyrimidine analogs as PfDHODH inhibitors.
• 3D-quantitative structure–activity relationship pharmacophore models were constructed using the structures of
known PfDHODH inhibitors.
Synthetic methodology to obtain PfDHODH inhibitors
• The compound DSM1 was identified as a PfDHODH inhibitor.
• Pyrazolopyrimidine derivatives were tested as inhibitors of PfDHODH.
• Dihydrothiophenone and dihydrofuranone derivatives were identified as PfDHODH inhibitors.
Future perspective
• New antimalarial compounds in development have been described.
• Develop potent and selective inhibitors of DHODH.
• X-ray crystallography contributes to the design of future generations of inhibitors.

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 Plasmodium falciparum dihydroorotate dehydrogenase: a drug target against malaria Review

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