3 P. falciparum DHODH
Dihydroorotate dehydrogenase (DHODH) is one of the most validated and druggable
targets. Miller et al. in 1968 first isolated L-dihydroorotate–ubiquinone reductase
complex (from the beef liver) and was determined to be a mitochondrial enzyme [47].
The enzyme was recognized as dihydroorotate dehydrogenase (DHODH) and was
further isolated from rat liver in 1976 by Chen et al. [48]. Its localization was
determined to be the outer surface of the inner mitochondrial membrane which allows
free diffusion of dihydroorotate (DHO) from cytosol into the mitochondria and
orotate from mitochondria to the cytosol for further conversion to uridine
monophosphate (UMP). Larsen et al. established in 1985 that the E. coli DHODH is a
flavoprotein which catalyzes the conversion of dihydroorotate (DHODH) to orotate
in the fourth and only redox reaction in de novo pyrimidine biosynthesis [49].
The DHODH enzymes can be classified into two different classes [50]:
Family I includes the cytosolic enzymes which utilize fumarate or NAD
+ as the
terminal electron acceptor and deprotonation of alpha hydrogen occurred in the
presence of cystein.
Family II includes membrane-bound enzymes that transfer electrons to ubiquinone
(CoQ) and deprotonation occur in the presence of serine. Both human and plasmodium contain family II mitochondrial enzymes. In the host cells, pyrimidine
biosynthesis occurs via salvage and de novo pathway, whereas in P. falciparum
pyrimidines are synthesized only via de novo pathway. Thus, lack of salvage
pathway in plasmodium makes it a vulnerable target [51].
McRobert and McConkey in 2002 reported the importance of DHODH enzyme
in P. falciparum by performing RNA interference assay [52]. In 2002, Baldwin et al.
conducted inhibitory studies of various known human DHODH inhibitors (Redoxal,
dichloroallyllawsone (DCL), three analogs of A77-1726, and brequinar analogs) on
malarial enzyme [53]. It was observed that the plasmodium enzyme showed 10
2
–10
4
folds higher IC 50 compared to the human enzyme. This study suggested that inhibition of DHODH enzyme is species specific and can be further explored to design
P. falciparum selective DHODH inhibitors. Boa et al. in 2005 identified brequinar
derivatives as non-selective and weakly selective PfDHODH inhibitors proving the
previous hypothesis and laying a base for further development of selective
PfDHODH inhibitors [54]. Baldwin et al. identified phenyl benzamide/
naphthamides as selective PfDHODH inhibitors in nanomolar range through
high-throughput screening [55]. Heikkilä et al. used de novo design technology to
identify six molecules as PfDHODH inhibitors in micromolar range [56].
3.1 Functional Aspects of PfDHODH
Pyrimidines are essential metabolites that are precursors for DNA and RNA
biosynthesis. Cells acquire pyrimidines either through de novo synthesis starting
Structure-Based Design of PfDHODH Inhibitors …
189
Dihydroorotate dehydrogenase (DHODH) is one of the most validated and druggable
targets. Miller et al. in 1968 first isolated L-dihydroorotate–ubiquinone reductase
complex (from the beef liver) and was determined to be a mitochondrial enzyme [47].
The enzyme was recognized as dihydroorotate dehydrogenase (DHODH) and was
further isolated from rat liver in 1976 by Chen et al. [48]. Its localization was
determined to be the outer surface of the inner mitochondrial membrane which allows
free diffusion of dihydroorotate (DHO) from cytosol into the mitochondria and
orotate from mitochondria to the cytosol for further conversion to uridine
monophosphate (UMP). Larsen et al. established in 1985 that the E. coli DHODH is a
flavoprotein which catalyzes the conversion of dihydroorotate (DHODH) to orotate
in the fourth and only redox reaction in de novo pyrimidine biosynthesis [49].
The DHODH enzymes can be classified into two different classes [50]:
Family I includes the cytosolic enzymes which utilize fumarate or NAD
+ as the
terminal electron acceptor and deprotonation of alpha hydrogen occurred in the
presence of cystein.
Family II includes membrane-bound enzymes that transfer electrons to ubiquinone
(CoQ) and deprotonation occur in the presence of serine. Both human and plasmodium contain family II mitochondrial enzymes. In the host cells, pyrimidine
biosynthesis occurs via salvage and de novo pathway, whereas in P. falciparum
pyrimidines are synthesized only via de novo pathway. Thus, lack of salvage
pathway in plasmodium makes it a vulnerable target [51].
McRobert and McConkey in 2002 reported the importance of DHODH enzyme
in P. falciparum by performing RNA interference assay [52]. In 2002, Baldwin et al.
conducted inhibitory studies of various known human DHODH inhibitors (Redoxal,
dichloroallyllawsone (DCL), three analogs of A77-1726, and brequinar analogs) on
malarial enzyme [53]. It was observed that the plasmodium enzyme showed 10
2
–10
4
folds higher IC 50 compared to the human enzyme. This study suggested that inhibition of DHODH enzyme is species specific and can be further explored to design
P. falciparum selective DHODH inhibitors. Boa et al. in 2005 identified brequinar
derivatives as non-selective and weakly selective PfDHODH inhibitors proving the
previous hypothesis and laying a base for further development of selective
PfDHODH inhibitors [54]. Baldwin et al. identified phenyl benzamide/
naphthamides as selective PfDHODH inhibitors in nanomolar range through
high-throughput screening [55]. Heikkilä et al. used de novo design technology to
identify six molecules as PfDHODH inhibitors in micromolar range [56].
3.1 Functional Aspects of PfDHODH
Pyrimidines are essential metabolites that are precursors for DNA and RNA
biosynthesis. Cells acquire pyrimidines either through de novo synthesis starting
Structure-Based Design of PfDHODH Inhibitors …
189