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Modeling & 3D-Characterisation of Plasmodium falciparum DHFR & Its

Inhibitors

Introduction:
Malaria is an endemic disease in several regions of Africa, Latin America,
Asia and Oceania. It can cause Anemia, Pulmonary edema, Renal failure, Jaundice,
Shock and Cerebral complications. If not treated in a timely and adequate manner, it
can result in death. Malaria is caused by protozoan species from the genus
Plasmodium, which is transmitted by infected females of anopheles mosquitoes. Only
four species of Plasmodium cause the disease in humans: P.falciparum, P.vivax,
P.malariae, P.ovale.It is usual to nominate malarial infections according to the species
of the parasites involved. It is worthy of note that concurrent infections by more than
one species are found in regions where malaria is endemic.
Falciparum malaria has an incubation period of 1 to 3 weeks. It is the most
severe type of disease, being responsible for a considerable number of lethal cases. It
is believed that it’s pathogenecity results from it’s rapid rate of asexual reproduction
in the host and it’s ability to sequester in small blood vessels as well as it’s capacity to
utilize erythrocytes of all ages for reproduction.
As a consequence, most morbidity and mortality are observed in children
under five years old. Most children that survive gradually develop a type of “Partial
Immunity”, which protects them from the severe form of the disease. Even so, a small
“Partial Immunity”, young women, who are at risk from severe anemia during
pregnancy. On the other hand, in South America, Southeast Asia, India and China,
falciparum malaria is less common than in Africa.
In Brazil, the spreading human activities in the Amazon region has caused an
increase in the number of registered cases of malaria, due to the excellent climatic
conditions of this region for vector proliferation, as well as the lack of adequate vector
control planning and infrastructure. Consequently more than 99% of the cases of
malaria in Brazil are registered in the Amazon region.
Up to date, the creation of an effective vaccine against malaria has not been
possible. Vaccines can prevent several infections caused by viruses and bacteria,
microorganisms that generally can be easily cultivated invitro and have relatively
simple life cycles. On the other hand, the difficulty of generating large amounts of

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Plasmodium parasites in invitro cultures and the complexity of their life cycle, which
includes several morphologically and antigenically different parasites forms, have
hampered the development of a malaria vaccine.
A new anti-malarial treatment, a combination of Chlorproguanil and Dapsone
known as ‘Lapdap’ which was developed by TDR and partners, have been approved
by the UK Medicines and Health care Products Regulatory Agency (MHRA) for
treatment of uncomplicated Plasmodium falciparum, the most life threatening malaria
parasite, in adults and children more that three months of age. Lapdap is effective
against drug-resistant parasites and is anticipated to be available in several African
countries by the end of 2003, assuming that national approval is granted.
While an effective vaccine is not available, other strategies should be
employed against malaria. The main strategies for controlling malaria are educational
programs, chemotherapy and vector control. This last strategy is conducted by the use
of insecticides. The life cycle of protozoa’s include Asexual and Sexual
reproductions. Asexual life cycle is carried out in humans, while the Sexual life cycle
is carried out in Mosquitoes.
Up to date, four Plasmodium species have been reported to cause malaria,
which show polymorphism. The key morphological differences between human
Plasmodium species in blood smear are-

Sr.no P.falciparum P. vivax P. ovale P. malariae

Numerous rings Smaller rings No trophozoites or Crescent shaped
1 schizonts gametocytes
Enlarged Schuffner’s Ameboid trophozoite Compact
2 erythrocytes dots similar to P. vivax trophozoites
Fewer Merozoites Elongated Compact parasite Merozoites in
3 in schizonts erythrocytes rosette

Active Role of P. falciparum DHFR

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DHFR is a key enzyme in the metabolism of all cells. Inhibition of DHFR activity
depletes the cellular pool of tetrahydrofolate, a cofactor that is essential for both DNA
and protein synthesis, and specific inhibitors have been designed for both prokaryotic
and eukaryotic pathogens. Pyrimethemine is a specific competitive inhibitor of DHFR
from falciparum and with Sulfadoxine is one of component of the anti malarial drug,
Fansidar. In P. falciparum, extensive field and laboratory studies have shown that
resistance to Pyrimethamine is caused by point mutations in the DHFR gene. How
ever, in contrast to P.falcipuram; P.vivax cannot be maintained in culture and so the
mechanism of resistance to Pyrimethamine has not been established for this species.
WR 99210 is a novel inhibitor of DHFR, effective even against the most
Pyrimethamine resistant P.falciparum strains. It is found that it is also an extremely
effective inhibitor of the P.vivax DHFR, and mutation that confer high level
resistance to Pyrimethamine render the P.vivax enzyme exquisitely sensitive to
WR99210. These data suggest that Pyrimethamine and WR99210 would exert
opposing selective forces on the P.vivax population.
Pyrimethamine and Cycloguanil are potent inhibitors of plasmodium
falciparum DHFR; that were effectively used for the treatment of P.falciparum
malaria until the widespread appearance of resistant parasites reduced their utility.
Recently; sequencing to the DHFR gene from Pyrimethamine and Cycloguanil
resistant P.falciparum isolates revealed linkages between point mutations of the gene
and the degree of drug resistance of the parasites. Single and multiple mutations at
residues 16, 51, 59, 108 and 164 conferred cross-resistance to both the antifolates
Pyrimethamine and Cycloguanil where as mutation of residue 16 especially conferred
resistance to Cycloguanil.
In P. falciparum, dihydrofolate reductase and thymidylate synthase activities
are conferred by a single 70kDa bifunctional polypeptide (DHFR-TS; dihydrofolate
reductase thymidylate synthase)which assembles into a functional 140kDa
homodimer. In mammal, the two enzymes are smaller distinct molecules encoded on
different genes. A 27kDa amino domain of malarial DHFR-TS is sufficient to
provide DHFR activity, but the structural requirements for TS function have not been
established. Although the 3’end of their DHFR-TS has high homology to TS
sequences from other species, expression of this protein fragment failed to yield
active TS enzyme, and it failed to complement TS (-) E.coli unexpectedly, even
partial 5’deletion of full length DHFR-Ts gene abolished TS function on the 5’-end.

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Thus, it was hypothesized that the amino end of the bi functional parasite protein
plays an important role in TS function. When the 27 kDa amino domain was
provided in trans, a previously inactive 40kDa carboxyl domain from malarial DHFR-
TS regained its TS function. Physical characterization of this “split enzymes”
revealed that the 27 and the 40 kDa hybrid complex. Thus, in malarial DHFR-TS,
there are physical interactions between the DHFR domain and the TS domain and
these interactions are necessary to obtained a catalytically active TS. Interference
with these essential protein-protein interactions could lead to new selective strategies
to treat malaria resistant to traditional DHFR-TS inhibitors.
Crystallization and characterization of pfDHFR-TS enzyme should help in
developing a novel screening system for new drugs and a better understanding of the
enzyme and resistance to inhibitors.
Material & methods
For the present work the bioinformatics softwares used includes SWISS PDB
viewer, Protein Explorer. The software helps to create the 3-D structure
characterization and it also helps to locate the active site of the protein molecule.
After active characterization the available inhibitors were tested towards their
mechanism of action with the active of DHFR enzyme of Plasmodium falciparum.
After the active site characterization the result obtained are as follows.
Results & Discussion

Fig 3-D Ribbon Structure of Plasmodium falciparum DHFR Generated Through Protein Explorer

The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the
quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor

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WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial
pyrimethamine, reveal features for overcoming resistance. In contrast to
pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits
well in the active site, thereby contributing to binding. The single-chain bifunctional
PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved
in dimerization and domain organization. Moreover, positively charged grooves on
the surface of the dimer suggest a function in channeling of substrate from TS to
DHFR active sites. These features provide possible approaches for the design of new
drugs to overcome antifolate resistance.
References
1) Warhurst D: Drug resistance in Plasmodium falciparum malaria.
Infection 1999, 27:S55-58.

2) Adagu SI, Okoyeh JN, Lege-Oguntoye L, Ogala WN, Ogunrinde GO, Faji JT, Sani AH.
Efficacy of a 3-day oral regimen of quinine in an area of northern Nigeria with low-grade
resistance of Plasmodium falciparum to chloroquine and sulphadoxine-pyrimethamine. J
Trop Med Hyg 1995 Oct;98(5):296-8.

3) Wernsdorfer W: Epidemiology of drug resistance in malaria.

Acta Trop 1994, 56:143-156.

4) Radloff PD, Philipps J, Nkeyi M, Hutchinson D, Kremsner PG: Atovaquone and

proguanil for Plasmodium falciparum malaria.
Lancet 1996, 347:1511-1514.

Bioinformatics Soft wares Used

1) SPDB Viewer
2) Protein Explorer

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