Anal. Chem. 2007, 79, 5006-5012
Immobilization of Malarial (Plasmodium
falciparum) Dihydrofolate Reductase for the
Selection of Tight-Binding Inhibitors from
Combinatorial Library
Chawanee Thongpanchang,* Supannee Taweechai, Sumalee Kamchonwongpaisan,
Yongyuth Yuthavong, and Yodhathai Thebtaranonth
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency,
113 Thailand Science Park, Pahonyothin Road, Klong 1, Klongluang, Pathumtani 12120, Thailand
Based on modeling of wild type and mutant PfDHFRs, a number
of inhibitors were synthesized and screened against the enzymes
individually and found to be effective against both wild type and
some mutant PfDHFRs.10-13
A simple procedure for selection of tight-binding inhibitors
of mutant dihydrofolate reductases from Plasmodium
falciparum (PfDHFRs) based on preferential binding to
the enzyme immobilized on a Sepharose column has been
described. PfDHFRs with a cysteine residue at the C-
terminal have been prepared in order to immobilize to a
thiopropyl-Sepharose gel via S-S linkage. The amount of
immobilized DHFRs was estimated to be 4-5 mg/g of
dried gel, and the activities of bound DHFRs were
comparable to that of free enzymes. The prepared im-
mobilized enzyme has been used for the selection of tight-
binding inhibitors from combinatorial libraries, based on
the affinities of each ligand with the enzyme. Free ligands
were then identified and analyzed quantitatively by high-
performance liquid chromatography-mass spectrometry,
and the components with high binding affinity of the
library could thus be realized. Results could be confirmed
by quantitative analysis of the bound ligands released from
the enzyme by guanidine hydrochloride treatment.
The advent of combinatorial chemistry techniques offers the
means for rapid generation of a large number of structurally
related compounds. However, identification of potential leads from
the combinatorial libraries required an effective selection system
that can single out the tight-binding inhibitors for further
characterization and development. Recently, Kamchonwongpaisan
et al. have developed an ultrafiltration method for the selection,
based on the dissociation constant of the enzyme with each
inhibitor.14 In the stoichiometric selection, the limitation of this
technique is its dependence on the solubility of the target enzyme,
which affected the compound concentration in the libraries: i.e.,
the larger the size of the library, the lower the concentration of
each inhibitor.14
It has been reported that immobilized enzymes are useful tools
in many areas such as for enzyme sensors, enzyme reactors, or
enzyme catalysis in organic synthesis.15-19 Dihydrofolate reductase
enzymes were also immobilized onto the solid support in different
Plasmodium falciparum dihydrofolate reductase-thymidylate
synthase (PfDHFR-TS) is a validated target of antifolate antima-
larial drugs1-3 such as pyrimethamine (Pry) and cycloguanil (Cyc),
which are commonly used clinically for the treatment of malaria
infection. Since the emergence of the resistant strain of malaria
parasites to these inhibitors became widespread, there has been
an urgent need to search for new drugs to combat the resistant
malaria. The molecular mechanism for resistance to PfDHFR
inhibitors has been shown to be due to point mutation at various
sites of the parasite gene sequence, leading to the decrease in
binding affinity of inhibitor to the enzyme. Mutation of one or
more residues at amino acid positions 16, 51, 59, 108, and 164 of
PfDHFR were identified to be involved in antifolate resistance.4-9
(6) Foote, S. J.; Galatis, D.; Cowman, A. F. Proc. Natl. Acad. Sci. U.S.A. 1990,
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* To whom correspondence should be addressed. Tel.: 662-5646700 Ext 3559.
Fax.: 662-5646632. E-mail: chawanee@biotec.or.th.
(13) Kamchonwongpaisan, S.; Quarrell, R.; Charoensetakul, N.; Ponsinet, R.;
Vilaivan, T.; Vanichtanankul, J.; Tarnchompoo, B.; Sirawaraporn, W.; Lowe,
G.; Yuthavong, Y. J. Med. Chem. 2004, 47, 673-680.
(14) Kamchonwongpaisan, S.; Vanichtanankul, J.; Tarnchompoo, B.; Yuvaniyama,
J.; Taweechai, S.; Yuthavong, Y. Anal. Chem. 2005, 77, 1222-1227.
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(17) Iwakura, M.; Nakamura, D.; Takenawa, T.; Mitsuishi, Y. Protein Eng. 2001,
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(2) Nzila, A. J. Antimicrob. Chemother. 2006, 57, 1043-1054.
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(4) Peterson, D. S.; Walliker, D.; Wellems, T. E. Proc. Natl. Acad. Sci. U.S.A.
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5006 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007
10.1021/ac070215s CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/26/2007