Faculty of Science - Mahidol University

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A simple method for screening combinatorial and other
libraries of inhibitors of malarial (Plasmodium falciparum)
dihydrofolate reductase (PfDHFR) has been developed, based on
the affinities of the inhibitors with the enzyme. In the presence of
limiting amounts of the enzyme, a number of inhibitors in the library
were bound to extents reflecting the relative binding affinities.
Following ultrafiltration and guanidine hydrochloride treatment to
release bound inhibitors, the amounts of free and bound inhibitors
could be determined by high-performance liquid chromatography
and liquid chromatography-mass spectrometry. The differences in
the patterns reflected the binding of high-affinity components
compared with the other members in the library. A good correlation
was found between the inhibition constants (K i values) and the extent
of binding of inhibitors to wild-type, double (C59R+S108N) and
quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well
as human DHFR. In addition to identifying lead components of the
libraries with high affinities (low K i values) and stabilities (low k off
rates), this simple method also provides an alternative way for quickly
and accurately calculating enzyme binding affinities of inhibitors
in combinatorial chemical libraries.
(Published in Anal Chem 2005; 77: 1222-7. Supported by EU INCO-
DEV, Wellcome Trust, MMV Program, WHO/TDR, Thailand Tropical
Diseases Research Program, and BIOTEC, NSTDA, Thailand.)
EFFECT OF METALLOPROTEASE INHIBITORS
ON INVASION OF RED BLOOD CELL BY
PLASMODIUM FALCIPARUM. (NO. 727)
Kitjaroentham A, Suthiphongchai T, Wilairat P.
Department of Biochemistry, Faculty of Science, Mahidol
University, Bangkok.
Key words : invasion, metalloprotease inhibitor, Plasmodium
falciparum.
For successful invasion, the malaria merozoite needs to
attach to the red blood cell membrane, undergo reorientation, form
a junction of the apical end with the host membrane, and internalize.
Malaria proteases have been implicated in the invasion process, but
their specific cellular functions remain unclear. To demonstrate the
involvement of metalloprotease in the process of Plasmodium
falciparum merozoite entry into host red blood cell, schizont-infected
red blood cells and parasitophorous vacuolar membrane-enclosed
merozoite structures were treated with 1,10-phenanthroline, a metal
chelator, resulting in a reduction of invasion with IC(50) value of
25 and 29 μM, respectively. Absence of an accumulation of schizont
stages after treatment with 1,10-phenanthroline indicated that the
inhibitory effect was not due to suppression of merozoite release
from red blood cells, but on the invasion step. Although treatment
with GM6001, a well-known inhibitor of the mammalian matrix
and disintegrin metalloprotease family, was less effective,
nevertheless this study points to the importance of metal-requiring
protease in the process of invasion of host red blood cell by the
malaria parasite.
(Published in Acta Trop 2005 Sep 14; [Epub ahead of print].
Supported by Thailand Research Fund.)
MAPPING ANTIMALARIAL PHARMACOPHORES
AS A USEFUL TOOL FOR THE RAPID DISCOVERY
OF DRUGS EFFECTIVE IN VIVO : DESIGN,
CONSTRUCTION, CHARACTERIZATION, AND
PHARMACOLOGY OF METAQUINE (NO. 728)
Dascombe MJ 1 , Drew MG 2 , Morris H 3 , Wilairat P 4 ,
Auparakkitanon S 4 , Moule WA 3,5 , Alizadeh-Shekalgourabi S 3,5 ,
Evans PG 2,3 , Lloyd M 3 , Dyas AM 3 , Carr P 5 , Ismail FM 3 .
1 Faculty of Life Sciences, The University of Manchester,
Manchester, UK; 2 School of Chemistry, University of Reading,
Reading, UK; 3 The Medicinal Chemistry Research Group, The
School of Pharmacy and Chemistry, Liverpool John Moores
University, Liverpool, UK; 4 Department of Biochemistry, Faculty
of Science, Mahidol University, Bangkok; 5 Department of
Chemistry, University of Hertfordshire, Hatfield, UK.
Key words : antimalarial pharmacophore, metaquine, Plasmodium
falciparum.
Resistant strains of Plasmodium falciparum and the
unavailability of useful antimalarial vaccines reinforce the need to
develop new efficacious antimalarials. This study details a
pharmacophore model that has been used to identify a potent, soluble,
orally bioavailable antimalarial bisquinoline,metaquine (N,N’-bis(7chloroquinolin-4-yl)benzene-1,3-diamine)
(dihydrochloride), which
is active against Plasmodium berghei in vivo (oral ID 50 of 25 μmol/
kg) and multidrug-resistant Plasmodium falciparum K1 in vitro (0.17
μM). Metaquine shows strong affinity for the putative antimalarial
receptor, heme at pH 7.4 in aqueous DMSO. Both crystallographic
analyses and quantum mechanical calculations (HF/6-31+G) reveal
important regions of protonation and bonding thought to persist at
parasitic vacuolar pH concordant with our receptor model.
Formation of drug-heme adduct in solution was confirmed using
high-resolution positive ion electrospray mass spectrometry.
Metaquine showed strong binding with the receptor in a 1:1 ratio
(log K = 5.7 +/- 0.1) that was predicted by molecular mechanics
calculations. This study illustrates a rational multidisciplinary
approach for the development of new 4-aminoquinoline
antimalarials, with efficacy superior to chloroquine, based on the
use of a pharmacophore model.
(Published in J Med Chem. 2005; 48: 5423-36.)
INDUCTION OF HUMAN CHOLESTGEROL
7ALPHA- HYDROXYLASE IN HEPG2 CELLS
BY 2,4,6-TRIHYDROXYACETOPHENONE (NO. 729)
Charoenteeraboon J 1 , Nithipatikom K 2 , Campbell WB 2 ,
Piyachaturawat P 3 , Wilairat P 1 , Rongnoparut P 1 .
1 Department of Biochemistry, Faculty of Science, Mahidol
University, Bangkok; 2 Department of Pharmacology and
Toxicology, Medical College of Wisconsin, Wisconsin, USA;
3 Department of Physiology, Faculty of Science, Mahidol
University, Bangkok.
Faculty of Science
Key words : cholesterol 7alpha-hydroxylase, HepG2 cell, 2,4,6trihydroxyacetophenone.
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