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(Last
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Vorschlag: Typ III PKS in 4-Hydroxycoumarin Biosynthese
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4-Hydroxycoumarin
synthesis by a biphenyl synthase
References: Liu et al. (2009); overview in
Beerhues and Liu (2009)
Biphenylsynthase (BIS), a type III PKS
cloned some years ago (Liu et al., 2007),
is an STS-type enzyme catalyzing three condensations of malonyl-CoA with the
unusual starter molecule benzyol-CoA, synthesizing a biphenyl residue, see Fig.
1A (mehr... in another page). Recent work (Liu
et al., 2009) now described two more BIS-cDNAs (BIS2 and BIS3), and
investigated the activities with the substrate 2-hydroxybenzoyl-CoA in more
detail. The earlier work (2007) had shown already that BIS1 accepted this
molecule, but with only about 52% efficiency, when compared with benzoyl-CoA,
and carried out only one condensation, with 4-hydroxycoumarin as release product
(Fig. 1B). The present data now describe that BIS2 and BIS3 had a different
substrate preference: they also had BIS activity, but actually preferred
2-hydroxybenzoyl-CoA (100%) against benzoyl-CoA (65% and 66%).
That result of course raised the question whether this might indeed a
function of BIS in the plant. That was investigated with a tissue culture of the
host plant, Sorbus aucuparia. Elicitation, a treatment known to
induce BIS1, also induced BIS3, but not BIS2. After about 12 hours, the expected
aucuparin and related biphenyls started to accumulate, but no 4-hydroxycoumarin was detected, and
it was also not detected when 2-hydroxybenzoic acid was fed
to the cultures. However, interestingly, the induced cultures synthesized
4-hydroxycoumarin when fed with a NAC-derivatíve of 2-hydroxybenzoyl-CoA (the
NAC = N-acetylcysteamine mimics the activation by Coenzyme A). This showed that
BIS indeed can carry out the reaction in vivo, but that at least in the
case of Sorbus aucuparia, the cells do not provide the substrate nor its
activation to the CoA-ester that is required for the reaction to 4-hydroxycoumarin.
Another interesting question was whether the previously described
benzophenone synthase (BPS) would do the same as BIS: the BPS also uses
benzoyl-CoA and carries out three condensations with malonyl-CoA, but folds the
tetraketide with a CHS-type ring-folding rather than with an STS-type
ring-folding (mehr...). Somewhat
surprisingly, at least to me, the published data show that 2-hydroxybenzoyl-CoA
was not a substrate at all for the wild-type BPS (Liu
et al., 2003) or its mutant that could carry out only two condensations (Klundt
et al., 2009) (mehr...).
Fig. 1.
Reactions of Biphenylsynthase with two different
substrates.
So, what could be significant about the finding that plant
biphenyl synthase (BIS) can perform this reaction? Could it be more than just a
side reaction of BIS with a non-physiological substrate? After all, such
derailment reactions are well-known from type III PKS (mehr...).
Yes, it could be significant, because of the finding that some plants do contain
4-hydroxycoumarin and derivatives (see for example Valle et al., 1987;
Arnoldi et al., 2004;
Aliotta et al., 1994;
Appendino et al., 1988).
So far these seem to be only a few plants, but there may be many more,
considering how little is known in many plants about their natural products.
Their biosynthesis has not been analyzed, and it is an interesting idea that plant type III PKS might be involved.
It is also noteworthy that this BIS
reaction formally corresponds to the benzalacetone synthase (BAS) reaction that
is proposed as possibility for a type III PKS activity in the biosynthesis of
quinolines: Mehr...
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Another (standard?) pathway to 4-hydroxycoumarin
The idea discussed above is novel,
because so far it is usually believed that 4-hydroxycoumarin and
derivatives are synthesized via a different pathway, as shown in a simplified
scheme in Fig. 2.
Fig. 2.
Formation of 4-hydroxycoumarin by microbial
processes
These reactions are catalyzed by fungal
enzymes (see for example Bye and King, 1970),
and the anticoagulant effects of 4-hydroxycoumarin and derivatives were discovered as early as
in the 1920; they were the reason for a previously unrecognized cattle disease
in the northern United States and Canada: the substances were present in
moldy silage, i.e. after certain molds had colonized the harvested plants. Actually,
the nature of the responsible substance was discovered much later, in 1940: have
a look at the English Wikipedia
page on warfarin for this and more interesting information.
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Medical interest
in 4-hydroxycoumarin and derivatives
If you search the large library
databases, for instance "Scopus", with the keyword '4-hydroxycoumarin',
you'll get almost a thousand references! So, what is the interest in these compounds?
There are not only a huge number of them and closely related compounds (see e.g.
McGlacken and Fairlamb, 2005),
but there is also considerable medical interest. Of course, this page is not adequate for a competent discussion, but it should
be noted that there is intensive work going on for years to explore the
potential benefits in use as anticoagulants (see
Au and Rettie, 2008, for a recent
review), and treatment of cancer (see for example
Kostova, 2007, for recent
review).
Most people will not be interested to
look at all those details, but many will be familiar with the wide-spread use of
4-hydroxycoumarins as anti-coagulants, i.e. medication that causes a slower
blood clotting (the often used word "blood thinning" is not correct). The number of people needing such medication is quite large, and
so is of course the market for these substances. All of them are based on the
original finding that is described in the the English Wikipedia
page on warfarin: it is worth looking at. Fig. 3 shows the structure of some
of the molecules, and below is a brief discussion of some of their properties.
Fig. 3.
4-Hydroxycoumarin and some derivatives. See text
for brief comments.
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All these substances are anti-coagulant
drugs, i.e. they inhibit blood coagulation by blocking the synthesis of
coagulation factors II, VII, and X, by inhibiting vitamin K epoxide
reductase. It is noteworthy that two factors are mainly responsible for
different responses to such drugs in people:
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VKORC1 (Vitamin K epoxide reductase complex, subunit 1): this
protein is responsible for reducing vitamin K 2,3-epoxide to the
enzymatically active form. This is the target of warfarin and related
compounds, and therefore mutations in this gene can be responsible for
higher or lower sensitivity to the drugs,
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CYP2C9 (Cytochrome P450 2C9): this is one of the most important P450
activities for the oxidation of many xenobiotic and endogenous compounds; it
makes up about 18% of the P450 in the liver. It is alos involved in the
metabolism of 4-hydroxycoumarins; e.g. warfarin is a substrate of this
protein. The gene is highly polymorphic, and more than 50 single nucleotide
polymorphisms are known, with several of them leading to reduced enzyme
activity when compared to the wild-type enzyme. Therefore this protein can
be responsible for increased sensitivity to the drug, in those cases where
the activity of the protein is lower, and thus the removal of the drug.
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Dicoumarol is non-enzymatically formed from two molecules of
4-hydroxycoumarin, by reaction with formaldehyde which is usually present in
silage from microbe-catalyzed degradation processes. It is well known for
its anticoagulant properties, and was the main reason for the toxicity of
moldy silage.
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Warfarin (also known under the brand names Coumadin, Jantoven,
Marevan, Lawarin, and Waran) is a synthetic derivative of 4-hydroxycoumarin,
and the anticoagulant most often prescribed in North America. It was
initially marketed as poison for rats and mice, but was admitted for medical
purposes already in the 1950s. Its half-life in blood plasma is 2.5 days,
and that is an important difference to phenprocoumon (see below).
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Phenprocoumon (several tradenames, e.g. Marcumar and Falithrom) is
the anticoagulant drug most often prescribed in Europe. The link given here
in the name of the drug refers to the German Wikipedia page: the
English Wikipedia page
does not contain much more than the name, basic property, and structure. It
is also a synthetic derivative of 4-hydroxycoumarin, and the structure is
slightly different from that of warfarin. This small difference, however,
has a drastic effect on the half-life of the drug in blood plasma: it is 5-6
days, in contrast to the 2.5 days for warfarin. This should be considered in
at least two cases: a) do not mix warfarin and phenprocoumon, and b) if you
travel between between North America and Europe and run out of your standard
prescription: you cannot simple replace one with the other. The shorter
half-life of warfarin requires higher dosis for a given extent of
anti-coagulant effect.
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References
Liu, B.,
Raeth, T., Beuerle, T., Beerhues, L., 2009. A novel 4-hydroxycoumarin
biosynthetic pathway. Plant Molecular Biology, Sep 15 [Epub ahead of print].
Coumarin forms in melilotoside (trans-ortho-coumaric acid
glucoside)-containing plant species upon cell damage. In moldy
melilotoside-containing plant material, trans-ortho-coumaric acid is
converted by fungi to 4-hydroxycoumarin, two molecules of which
spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol
causes internal bleeding in livestock and is the forerunner of the warfarin
group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin
formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from
elicitor-treated Sorbus aucuparia cell cultures. The encoded
isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter
substrate and catalyzed a single decarboxylative condensation with
malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia
cell cultures were fed with the N-acetylcysteamine thioester of
salicylic acid, 4-hydroxycoumarin accumulated in the culture medium.
Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted
in the formation of 3,5-dihydroxybiphenyl which is the precursor of the
phytoalexins of the Maloideae.
Return
Beerhues,
L., Liu, B., 2009. Biosynthesis of biphenyls and benzophenones - evolution
of benzoic acid-specific type III polyketide synthases in plants.
Phytochemistry 2009, Aug 21 [Epub ahead of print].
Type III polyketide synthases (PKSs) generate a diverse array of
secondary metabolites by varying the starter substrate, the number of
condensation reactions, and the mechanism of ring closure. Among the starter
substrates used, benzoyl-CoA is a rare starter molecule. Biphenyl synthase
(BIS) and benzophenone synthase (BPS) catalyze the formation of identical
linear tetraketide intermediates from benzoyl-CoA and three molecules of
malonyl-CoA but use alternative intramolecular cyclization reactions to form
3,5-dihydroxybiphenyl and 2,4,6-trihydroxybenzophenone, respectively. In a
phylogenetic tree, BIS and BPS group together closely, indicating that they
arise from a relatively recent functional diversification of a common
ancestral gene. The functionally diverse PKSs, which include BIS and BPS,
and the ubiquitously distributed chalcone synthases (CHSs) form separate
clusters, which originate from a gene duplication event prior to the
speciation of the angiosperms. BIS is the key enzyme of biphenyl metabolism.
Biphenyls and the related dibenzofurans are the phytoalexins of the
Maloideae. This subfamily of the Rosaceae includes a number of economically
important fruit trees, such as apple and pear. When incubated with
ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a single decarboxylative
condensation with malonyl-CoA to form 4-hydroxycoumarin. A well-known
anticoagulant derivative of this enzymatic product is dicoumarol.
Elicitor-treated cell cultures of Sorbus aucuparia also formed
4-hydroxycoumarin when fed with the N-acetylcysteamine thioester of
salicylic acid (salicoyl-NAC). BPS is the key enzyme of benzophenone
metabolism. Polyprenylated benzophenone derivatives with bridged polycyclic
skeletons are widely distributed in the Clusiaceae (Guttiferae). Xanthones
are regioselectively cyclized benzophenone derivatives. BPS was converted
into a functional phenylpyrone synthase (PPS) by a single amino acid
substitution in the initiation/elongation cavity. The functional behavior of
this Thr135Leu mutant was rationalized by homology modeling. The
intermediate triketide may be redirected into a smaller pocket in the active
site cavity, resulting in phenylpyrone formation by lactonization.
Return
Liu, B., Raeth, T., Beuerle, T., Beerhues, L., 2007.
Biphenyl synthase, a novel type III polyketide synthase. Planta 225,
1495-1503.
Biphenyls and dibenzofurans are the phytoalexins of the Maloideae,
a subfamily of the economically important Rosaceae. The carbon skeleton of
the two classes of antimicrobial secondary metabolites is formed by biphenyl
synthase (BIS). A cDNA encoding this key enzyme was cloned from
yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a
novel type III polyketide synthase (PKS) that shares about 60% amino acid
sequence identity with other members of the enzyme superfamily. Its
preferred starter substrate is benzoyl-CoA that undergoes iterative
condensation with three molecules of malonyl-CoA to give
3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not
accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate,
however, was the preferential starter molecule for chalcone synthase (CHS)
that was also cloned from S. aucuparia cell cultures. While BIS
expression was rapidly, strongly and transiently induced by yeast extract
treatment, CHS expression was not. In a phylogenetic tree, BIS grouped
together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA
as starter molecule but cyclizes the common intermediate via intramolecular
Claisen condensation. The molecular characterization of BIS thus contributes
to the understanding of the functional diversity and evolution of type III
PKSs.
Return to text, more...
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Klundt, T., Bocola, M., Lütge, M., Beuerle, T.,
Liu, B., Beerhues, L., 2009. A single amino acid substitution converts
benzophenone synthase into phenylpyrone synthase. Journal of Biological
Chemistry Aug 26. [Epub ahead of print]
Benzophenone metabolism provides a number of plant natural products
with fascinating chemical structures and intriguing pharmacological
activities. Formation of the carbon skeleton of benzophenone derivatives
from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by
benzophenone synthase (BPS), a member of the superfamily of type III
polyketide synthases. A point mutation in the active site cavity (Thr135Leu)
transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic
change in both substrate and product specificities of BPS was rationalized
by homology modeling. The mutation may open a new pocket which accommodates
the phenyl moiety of the triketide intermediate but limits polyketide
elongation to two reactions, resulting in phenylpyrone formation.
3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor
substrate for PPS. The aryl moiety of the triketide intermediate may be
trapped in the new pocket by hydrogen bond formation with the backbone,
thereby acting as an inhibitor. PPS is a promising biotechnological tool for
manipulating benzoate-primed biosynthetic pathways to produce novel
compounds.
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Liu, B., Falkenstein-Paul, H., Schmidt, W., Beerhues,
L., 2003. Benzophenone synthase and chalcone synthase from Hypericum
androsaemum cell cultures: cDNA cloning, functional expression, and
site-directed mutagenesis of two polyketide synthases. Plant Journal 34,
847-855.
Benzophenone derivatives, such as polyprenylated
benzoylphloroglucinols and xanthones, are biologically active secondary
metabolites. The formation of their C13 skeleton is catalyzed by
benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell
cultures of Hypericum androsaemum. BPS is a novel member of the
superfamily of plant polyketide synthases (PKSs), also termed type III PKSs,
with 53-63% amino acid sequence identity. Heterologously expressed BPS was a
homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter
substrate was benzoyl-CoA that was stepwise condensed with three
malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept
activated cinnamic acids as starter molecules. In contrast, recombinant
chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures
preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic
acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a
phylogenetic tree, the two PKSs occurred in different clusters. One cluster
was formed by CHSs including the one from H. androsaemum. BPS grouped
together with the PKSs that functionally differ from CHS. Site-directed
mutagenesis of amino acids shaping the initiation/elongation cavity of CHS
yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over
4-coumaroyl-CoA.
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Bye, A., King, H. K., 1970. The
biosynthesis of 4-hydroxycoumarin and dicoumarol by Aspergillus fumigatus
Fresenius. Biochemical Journal 117, 237-245.
A strain of
Aspergillus fumigatus Fresenius, isolated from
spoiled hay, converts melilotic acid (o-hydroxyphenylpropionic acid) and
o-coumaric acid into 4-hydroxycoumarin and dicoumarol. The sequence is shown
to be melilotic acid (I) -> coumaric acid (IV) -> ß-hydroxymelilotic acid
(II) -> ß-oxomelilotic acid (III) -> 4-hydroxycoumarin (VI), on the basis of
(1) studies on the formation of postulated intermediates, (2) experiments
with isotopically labelled materials and (3) sequential enzyme induction. In
the presence of semicarbazide, o-coumaraldehyde is formed from o-coumaric
acid: there is no evidence, however, that this lies on the normal metabolic
pathway.
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Valle, M. G., Appendino, G., Nano, G. M., Picci,
V., 1987. Prenylated coumarins and sesquiterpenoids from Ferula
communis. Phytochemistry 26, 253-256.
From the latex of Ferula communis, two 4-hydroxycoumarin
derivatives were isolated bearing a farnesylic and a
12-hydroxyfarnesylic residue, respectively, at C-3. Prenylated coumarins,
which represent toxic principles of the plant, were absent in other
samples, which gave, besides known compounds, a germacrane alcohol (hallohedycariol)
and a daucane ester (siol p-hydroxybenzoate).
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Arnoldi, L., Ballero, M., Fuzzati, N., Maxia,
A., Mercalli, E., Pagni, L., 2004. HPLC-DAD-MS identification of
bioactive secondary metabolites from Ferula communis roots.
Fitoterapia 75, 342-354.
A simple HPLC method was developed to distinguish between 'poisonous'
and 'non-poisonous' chemotypes of Ferula communis. The method was
performed on a C8 reverse phase analytical column using a binary eluent
(aqueous TFA 0.01%-TFA 0.01% in acetonitrile) under gradient condition.
The two chemotypes showed different fingerprints. The identification of
five coumarins and eleven daucane derivatives by HPLC-diode array
detection (HPLC-DAD) and HPLC-MS is described. A coumarin, not yet
described, was detected.
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Aliotta, G., Cafiero, G., De Feo, V., Sacchi,
R., 1994. Potential allelochemicals from Ruta graveolens L. and
their action on radish seeds. Journal of Chemical Ecology 20, 2761-2775.
An aqueous extract of Ruta graveolens L. (250 g/liter) was
tested for its allelopathic activity in vitro on radish
germination and radicle growth in light and darkness. It caused a delay
in the onset and a decrease in the rate of germination (40%) in the
light. The photoinhibition of germination was accompanied by an
inhibition of water uptake into the seed. Furthermore, the inhibition of
radicle growth was slightly higher in the light than in darkness. Three
potential allelochemicals, biologically active in the light, were
isolated from the extract: 5-methoxypsoralen (5-MOP), 8-methoxypsoralen
(8-MOP), and 4-hydroxy-coumarin at concentrations of 10-4 M,
2×10-4 M, and 0.4 ×10-5 M respectively. At a
concentration of 2×10-4 M, 5-MOP was the most potent
inhibitor, decreasing radish germination to 32% and radicle growth to
17% with respect to control. Microscopic observations of radish seeds
treated with 5-MOP suggest that this substance changes the swelling of
the seed coat and aleurone layer, which precedes radicle protrusion.
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Appendino, G., Tagliapietra, S., Gariboldi,
P., Mario Nano, G., Picci, V., 1988.
w-Oxygenated prenylated coumarins from
Ferula communis. Phytochemistry 27, 3619-3624.
From the toxic variety of Ferula communis, derivatives of the
prenylated coumarins ferulenol and ferprenin bearing an oxygen function
(hydroxyl, acetoxyl, aldehydic carbonyl) at the omega-composition have
been isolated. The structures of the coumarins were established by
spectral methods and by chemical reactions. Photooxygenation of
ferulenol and (E) omega-hydroxyferulenol gave o-hydroxyphenylglyoxylic
esters, resulting from the oxidative decarbonylation of the
4-hydroxycoumarinic nucleus and loss of the prenyl side chain. Ethyl
o-hydroxyphenylglyoxylate was also isolated from the plant extract,
suggesting that a reaction of this type might be responsible for the
degradation of the prenylated coumarins in plant samples and extracts of
Ferula communis.
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McGlacken, G. P., Fairlamb, I. J. S., 2005.
2-Pyrone natural products and mimetics: isolation, characterisation and
biological activity. Natural Product Reports 22, 369-385.
The review summarises natural products containing the 2-pyrone
moiety. An emphasis has been placed upon the biological activity associated
with 2-pyrones, particularly with respect to potential therapeutic or
anti-microbial agents. Where appropriate, non-natural 2-pyrone analogues are
discussed, particularly those derived from natural product lead compounds.
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Kostova, I., 2007. Studying plant-derived
coumarins for their pharmacological and therapeutic properties as potential
anticancer drugs. Expert Opinion on Drug Discovery 2, 1605-1618.
Coumarins have attracted intense interest in recent years because
of their diverse pharmacological properties. Among these properties, their
anticancer effect was most extensively examined. In this review, their broad
range of effects on the tumours as shown by various in vitro and in vivo
experiments as well as clinical investigations is discussed. Studies have
indicated that coumarins elicit inhibitory effects on cell growth of various
carcinoma cell lines and may be potential candidates for cancer therapy.
These natural compounds have served as valuable leads for further design and
synthesis of more active analogues. In view of the relative simplicity of
the coumarin compounds and their mechanism of action, the coumarin
pharmacophore may serve as an important model on which to develop new
patterns in cancer chemotherapy. The aim of this review is to examine in
detail the properties of the title compounds as anticancer agents. In view
of their comparatively low toxicity, relative cheapness, presence in the
diet and occurrence in various herbal remedies, it appears important to
evaluate their anticancer potentialities. Moreover their synergistic
activity in combination therapy with other well-unknown anticancer drugs
could be the basis for the development of rational approaches to new forms
of cancer chemotherapy.
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Au, N., Rettie, A. E., 2008. Pharmacogenomics of
4-hydroxycoumarin anticoagulants. Drug Metabolism Reviews 40, 355-375.
Oral anticoagulants of the 4-hydroxycoumarin class, typified by
warfarin, are used worldwide to treat thromboembolic disease. These drugs
show the beneficial attributes of high efficacy and low cost, but patient
management can be complicated by their narrow therapeutic index and wide
inter-individual variability in dosing. Our understanding of the latter
complication has improved significantly in recent years due to intense
investigation of genetic factors influencing drug pharmacokinetics (CYP2C9)
and pharmacodynamic response (VKORC1). In particular, the discovery of
polymorphisms in the VKORC1 gene that strongly impact oral anticoagulant
dose has heightened expectations that genetic testing for a relatively small
cadre of warfarin-response genes might substantially enhance patient care in
this area, especially during the initiation phase of therapy. However,
enthusiasm for genotype-based dosing of oral anticoagulants must be balanced
against the ready availability of both a simple phenotypic test (prothrombin
time) and an antidote to over-anticoagulation (vitamin K). Wide-spread
acceptance of genetically based tests for establishing therapy with warfarin
and its congeners will likely require additional evidence that such an
approach offers protection against a variety of negative anticoagulation
outcomes, especially severe bleeding, as well as offering utility across
many racial populations. This article will review recent events in these and
other related areas.
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