26' Deoxyribonucleotide biosynthesis and nucleotide catabolism drug design and testing

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26. Deoxyribonucleotide biosynthesis and
nucleotide catabolism drug design and testing
40 on the material not covered in the hour exams
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Ribonucleotide reductase reduces ADP, GDP, CDP
UDP to the deoxyribonucleotides
ribonucleotide reductase
Two cysteines in the enzyme serve as the
reductant.
NADP
NADPH
DNA synthesis also requires thymine, which is
made from dUDP.
Ribonucleotide reductase is located in the
cytosol.
Regeneration of the reduced enzyme requires
several steps
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Each of the catalytic sites in ribonucleotide
reductase has a binuclear Fe center and a stable
tyrosyl radical
The enzyme has two types of subunits, R1 and R2.
The catalytic sites are at the R1-R2 interfaces
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Ribonucleotide reductase generates free-radical
intermediates
1
NDP
The tyrosine radical generates another radical
(X?) close to the substrate.
2
3
dNDP
Most electron-transfer reactions of NADH involve
hydride ion transfer.
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Regulation of ribonucleotide reductase at two
allosteric sites balances the rates of formation
of the different deoxyribonucleotides
dTTP inhibits reduction of CDP UDP, and
activates reduction of GDP.
ATP activates reduction of CDP UDP.
dATP inhibits and ATP activates the enzyme
with all substrates.
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At the activity-regulation site, dATP inhibits
RNR and ATP increases Vmax for the reactions of
all the ribonucleotides
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At the specificity site, ATP, dATP, dTTP and dGTP
tune the relative affinities of RNR for CDP, UDP,
GDP and ADP

ATP
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Two sets of redox proteins carry electrons from
NADPH to ribonucleotide reductase (RNR)
RNR
RNR
thioredoxin
glutaredoxin
thioredoxin reductase (FAD)
NADP
NADPH
glutathione
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Structures of reduced and oxidized thioredoxin in
solution
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Thymidylate (dTMP) can be synthesized from either
CDP or UDP
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In the process of methylating dUMP, thymidylate
synthase oxidizes N5,N10-methylenetetrahydrofolate
to dihydrofolate
N5,N10-methylene-tetrahydrofolate
7,8-dihydrofolate
dTMP
dUMP
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Dihydrofolate reductase regenerates
tetrahydrofolate, using NADPH as the reductant
dihydrofolate reductase
7,8-dihydrofolate
tetrahydrofolate
NADPH H NADP
Serine
serine hydroxymethyl transferase (PLP)
Glycine
N5,N10-methylene-tetrahydrofolate
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Enzymes of nucleotide biosynthesis provide
targets for cancer chemotherapy
methotrexate
Analog of DHF. Inhibits dihydrofolate reductase
(in vivo)
Analog of dUMP. Inhibits thymidylate synthase.
5-fluorouracil
FdUMP
Analog of Gln. Inhibits glutamine
amidotransferases (steps 1 4 of purine
biosynthesis, CTP synthetase,
carbamoylphosphate synthetase II).
azaserine (O-diazoacetyl-L-serine)
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Primates oxidize purines to uric acid for
excretion
uric acid
adenosine
guanosine
Birds, reptiles and insects also excrete uric
acid. Fish and most terrestrial mammals oxidize
uric acid further before excretion.
uric acid has several tautomeric forms
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Formation of uric acid from purines
adenosine
inosine
hypoxanthine
adenosine deaminase
O2 H2O
xanthine oxidase
H2O2
xanthine
guanosine
uric acid
O2 H2O
Xanthine oxidase contains FAD, a Mo complex, and
two Fe-S centers. Its substrates are the free
purines, not the nucleosides or nucleotides.
H2O2
xanthine oxidase
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Mutations in adenosine deaminase cause Severe
Combined Immunodeficiency Disease
deoxy-adenosine
deoxyinosine
uric acid
adenosine deaminase
In the absence of adenosine deaminase, dATP
accumulates to high levels. dATP inhibits
ribonucleotide reductase, which prevents
synthesis of other deoxyribonucleotides and cuts
off DNA synthesis. Lymphocytes are particularly
susceptible to this inhibition. Untreated SCID
is fatal. SCID was the first human disorder to
be addressed by gene therapy.
dATP
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see Garrett Grisham, pp. 398-400
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Deposition of sodium urate crystals in tissues
causes gout
Impaired excretion of uric acid results in high
levels of uric acid in body fluids. Crystals of
sodium urate form in the toes, kidney and other
tissues, causing painful inflamation.
Gout can be treated with allopurinol, an analog
of hypoxanthine that inhibits xanthine oxidase.
sodium urate
allopurinol
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Salvage pathways regenerate nucleotides from
free purine and pyrimidine bases
adenine phosphoribosyl transferase
adenine
PPi
AMP
A similar enzyme (hypoxanthine-guanine
phosphoribosyl transferase) works on hypoxanthine
and guanine. Mutations in this enzyme lead to
Lesch-Nyhan syndrome.
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Lesch-Nyhan syndrome
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Lesch-Nyhan Syndrome can be diagnosed prenatally
Fetal fibroblasts obtained by amniocentesis are
cultured in the presence of 3H-hypoxanthine.
Cells from normal individuals convert
hypoxanthine into IMP, which procedes to AMP and
GMP and is incorporated into DNA.
normal
Lesch-Nyhan
IMP
hypoxanthine
Blocked in Lesch-Nyhan syndrome
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Drug design cycle using protein structural
information
X-ray, NMR, electron microscopy
3D Structures of Target Proteins Protein-Drug
Complexes
Functional Studies
Quantum Chemistry, Molecular Modeling, Chemical
Intuition
Data Base of Protein-Drug Complexes
Chemical Synthesis, Combinatorial Chemistry
Further Development
For examples, see www.bmsc.washington.edu/WimHol/
summary
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Clinical trials of drugs are conducted in four
phases
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Double-blinded tests with placebo controls are
required in Phase III
key to assignments kept secret