PHAR2811 lecture
Nucleotides as drug targets
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Nucleic acids as drug targets
• Nucleic acids are almost too important to
have analogues
• ATP, GTP, CTP, UTP
• NAD/NADH
• Bases of RNA and DNA
Strategies: HIV
• Inhibit reverse transcriptase produced
by HIV by having nucleosides with no
3’OH
• AZT, 2’, 3’ dideoxycytidine, 2’, 3’
dideoxyinosine
HIV reverse transcriptase inhibitors
O
NH 2
NH
N
HO
N
O
N
HO
O
H
O
H
H
H
H
N
H
H
H
H
H
H
N+
N-
AZT
2' 3' dideoxycytidine
O
Anti-viral drugs
Nucleosides need to be
phosphorylated once they
enter the cell…only done
by viral thymidine kinase
O
O
N
NH
N
N
NH
N
NH 2
HO
N
O
H
O
H
N
NH 2
HO
H
H
OH
Modified
“non-sugar”
H
deoxyguanosine
Aciclovir
Strategies: Anti cancer drugs
• Identifying pathways much more active in
proliferating cells
• DNA synthesis only occurs when cells
divide
– De novo nucleotide metabolism
– Thymidine formation
– Cytoskeleton, spindle formation
De novo synthesis vs salvage
• In non-dividing or slowly dividing cells
salvage pathways supply most of the
nucleotides needed (much less energy)
• In rapidly proliferating cells de novo
synthesis of nucleotides becomes
important (only done when absolutely
necessary…very expensive energy wise)
De novo synthesis vs salvage
• De novo synthesis is starting from the
beginning. Purine synthesis starting from
small precursors: PRPP, 2 X glutamine
(N), glycine (-C-C-N-), 2 X folate (C), CO2,
aspartate (N)
• Salvage is using or recycling species;
using purine nucleotides and –sides
already made
Purine Biosynthesis
O
N
NH
Starting
material
N
O
-O
P
O
O
H
O-
H
H
H
OH
OH
N
Purine Biosynthesis
O
glycine
CO2
aspartate
N
NH
folate
folate
N
sugar
N
glutamines
De novo pyrimidine synthesis
O
NH
Step 3:
aspartate
N
O
-O
P
O
Step 2:
glutamine
O
Step 1:
HCO3
O
H
O-
Added as
PRPP
H
H
H
OH
OH
Anti cancer drugs: Methotrexate
• Methotrexate, one of the earliest anticancer drugs, inhibits folate metabolism
• Folate provides methyl groups for
biosynthetic reactions
– It is essential for the conversion of dUMP to
TMP
– It provides carbon for the purine ring.
Folate
Methotrexate
COOH2N
N
H
N
CH 2
O
CH 2
HN
N
H
O
NH2
6 methyl pterin
CH 2
NH
C
NH
CH
COO-
CH3
p-amino benzoic acid
glutamate
Folate analogues
O
NH 2
S
NHR
Sulfonamides
O
NH 2
COO-
p amino benzoic acid
Fluorine substituted pyrimidines
• 5FU, 5 fluorouracil is an analogue of
uracil, 5FC (5 fluorocytosine) 5FO (5
fluoroorotate)
• There is a F attached to carbon 5 of the
pyrimidine ring instead of an H
• This is the same C that has the methyl
group attached in thymine formation
• F is very electronegative, small and the
C-F bond is very unreactive
Fluoro-pyrimidines
NH 2
O
F
F
N
N
H
NH
O
5-Fluorocytosine
This is an anti-fungal
treatment…fungi can
convert to 2 deoxy5FU
-OOC
N
H
O
5-Fluoroorotate
Anti-malarial..the
malaria parasite can
take up orotate to
make pyrimidines
Formation of thymine
O
O
Thymidylate synthase
NH
H3C
NH
N
O
O
N
O
-O
P
O
O
H
O-
-O
P
O
O
H
H
H
OH
H
dUMP
O-
H
H
OH
H
H
H
TMP
O
ANTI-CANCER DRUGS
O
O
F
F
NH
NH
N
O
N
H
HO
O
H
H
H
5-fluorouracil
H
OH
H
5-fluorodeoxyuridine
O
5 fluorouridine
O
O
Rapidly
phosphorylated
once in the cell
F
NH
N
F
NH
O
N
O
HO
-O
O
H
H
OH
H
H
P
O
O
H
O-
H
FdUridine
H
H
H
OH
H
FdUMP
O
5FU: a suicide inhibitor
A form of folate (THF) is
linked to C5 via the
methyl group it is
donating
folate
N
O
CH 2
F
NH
The enzyme reacts
with C 6 on the ring
and forms a
covalent bond
:B- Enzyme
S
CH
N
O
-O
P
O
O
H
O-
H
H
H
OH
H
O
The end result
• The enzyme – F5dUMP – folate (THF)
gets stuck in this complex unable to
progress through the reaction because of
the F!
• The enzyme commits suicide
• Because DNA synthesis needs TTP it
grinds to halt!
• DNA synthesis is the only place you see
TTP.
Review of DNA Structure
• DNA is a biopolymer made up of
nucleotides:
– the sugar; deoxyribose,
– the phosphate,
– the base: adenine, thymine, guanine or
cytosine.
DNA as the store of genetic
information
•
•
•
•
•
The removal of the OH at position 2’
The formation of thymine from uracil
Two strands gives a template for repair
Two copies of the information
The information carrying face is buried in
the middle of the two strands
Some useless statistics to drive
home the point:
• E. coli has one single circular
chromosome containing one long DNA
molecule, 1.3 mm in length. The bacterium
it has to fit in is a cylinder of diameter ~1
um and length 3 um. In other words the
bacterial dimensions seem to be 1/1000 th
of the length of the DNA (mm  um).
Some useless statistics to drive
home the point:
• The full human genome contains 2 metres
of DNA (this is all 46 chromosomes worth!) in
each cell.
• The 2 metres of DNA has to be packaged
into a nucleus with a diameter of ~6 um.
This makes packing the family station
wagon to go camping look like a breeze!!
Another useless fact:
• There are about 1013 cells in your average
human (some have more, some less!!).
• The distance from the earth to the sun is
1.5 X 1011 m.
• This means there is enough DNA in the
average human to stretch from the earth to
the sun and back about 50 times!!
How is this amazing packaging
achieved?
• Chromosomes!!
• Geneticists for years have predicted the
existence of chromosomes; both from
microscopy and from the observation that
certain genes did not inherit in the
standard Mendelian pattern.
Chromosomes!!
Prokaryotes
• The genome of prokaryotes is extremely
efficient.
• There are 4.6 million base pairs in your
average E. coli
• If the average bacterial protein has a
molecular weight of ~40,000 D how many
different proteins does the average E. coli
make?
Prokaryotes
• To do this calculation you need to know:
• The average mol. Wt. of an amino acid
~100
• This means the average protein has 400
amino acids
• Which means 1200 bases + promoter and
terminator sequences  ~1500 bp.
• 4.6 X 106/1500 = ~3000 different proteins.
Prokaryotes versus Eukaryotes
• Prokaryotes have no room for redundant
sequences.
• Their survival depends on rapid
proliferation when nutrients are available
• Complex multi-cellular eukaryotes depend
for survival on quick responses, adjusting
to changes in the environment.
Prokaryotes versus Eukaryotes
• E. coli can divide every 20 min if
conditions are optimal
• The human cell takes 18 to 24 h to go
through the cell cycle once.
• The human genome only has about 2%
coding regions.
• The gene density is much lower!!
Chromosome Characteristics
• Chromosomes vary in number between
species. The chromosome number is a
combination of the haploid number (n) X
the number of sets. Algae and fungi are
haploid; most animals and plants are
diploid. The number of pairs of
chromosomes in different species’
genomes is bizarre.
What do these life forms have in
common?