phar2811 lecture nucleotides as drug targets commonwealth of australia copyright regulation warning...
TRANSCRIPT
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PHAR2811 lecture
Nucleotides as drug targets
COMMONWEALTH OF AUSTRALIA Copyright Regulation
WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of
the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright
under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection
under the Act.
Do not remove this notice
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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
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Strategies: HIV
• Inhibit reverse transcriptase produced by HIV by having nucleosides with no 3’OH
• AZT, 2’, 3’ dideoxycytidine, 2’, 3’ dideoxyinosine
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HIV reverse transcriptase inhibitors
O
HN
HH
HH
HO
N
NH
O
O
N+
N-AZT
O
HH
HH
HH
HO
N
N
NH2
O
2' 3' dideoxycytidine
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Anti-viral drugs
NH
N
N
O
NH2N
O
HO
NH
N
N
O
NH2N
O
HOH
HH
HH
HO
deoxyguanosine Aciclovir
Modified “non-sugar”
Nucleosides need to be phosphorylated once they enter the cell…only done by viral thymidine kinase
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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
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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)
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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
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Purine Biosynthesis
NH
NN
N
O
O
OHOH
HH
HH
OP-O
O-
O
Starting material
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Purine Biosynthesis
NH
NN
N
O
sugar glutamines
glycine
folate
folate
CO2
aspartate
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De novo pyrimidine synthesis
NH
O
ON
O
OHOH
HH
HH
OP-O
O-
OStep 1: HCO3
Step 2: glutamine
Step 3: aspartate
Added as PRPP
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Anti cancer drugs: Methotrexate
• Methotrexate, one of the earliest anti-cancer 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.
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Folate
NH
HNN
HN
O
H2N
CH2 NH C NH CH COO-
CH2
CH2
COO-
O
6 methyl pterin p-amino benzoic acid glutamate
NH2
CH3
Methotrexate
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Folate analogues
NH2 S
O
NHR
O
NH2 COO-
Sulfonamides
p amino benzoic acid
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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
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Fluoro-pyrimidines
N
NH
NH2
O
F
5-Fluorocytosine
NH
NH
O
O
F
-OOC
5-Fluoroorotate
This is an anti-fungal treatment…fungi can convert to 2 deoxy5FU
Anti-malarial..the malaria parasite can take up orotate to make pyrimidines
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Formation of thymine
NH
O
ON
O
HOH
HH
HH
OP-O
O-
O
NH
O
O
H3C
N
O
HOH
HH
HH
OP-O
O-
O
Thymidylate synthase
dUMP TMP
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ANTI-CANCER DRUGS
NH
O
ON
O
HOH
HH
HH
HO
5-fluorodeoxyuridine
F
NH
O
ONH
5-fluorouracil
F
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5 fluorouridine
NH
O
ON
O
HOH
HH
HH
OP-O
O-
O
FdUMP
FRapidly phosphorylated once in the cell
NH
O
ON
O
HOH
HH
HH
HO
FdUridine
F
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5FU: a suicide inhibitor
The enzyme reacts with C 6 on the ring and forms a covalent bond
CH
NH
O
O
F
N
O
HOH
HH
HH
OP-O
O-
O
S
:B- Enzyme
CH2
N
folate
A form of folate (THF) is linked to C5 via the methyl group it is donating
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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.
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Review of DNA Structure
• DNA is a biopolymer made up of nucleotides:– the sugar; deoxyribose, – the phosphate, – the base: adenine, thymine, guanine or
cytosine.
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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
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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).
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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!!
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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!!
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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.
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Chromosomes!!
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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?
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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.
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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.
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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!!
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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.
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What do these life forms have in common?