PHAR2811 lecture Nucleotides as drug targets COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated.

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

HN

HH

HH

HO

N

NH

O

O

N+

N-AZT

O

HH

HH

HH

HO

N

N

NH2

O

2' 3' dideoxycytidine

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

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

NH

NN

N

O

O

OHOH

HH

HH

OP-O

O-

O

Starting material

Purine Biosynthesis

NH

NN

N

O

sugar glutamines

glycine

folate

folate

CO2

aspartate

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

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.

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

Folate analogues

NH2 S

O

NHR

O

NH2 COO-

Sulfonamides

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

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

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

ANTI-CANCER DRUGS

NH

O

ON

O

HOH

HH

HH

HO

5-fluorodeoxyuridine

F

NH

O

ONH

5-fluorouracil

F

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

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

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?