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

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

Anti-viral drugs deoxyguanosineAciclovir 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), CO 2, aspartate (N) Salvage is using or recycling species; using purine nucleotides and –sides already made

Purine Biosynthesis Starting material

Purine Biosynthesis sugar glutamines glycine folate CO 2 aspartate

De novo pyrimidine synthesis Step 1: HCO 3 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 6 methyl pterin p-amino benzoic acidglutamate NH 2 CH 3 Methotrexate

Folate analogues 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 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

ANTI-CANCER DRUGS

5 fluorouridine Rapidly phosphorylated once in the cell

5FU: a suicide inhibitor The enzyme reacts with C 6 on the ring and forms a covalent bond 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 cells in your average human (some have more, some less!!). The distance from the earth to the sun is 1.5 X 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 10 6 /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?