Nucleotide drug targets.

The place of nucleotides.

While nucleotides are pivotal to metabolism it is interesting that there are very few
examples where drugs are designed around nucleotides. It is almost that they are too
important. Let’s consider the place of nucleotides. ATP is the energy currency of the
cell; you don’t get much more important that. ATP is adenine attached to ribose with 3
phosphates attached. One or two of these phosphates may be cleaved in a reaction to
yield energy (the phosphodiester bond is a quite high energy bond). Likewise GTP is
used in protein synthesis as an energy source to drive translation, UTP to activate
monosaccharides for polysaccharide biosynthesis and CTP is involved in supplying
energy for phospholipid synthesis. NAD (nicotinamide adenine dinucleotide) is 2
nucleotides joined together via their phosphates. One of the nucleotides contains an
adenine base and the other contains nicotinamide. The nicotinamide is able to carry a
hydride ion; a proton and 2 electrons or H-. It captures these hydride ions as fuels (fat
or carbs) are oxidised. The process of oxidation is ultimately to produce CO2 from
fuels and involves the removal of electrons and protons from carbons and replacement
with oxygen. The capturing of these high energy electrons by NAD enables the energy
to be ultimately converted to ATP.

Such pivotal roles mean any drug analogue that mimics these compounds would
have far reaching consequences.

The major examples are drugs used in HIV treatment and chemotherapy treatments
for cancers. AZT, dideoxycytidine and dideoxyinosine are used in HIV therapy
because the AIDS virus, as an RNA virus, requires reverse transcriptase (present in
the virus and injected into the host cell on infection) to convert its RNA genome to
DNA. Reverse transcriptase has a higher affinity for the deoxynucleotides than the
host cell DNA polymerases. The modified 3’OH prevents elongation.

Chemotherapy for cancer involves identifying pathways that are much more active in
rapidly proliferating cells (usually cancer cells) and targeting some part of this
pathway.

The most targeted process is DNA synthesis which happens on a grand scale only in
cell division. Rapidly proliferating cells do a lot more DNA synthesis than other
cells.

The main strategies have been to target selected parts of the process; de novo
nucleotide synthesis and thymidine conversion and more recently cytoskeleton
components involved in the chromosome separation at mitosis (spindle formation
etc).

Lets consider de novo biosynthesis with reference to purines. This term means
making from scratch i.e. small precursors. The purine rings are made from glutamine,
formate via folate, glycine, aspartate and CO2 starting with a sugar molecule (see
slides). But most of the time in non-dividing and slowly dividing cells purines are
 salvaged from degradation of other nucleotides, RNA etc and recycled rather than
making them from scratch. It is much cheaper energy-wise. Only when cells divide
rapidly does de novo synthesis become significant. So it is a good target.

Likewise de novo pyrimidine synthesis. Unlike purines, pyrimidine biosynthesis starts

by making the base then the sugar is added. The process starts as HCO3- which has an
amino (-NH3+) added from the sidechain of glutamine (and a phosphate from ATP),
producing a compound known as carbamoyl phosphate. This reaction is catalysed
carbamoyl phosphate synthase (CPSII) of which there are 2 types, used for very
different pathways and tightly regulated. An aspartate is then added to the carbamoyl
phosphate, which cyclises to produce orotate (see slides). The ribose phosphate is then
added as PRPP, giving rise to the pyrimidine nucleotides we all know and love. While
drugs have targeted the aspartate transcarbamylase (PALA being the most famous)
they are not as widely used as the purine inhibitors. Lets look further into the unique
steps in DNA synthesis.

Thymine is only found in DNA. TTP is only ever used to make DNA so its synthesis
from dUMP is a good target. The enzyme that achieves this is thymidylate synthase
which requires folate (THF) to donate the methyl group. The first anti-cancer drug
methotrexate acted on both thymidylate synthase and on de novo purine synthesis.
Because both processes require folate this powerful competitive inhibitor of
dihydrofolate reductase was and is used to treat many cancers, particularly childhood
leukemias. It mimics dihydrofolate and binds to its cognate enzyme (the reductase)
with > 1 000 fold the affinity of the natural substrate, making it all but irreversible.
The other interesting anti-cancer drug is 5-Fluorouracil and the nucleoside 5-
fluorourydine. This drug specifically targets thymidylate synthesis:

O
O

NH Thymidylate synthase H3C

NH

N O
O
N O
O
-O P O
O
-O P O
H H O
O-
O- H H
H H
OH H H H
OH H

dUMP TMP

5-Fluorouracil is rapidly converted to the nucleoside then phosphorylated in cells.

Fluorine is attached to carbon5 of the uracil ring. This is the carbon that has the
methyl group attached in the conversion to thymine. The fluorine stuffs this reaction
up! But it is known as a suicide inhibitor because it causes the enzyme to “commit
suicide”. The enzyme binds to the substrate (and actually forms a covalent bond with
C6), the folate begins to donate the methyl group and then the enzyme must abstract
or take the proton from C5, but there is a fluorine atom there instead of an H. At this
point the enzyme gets stuck and locked up in covalent complex with folate and the
substrate (see the slides for the mechanism).
O
O
F
NH F
NH

N O
N O
H
HO
O
H H
5-fluorouracil
H H
OH H

5-fluorodexouridine

The substitution of the fluorine for the hydrogen on C5 is used in a couple of other
drugs. 5-Fluorocytosine is an antifungal agent. Fungi, unlike mammals, can convert
this modified base to the 2 deoxy 5FU monophosphate or fluorinated dUMP which
will inhibit replication as above.
NH2 O

F F
N NH

N O -OOC N O
H H

5-Fluorocytosine
5-Fluoroorotate

Another drug synthesised by fluorine substitution is 5-Fluoroorotate. This anti-

malarial drug owes its effectiveness to the ability of the malarial parasite,
plasmodium, to take up exogenous orotate from the surroundings, unlike mammals.
As orotate is the precursor for UMP and CMP, the uptake of 5FO results in the
formation of d-5FUMP, which again acts as a suicide inhibitor on thymidylate
synthase as above.
Structure of the Genome
Lecture Synopsis: Review DNA structure. What does the DNA look like in a cell?
Chromosome length, diversity and packaging e.g. histones . Heterochromatin and
euchromatin and their relationship to transcription.

Review of DNA structure

DNA is a biopolymer made up of nucleotides;

 the sugar; deoxyribose,
 the phosphate,
 the base: adenine, thymine, guanine or cytosine.

The nucleotides are able to base pair; Adenine to Thymine and Guanine to Cytosine.
They are known as complementary or forming Watson and Crick canonical base pairs.

The nucleotides are joined via a phosphodiester bond, forming a polymer which has a
5’ phosphate (-PO4) “head” and a 3’hydroxyl (OH) “tail”.

DNA exists in the cell as a double stranded structure; the base sequence of each strand
is complementary to the other; one strand in the 5’ to 3’ orientation and the other in
the 3’ to 5’ orientation. The strands base pair throughout the full length of the
structure.

DNA is a specialised structure that functions as the genetic store of the cell; the
template. The absence of the OH at position 2’ of the ribose is a modification unique
to DNA which enhances the stability of the backbone to base attack (RNA, which
retains the OH at position 2’ is much more susceptible to base attack). The thymine
(methylated uracil) ensures that corruptions to the code brought about by spontaneous
deamination of cytosine can be corrected. Thymine also only exists in DNA.

Other structural features which contribute to DNA’s role as genetic

storehouse are:

The double stranded structure provides protection to the information containing face
of the bases, an extra copy of the information and a template for repair. The outside of
the DNA, with its predominating phosphate groups and sugar is very hydrophilic. The
bases, buried in the interior, are much more hydrophobic and the information in the
very heart of the molecule is polar. The exterior properties make it very difficult for
potential mutagenic compounds to penetrate the hydrophilic outer shell, move through
the hydrophobic interior to the polar information centre.
DNA Packaging

The genome of any organism, be it a eukaryote or a prokaryote contains a lot of

information so the DNA becomes extremely long.

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 m and length
3 m. In other words the bacterial dimensions seem to be 1/1000 th of the length of
the DNA (mm  m). The DNA is packaged as loops that are then supercoiled and
associate with proteins forming a dense structure termed the scaffold.

The full human genome contains 2 metres of DNA (this is all 46 chromosomes
worth!) in each cell. There are about 1013 cells in your average human (some have
more, some less). Therefore there must be 2 X 1013 m of DNA. Another useless fact:
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!!

The 2 metres of DNA has to be packaged into a nucleus with a diameter of ~6 m.
This makes packing the family station wagon to go camping look like a breeze!!

How is this amazing packaging achieved?

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. Up until now you have had this view that genes are sections of
double stranded, double helical DNA that code for one polypeptide chain. This is a
very precise and accurate definition but gives no idea of how it exists in the cell. You
have been taught that the hereditary material is DNA yet it appears as chromosomes.

Prokaryotes:

The genome of prokaryotes is extremely efficient. Survival depends on the ability to

divide rapidly when nutrients are available so there is no room for extra non-coding
stretches of sequence. Using the quintessential prokaryote example, Eschericha coli,
affectionately known by all as E. coli, this organism contains 4.6 million nucleotide
pairs or base pairs (bp). Consider your average E. coli bacterium: each protein on
average has a molecular weight of ~35 000, thus requiring ~350 amino acid residues
(assuming the average molecular weight of an amino acid residue is 100). This in turn
will need 1 050 base pairs which after including intergenic sections, promoter regions
and termination sections will give a final “gene” of 1 500 bp. If the bacterial genome
contains ~4.6 million bp then the bacterium can code for ~ 3 000 proteins. This is
within the “ball-park” estimate of the number of total number of proteins produced by
E. coli.

Despite this efficiency the DNA even for E. coli is quite long and as mentioned earlier
requires scaffold proteins to package it into the cell. The drawings I usually do of a
neat little circle sitting happily inside a cell may be a tad simplistic!

BUT as mentioned earlier this is nothing compared to eukaryotic DNA. For a start
eukaryotes are not as efficient with their code. Evolutionary imperatives for
multicellular organisms are not driven by the ability to colonise when ever and where
ever they can. The multicellular organism will be successful if it can adapt to its
environment; if its organisation responds quickly etc. In fact to have uncontrolled
proliferation, such as that seen with bacteria, in a multicellular organism is termed
cancer!! Because dividing rapidly is not a top priority eukaryotes, particularly higher
forms, can afford redundancy in the code. E. coli double every 20 min under optimal
conditions, human cells take 18 - 24 h to complete one round of the cell cycle. This
redundancy is seen as extra non-coding sequence. In fact it is estimated that only ~2
% of the human genome is actually coding i.e is transcribed and translated into
protein. We will discuss the rest in the next lecture.