Nucleic acid synthesis as a drug target

Question 1

Roles of Nucleotides

Answer 1

Information storage (DNA,RNA)
Energy Storage (ATP)
Information Transfer (cAMP. GTP)
Co-factors (NAD(H), NADP(H), FAD, CoA.)

Question 2

Nucleobases

Answer 2

Pyrimidines (single ring) or purines (double ring)
Adenine, Thymine, Cytosine, Guanine

Nucleosides - adenosine, guanosine etc.

Question 3

Parasitic Protozoa do not possess purine biosynthetic pathways

Answer 3

They scavenge purines from their host
Using specific purine transporter proteins.
Inter-conservation of purines using purine salvage enzymes

Question 4

Multiple purine transporters in bloodstream Trypanosoma Brucei

Answer 4

Overlapping substrate selectivity.
Substrates of T.brucei P2 adenosine transporter include:
Adenosine, adenine (purines), pentamidine, melarsoprol, isometamidium (trypanocides).

Question 5

Nucleotide biosynthesis

Answer 5

Nucleosides converted to nucleobases by nucleoside phosphorylase.
Nucleobases converted to nucleotides by phosphoribosyl transferases.

Question 6

Allopurinol

Answer 6

Used in the treatment of gout.
It is a hypoxanthine analogue and it inhibits xanthine oxidase, stopping hypoxanthine being cpnverted into uric acid.
It is also a substrate of hypoxanthine phosphoribosyl transferase in trypanosomatids and has been used clinically in the treatment of leishmaniasis.

Question 7

Differences between protozoa

Answer 7

Adenosine is the primary precursor of all purine nucleotides in Trichonmonas vaginalis.
in this case there is no role for hypoxanthine as there is no phosphoribosyl transferases.

Question 8

Polyamines

Answer 8

Essential for DNA and RNA stability and replication.
polyamines are cellular cations present in high abundance.
They bind many cellular anions i.e DNA and RNA and stabilise them by neutralising the negative charge.
They appear to be crucial to proliferating cells, and therefore polyamide metabolism is a good drug target in rapidly proliferating prtotozoa such as trypanosomes.

Question 9

Trypanothione

Answer 9

the Trypanosomatids the roles of glutathione are taken on by trypanothione molecules.
It comprises of 2 glutathione moieties linked by a spermidine moiety.
In many cells glutathione is a key low molecular weight thiol that is involved in maintaining the intracellular redox environment.
Trypanothione is synthesised from the reduction of trypanothione disuphide + spermidine by trypanothione reductase.

Question 10

Trypanosomes treated with Eflornithine

Answer 10

In the trypanosome-
Ornithine levels increase.
Putrescine, spermidine and trypanothione levels fall.
De-carboxylated s-adenosylmethionine levels increase.
Ornithine decarboxylase is the target for Eflornithine.

Question 11

Sulphonamides (sulfa)

Answer 11

Prontosil dye - effective against streptococci.

First group of chemical substances systematically used to treat and prevent bacterial infections in humans.

Question 12

The folate pathway

Answer 12

S-adenosylmethionine is one key methyl donor in the cell, another is folate (or its derivatives).
The folate pathway has been shown to be important in a number of protozoa, particularly apicomplexans like plasmodium and toxoplasma.
Mammals obtain folate from green veggies (folliage).
Apicomplexan parasites make their own folate from GTP.
Folate is an essential co-factor, structurally related to purines.

Question 13

Folates in Plasmodium

Answer 13

Folates are synthesised from GTP and PABA, they are essential for nucleotide metabolism.
Dihydropteroate synthase - can be inihibited by PABA analogues such as sulphonamides.
Dihydrofolate reductase - can be inhibited by folate analogues such as pyrimethimine and cycloguanil.

Question 14

Fansidar (sulphadoxine-pyrimethamine)

Answer 14

Introduced in 1960s as a synergistic anti-malarial drug
Reports of resistance within a few years.
Used as a second-line drug in areas where Chloroquine is still used.
In areas that are Choroquine resistant, it is a first line anti-malarial.
Many hyperendemic and holoendemic areas that are now Fansidar resistant.
Artemisinins are taking over as first line drug in many places.

Question 15

Mechanism of Fansidar resistance

Answer 15

Many theories, potentially many causes -
Amplification/overexpression of the DHFR-TS gene
Modification of transport of various substrates involved in folate biosynthesis.
Drug efflux
P.Vidax inherant resistance to anti-folates due to conformation of active site cavity of PvDHFR-TS.
Point mutations resulting in the substitution of amino acid residues that form the active site of DHFR-TS enzyme, leading to decreased affinity between enzyme and anti-folates.

Question 16

DHFR polymorphims in P. falciparum

Answer 16

Mutations in DHFR confer resistance to pyrimethamine and other DHFR inhibitors (sulphonamides).
Ser108Asn causes moderate pyrimethamine resistance in vitro, with higher resistance resulting from Asn51Ile and/or Cys59Arg.
The addition of Ile164Leu causes highest level of resistance.
Multi-mutations diminish the catalytic efficiency of the enzyme and therefor unfavourable to the enzyme in abscence if drug pressure.
DHFR Ser108Asn is used as a marker for SP resistance as it is selected for by SP treatment.
Asp108Tyr in DHFR reduces affinity to drugs, without affecting normal enzyme function.
Point mutations in cytochrome B underlie resistance to atovaquone.

Question 17

Trimethoprim

Answer 17

An inhibitor of bacterial DHFR
50,000 times more active against bacterial DHFR than mammalian.
Synergistic with sulphamethoxazole (sulphonamide), as they inhibit different parts of the same pathway.
Orally available, bacteriostatic antibiotic.
Mainly use in the prophylaxis and treatment of urinary tract infections.
Resistance can occur due to changes in the DHFR enzyme.