ANTIMETABOLITES

Many antimetabolites stop de novo RNA/DNA biosynthesis

 inhibit the formation of the nucleotides
 rate-limiting enzymes of nucleotide biosynthesis are primary targets
o they are usually false substrates for these enzymes
o preferential irreversibe (or pseudoirreversible) binding over the endogenous
substrate
o inhibition of these key enzymes is a highly efficient way to stop the biochemical
reaction sequence
o other enzymes required in DNA biosynthesis can also be inhibited
 chain elongation can be arrested, or the RNA/structure destabilised, through the
incorporation of false nucleotides into the growing strands
 if the DNA building blocks cannot be synthesised or efficient/stable RNA/DNA
formation is blocked, then RNA/DNA synthesis is stopped leading to cell death

When cells proliferate, they must replace nucleotides at the same rate as they are being
incorporated into DNA to maintain homeostasis. This can be achieved by:
 salvage pathways (e.g. recycling of cellular components after cell death)
 increased cellular uptake
 de novo synthesis
Rapidly proliferating cells (e.g. cancers) make significant use of the de novo pathway

De novo synthesis
 the synthesis of complex molecules from simple molecules, e.g. sugars, amino acids
 nucleotides are not needed in the diet as they can be constructed from small
precursor molecules e.g. formate and aspartate
 de novo pathways of nucleotides do not use free bases; the rings are assembled from
smaller units and attached which are attached to (deoxy)ribose/at stages of the
process
 agents targeting de novo synthesis of nucleotides can selectively inhibit the division
of rapidly proliferating cells
 DNA Homocysteine Methionine

Methyl-B 12
MS
dTTP
5-flurouracil 5-methyl-THF THF
ralitrexed
pemetrexed
NADP+ METHIONINE Glycine
CYCLE SHMT
dTDP
NADPH + H+ Serine

dUMP
NADPH + H+
NADP+
dTMP

MTHFD 5,10-methenyl-THF

FA DHF 5,10-methylene-THF
H2O
CYH
THYMIDYLATE PURINE Formate
CYCLE CYCLE
Glycine
DHFR SHMT SHMT
10-formyl-THF
NADPH + H+ Serine
GART
THF pemetrexed
NADP+ AICART
THF
GAR

methotrexate FGAR AICAR

RNA pemetrexed FAICAR
IMP

dATP ATP ADP AMP

dGTP GTP GDP GMP
RR 6-mercaptopurine
dATP
dGTP
FA: folate
DNA
gemcitabine DHF: dihydrofolate
azacitidine, cytarabine, clofarabine THF: tetrahydrofolate
fludarabine,
gemcitabine, trifluridine DNA: deoxyribonucleic acid
RNA: ribonucleic acid

AMP: adenosine monophosphate IMP: inosine monophosphate

ADP: adenosine diphosphate
ATP: adenosine triphosphate
AICART: AICAR formyltransferase
dAMP: deoxyadenosine monophosphate CYH: methylene-THF-cyclohydrolase
dADP: deoxyadenosine diphosphate DHFR: dihydrolate reductase
dATP: deoxyadenosine triphosphate GART: GAR-formyltransferase
MS: methionine synthase
GMP: guanosine monophosphate MTHFD: methylene-THF-dehydrogenase
GDP: guanosine diphosphate MTHFR: 5,10-methylene-THF-reductase
GTP: guanosine triphosphate RR: ribonucleotide reductase
SHMT: serine hydroxymethyltransferase
dGMP: deoxyguanosine monophosphate
TS: thymidylate synthase
dGDP: deoxyguanosine diphosphate
dGTP: deoxyguanosine triphosphate
AICAR: aminomidazole carboxamide ribonucleotide
dTMP: deoxythymidine monophosphate B12: vitamin B12 (cobalamin)
dTDP: deoxythymidine diphosphate
FAICAR: formyl-AICAR
dTTP: deoxythymidine triphosphate
FGAR: formyl-GAR
dUMP: deoxyuridine monophosphate GAR: glycinamide ribonucleotide
dUDP: deoxyuridine diphosphate NADP: nicotinamide adenine dinucleotide phosphate
dUTP: deoxyuridine triphosphate NADPH: reduced NADP
Folic acid
 an essential metabolite
 starting material for the mammalian folic acid cycle
 cannot be synthesised
o is obtained through diet
 fruit, vegetables, and fortified grains
 enzymatically reduced to dihydrofolate (DHF) and then tetrahydrofolate (THF)
catalysed by dihydrofolate reductase (DHFR)
o DHFR concentration in tumour cells is higher than healthy cells and reflects
importance in cell proliferation processes
o THF is processed to give one-carbon donor coenzyme precursors
o methyl and formyl donors that support de novo DNA synthesis
o each time these one‐carbon donor coenzyme precursors are used, the
carriers are converted back to DHF
o re-reduction of DHF to THF is key to maintaining the levels of the crucial
coenzyme precursors, which which must be re‐reduced to THF to maintain
levels of the one-carbon donors and it is this reduction, which is, which is
inhibited by the anti‐metabolite (more specifically, the anti‐folate) MTX.

Antifolates
 close in structure to folic acid
o methotrexate
 dihydrofolate reductase inhibitor
 competitive inhibitor to DHF
o raltitrexed
 thymidylate synthetase inhibitor
 competitive inhibitor to ‘methyl donor’
o pemetrexed
 less specific inhibitor of folate action and acts on thymidylate
synthetase, dihydrofolate reductase and glycinamide ribonucleotide
formyltransferase
Methotrexate
 tight binding competitive inhibitor of dihydrofolate reductase (DHFR)
 blocks the production of methyl and formyl donors to halt DNA replication and cell
proliferation
 similar in structure to folic acid but does not bind to DHFR in same way
 binds more tightly to DHFR and with greater affinity than either folic acid or
dihydrofolic acid (DFA) (at the same binding site)
 mainly due to increased basicity of the pterin component of methotrexate compared
to DFA
o bioisosteric change increase pKa for the cation from 2.6 (N-5) to 5.7 (N-1, N-
4)
o this increases percentage ionisation at physiological pH
o various resonance forms distribute positive charge through pterin ring
o ionised form has a much stronger interaction with the active site, through an
ionic bond to a glutamate of DHFR (and additional hydrogen bonding
interactions)
o as a result, methotrexate has a different orientation to folic acid/DFA in the
DHFR binding site
  inside the cell, methotrexate is polyglutamated
o enzymes folylpolyglutamate synthetase (FPGS) conjugates glutamic acids to
the γ‐carboxylic acid group and γ‐glutamyl hydrolase (γ‐GH) hydrolyses the
polyglutamates
o methotrexate(glu)n (n = 2–7) have equivalent DHFR inhibitory potency
o less readily effluxed from the cell
o enhances action

For severe methotrexate toxicity, folate replacement therapy with folinic acid is used
 does not need reduction by DHFR in order to act as a folate source
 generates the methyl and formyl folate cofactors by alternative mechanisms
 continued synthesis of pyrimidine and purine nucleotides is still possible in healthy
cells

Methotrexate resistance
 decreased uptake (uses reduced folate carrier)
 increased efflux
 altered levels of the drug target
5-Flurouracil
 acting as an inhibitor of thymidylate synthase (TS)
 mistakenly incorporated into DNA and RNA

 like uracil, 5FU rapidly equilibrates across cell membranes

 facilitated transport
 inside cells, 5FU is converted to 5‐fluorodeoxyuridine (5FdUMP)
o thymidine phosphorylase (TP), then
o thymidine kinase (TK) or orotate phosphoribosyltranserase (OP)
 also further converted to 5FdUTP
 hepatic metabolism of 5FU is a factor contributes to poor bioavailability
o dihydropyrimidine dehydrogenase (DPD) catalysed conversion of 5FU to the
inactive dihydrofluorouracil (DHFU)
o this then undergoes hydrolytic degradation
o can be overcome by the use of the 5FU oral prodrug capecitabine
 avoids first-pass DPD‐catalysed degradation
 is metabolised to 5FU after absorption from the gastrointestinal tract
 prodrug activation also involves thymidine phosphorylase which is
much more active in tumours than in normal tissue
 gives more tumour-selective generation of 5FU and hence
5FdUMP
 levels of active drug in the tumour can be up to 3.5-fold higher
than in surrounding tissue
o lower incidence of adverse effects compared to 5FU
therapy

 TS is a methyltransferase which catalyses the conversion of deoxyuridine

monophosphate (dUMP) to deoxythymidine monophosphate (dTMP).
 catalyses the transfer of a one‐carbon unit (a methyl group) from the ‘methyl donor’
cofactor N5,N10‐methylene‐FH4 (N5,N10‐CH2‐FH4) to dUMP
 the dUMP and ‘methyl donor’ bind in different but adjacent sites in TS
 dUMP to dTMP
o involves a nucleophilic attack by a cysteine residue (cysteine 195) at the TS
active site on the 6‐position of dUMP
 this enhances the nucleophilicity of the 5,6 C=C which reacts with the
electrophilic ‘methyl donor’
 forms a covalently bonded enzyme-substrate complex
o there is then deprotonation at the 5‐position by a basic residue (tyrosine 135)
 leads to cleavage of the enzyme–substrate complex
 dTMP is released from the enzyme

 5FdUMP is mistaken by TS for the natural substrate, dUMP

o initial nucleophilic attack by cysteine residue (cysteine 195) at the TS active
site on the 6‐position of 5FdUMP proceeds as normal
o release of the substrate and co‐factor from the ternary complex is inhibited
as there is no 5‐H to be abstracted by the tyrosine base
 fluorine is bioisosteric (similar size) to hydrogen
 C-F bond is strong
 electronegative element (has a partial negative charge)
 repels the basic residue which would normally abstract H‐5
 enzyme is irreversibly covalently attached to substrate
 binding of ‘methyl donor’ to TS is also enhanced
 a stable ternary complex is formed
o reduction in the cellular levels of dTMP, and so deoxythymidine triphosphate
(dTTP)
o elevation of dUMP levels (and so dUTP)
o dUTP and 5FdUTP can be mistakenly incorporated into DNA
o nucleotide excision repair enzymes become overwhelmed (NER)
o repair cycle fails leading to DNA strand breaks and cell death
o 5FdUTP can be mistakenly incorporated into RNA
o cellular metabolism and viability is affected as a result of disruptions to
RNA processing and function
DNA Polymerase/DNA Chain Elongation Inhibitors
There are a number of ribose-modified/base-modified DNA nucleoside/nucleotide
analogues
 all have complex and multifaceted mechanisms
 all include inhibition of DNA polymerase and/or DNA chain elongation

 actively transported into tumour cells

 converted to triphosphorylated nucleotides by specific kinases
o subsequently incorporated into the growing DNA chain
 this can arrest further elongation
 this can destabilise DNA structure

 may also inhibit the biosynthesis of essential deoxyribonucleotide
o e.g. gemcitabine is a ribonucleotide reductase inhibitor