In this second part of the lecture covering
the pharmacology of drugs used for treatment
of cancer, we’re gonna discuss Antimetabolites.
So in the last lecture on cancer drugs, we
covered agents that mostly affected cells
in the G1 phase of the cell cycle.
In this presentation we will shift our focus
to agents that affect the S phase of the cycle.
Now in order to gain better understanding
of how these agents work, first let’s take
a closer look at what exactly happens during
the S phase.
So as you may remember, during the S phase,
DNA is replicated.
DNA replication begins when an enzyme, topoisomerase
unwinds coiled DNA, and an enzyme DNA helicase,
breaks the bonds between complementary nucleotide
bases.
This exposes the bases, which are of two types;
purines and pyrimidines.
The purines are adenine and guanine, and the
pyrimidines are cytosine and thymine.
Now, once the DNA strand is open, with the
help of an enzyme called DNA polymerase, each
nucleotide base is then bonded to a new free-floating
partner.
Adenine pairs with thymine and guanine pairs
with cytosine to form a new strand of DNA,
which is essentially an exact copy of the
original version.
Now, the metabolic requirements for the nucleotides
and their bases can be met by both dietary
intake and synthesis from smaller precursors
known as de novo synthesis.
Cancer cells are actually highly dependent
on the de novo synthesis of nucleotides to
maintain sufficient pools to support DNA replication
and the production of RNA.
Now, although both purine and pyrimidine bases
are found in nucleotides, they are made in
different ways.
So, first let’s take a look at the synthesis
of purines, which begins with the formation
of nucleotide called inosine monophosphate
(IMP), from phosphoribosyl pyrophosphate (PRPP),
amino acids, and folate.
Now, IMP represents a branch point because
it can be converted into either adenosine
monophosphate (AMP) or guanosine monophosphate
(GMP).
Conversion of IMP to AMP requires a synthase
enzyme that converts IMP to adenylosuccinate
and then lyase enzyme that converts adenylosuccinate
into AMP.
AMP can be then reduced to deoxyAMP (dAMP),
and further converted to deoxyATP (dATP),
which can be incorporated into DNA.
Alternatively, conversion of IMP to GMP requires
dehydrogenase enzyme that catalyzes the conversion
of IMP to xanthosine monophosphate (XMP),
which serves as a substrate for the production
of GMP.
Likewise, GMP can be reduced to deoxyGMP (dGMP),
and then converted into deoxyGTP (dGTP), which
is incorporated into DNA.
Now let’s take a look at the synthesis of
pyrimidines.
So unlike purine synthesis, pyrimidine synthesis
begins with the formation of nitrogenous base
called orotate, which combines with phosphoribosyl
pyrophosphate (PRPP) to form uridine monophosphate
(UMP).
As a branch point in pyrimidine synthesis,
UMP can be phosphorylated to uridine triphosphate
(UTP), aminated to cytidine triphosphate (CTP)
and then reduced to deoxyCTP, which is incorporated
into DNA.
Alternatively, UMP can be reduced to deoxyUMP,
and then converted to deoxythymidine monophosphate
(dTMP) by an enzyme called thymidylate synthase.
In the final step, dTMP is converted to deoxythymidine
triphosphate (dTTP), which is incorporated
into DNA.
Now, it’s important to note here that thymidylate
synthase is a key enzyme that participates
in folate metabolism.
In order to convert dUMP to dTMP, thymidylate
synthase uses methylenetetrahydrofolate (MTHF)
as a methyl group donor, resulting in the
formation of dihydrofolate (DHF) that in turn
is reduced to tetrahydrofolate (THF) by dihydrofolate
reductase.
Finally, methylenetetrahydrofolate is regenerated
from tetrahydrofolate in a one-carbon transfer
reaction.
Now that we covered basic overview of purine
and pyrimidine biosynthesis, let’s move
on to discussing mechanism of action of antimetabolites.
So antimetabolites tend to exert greatest
cytotoxicity in the S-phase of the cell cycle
by simply interfering with the de novo synthesis
of purines and pyrimidines.
In order to understand how these drugs work,
first let’s go back to our overview of de
novo purine synthesis.
Perhaps one of the most well known antimetabolite
that interferes with this pathway is a drug
called Mercaptopurine (6-MP).
Mercaptopurine is a pro-drug that upon entry
into the cell it’s first converted to thioinosine
monophosphate (TIMP) by an enzyme called hypoxanthine-guanine
phosphoribosyltransferase (HGPRT).
HGPRT is an enzyme that plays a key role in
the recycling of the purine bases, hypoxanthine
and guanine.
Now, TIMP inhibits several chemical reactions
involving inosine monophosphate (IMP), including
the conversion of IMP to xanthosine monophosphate
(XMP), and the conversion of IMP to adenylosuccinate.
Furthermore, through series of reactions,
TIMP can be converted into thioguanosine triphosphate
(TGTP) which then can be incorporated into
RNA, as well as thio-deoxy-guanosine triphosphate
(TdGTP) which can be incorporated into DNA
thus leading to inhibition of both, DNA and
RNA synthesis, resulting in cell death.
Now, let’s move on to the next antimetabolite
drug that is Hydroxyurea.
The main target of Hydroxyurea is an enzyme
that is responsible for conversion of ribonucleotides
into deoxyribonucleotides, called ribonucleotide
reductase (RNR).
Hydroxyurea inhibits this enzyme, which results
in nucleotide deprivation, stalling of replication
fork progression and ultimately S phase cell
cycle arrest in proliferating cells.
Now, ribonucleotide reductase (RNR) is also
a major target of other purine antimetabolites
such as Cladribine and Fludarabine.
The first one, Cladribine, is activated mostly
in lymphocytes, when it’s converted to its
toxic metabolite 2-chlordeoxyadenosine triphosphate
(2-CdATP).
In proliferating cells, 2-CdATP not only interferes
with ribonucleotide reductase (RNR) but also
acts as a DNA polymerase inhibitor by competing
with the binding of dATP to that enzyme.
In addition to that, unlike other antimetabolite
drugs, Cladribine has cytotoxic effects on
resting lymphocytes.
In resting cells, Cladribine causes a single-strand
DNA breaks, inducing the DNA repair enzyme,
which in turn exhausts the intracellular pools
of nicotinamide adenine dinucleotide (NAD)
and adenosine triphosphate (ATP), thus causing
apoptotic cell death.
Now, moving on to our next antimetabolite
drug, Fludarabine.
So Fludarabine also requires conversion to
the active metabolite, in this case, arabinosyl-2-fluoroadenine
triphosphate (F-ara-ATP).
Once formed, F-ara-ATP inhibits ribonucleotide
reductase (RNR), and competes with dATP for
incorporation into the elongating DNA strand
by DNA polymerase thus terminating DNA synthesis
at the incorporation sites.
Lastly, F-ara-ATP, inhibits two additional
enzymes that are crucial for DNA synthesis,
that is; DNA primase, an enzyme that synthesizes
short RNA sequences called primers, which
serve as a starting point for DNA synthesis;
and DNA ligase 1, an enzyme required for joining
of short DNA fragments to create the lagging
strand during DNA replication.
Now, let’s switch gears and let’s move
on to discussing antimetabolites that affect
de novo synthesis of pyrimidines.
Here just like in the purine synthesis pathway,
Hydroxyurea inhibits ribonucleotide reductase
(RNR), which converts ribonucleotides into
deoxyribonucleotides.
Another antimetabolite that interferes with
pyrimidines synthesis is a drug called Gemcitabine.
So, upon entry into the cell, Gemcitabine
gets rapidly phosphorylated to form active
metabolites, gemcitabine diphosphate (dFdCDP)
followed by gemcitabine triphosphate (dFdCTP).
The first active metabolite, gemcitabine diphosphate
(dFdCDP) inhibits ribonucleotide reductase
(RNR) that converts cytidine diphosphate (CDP)
to deoxycytidine monophosphate (dCMP) in the
so-called salvage pathway through which nucleosides
are recycled.
As a result deoxycytidine triphosphate (dCTP)
pools are depleted, which in turn makes it
easier for the second active metabolite, dFdCTP,
to come in and insert itself into DNA.
Interestingly, insertion of dFdCTP into DNA
allows further insertion of an additional
base pair before DNA polymerase is inhibited
thus making DNA repair more difficult.
This action is called “masked termination”.
Now, another antimetabolite analogue of cytidine
is a drug called Cytarabine.
Similarly to Gemcitabine, upon entry into
the cell, Cytarabine is sequentially phosphorylated
by kinase enzymes to its active metabolite
cytarabine triphosphate (ara-CTP).
Ara-CTP is a potent inhibitor of DNA polymerases
and is also incorporated into replicating
strands, leading to termination of DNA synthesis
directly at the site of incorporation.
Now, finally let’s move on to discussing
antimetabolites that interfere with the last
critical step of pyrimidine synthesis, that
is conversion of dUMP to dTMP.
The first drug that we’re gonna discuss
is an agent called Fluorouracil (5-FU).
So, just like the other antimetabolites, Fluorouracil
requires few enzymatic conversions to form
its active metabolites; 5-fluoro-deoxyuridine-monophosphate
(5-FdUMP) and 5-fluorouridine-triphosphate
(5-FUTP).
The first one, 5-FdUMP acts as an inhibitor
of thymidylate synthase, the enzyme that methylates
dUMP to form dTMP.
The second metabolite, 5-FUTP mimics uridine
triphosphate (UTP), and is recognized as the
substrate and incorporated into RNA by RNA
polymerase, leading to inhibition of RNA synthesis
and function.
Now, historically, it was only possible to
give Fluorouracil (5-FU) intravenously as
the oral bioavailability was poor and unpredictable.
This has been overcome by development of the
well tolerated, more bioavailable, oral alternative
called Capecitabine.
After being absorbed through the intestine,
Capecitabine, which itself is nontoxic, undergoes
a series of enzymatic reactions to form active
drug that is Fluorouracil (5-FU).
The final step in this reaction is catalyzed
by thymidine phosphorylase, an enzyme that
happens to be concentrated primarily in tumor
tissues, which gives Capecitabine greater
selective toxicity than Fluorouracil (5-FU).
Now, let’s move onto our last antimetabolite
drug that is Methotrexate.
So Methotrexate enters cells through the reduced
folate carrier (RFC-1) and is intracellularly
converted to the active metabolite methotrexate
polyglutamate [MTX(Glu)n].
Polyglutamated Methotrexate, then inhibits
two key enzymes in the folic acid pathway;
dihydrofolate reductase (DHFR), which reduces
dihydrofolate (DHF) to tetrahydrofolate (THF),
and thymidylate synthetase, which converts
dUMP to dTMP.
As a result tetrahydrofolate and dTMP pools
become depleted, ultimately leading to cessation
of DNA synthesis.
Now when it comes to side effects associated
with antimetabolites, the ones that occur
commonly include; nausea, vomiting, mouth
sores, hair loss, and myelosuppression that
can lead to fatigue, anemia, bleeding, or
increased risk of infections.
And with that I wanted to thank you for watching,
I hope you enjoyed this video and as always
stay tuned for more.
