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.