Regulation of DNA synthesis

Molecular Pharmacology course
lecture No.2
regulation of DNA synthesis
(growth factors and growth factors’ inhibitors , licensing and geminin )
Dr. Omer Yahia Elhussein
B.sc. Pharmacy.
M.sc. Molecular Medicine

Control of nuclear
DNA replication

1- Growth factors and inhibitors
• Within eukaryotes , DNA
replication is controlled within
the cell cycle.
• Most bacteria do not go
through a well-defined cell
cycle and instead continuously
copy their DNA; during rapid
growth this can result in
multiple rounds of replication
occurring concurrently.
• The G1/S checkpoint regulates
whether eukaryotic cells enter
the process of DNA replication
and subsequent division.
• Cells which do not proceed
through this checkpoint are
quiescent in the "G0" stage
and do not replicate their DNA.Regulation of Cell Division in Normal Cells
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

2- Cycline dependent kinase complex
• DNA replication is controlled within
the context of the cell cycle.
• MPF (Maturation promoting factor)
drives the cell into mitosis.
• Cyclin B1–Cdk1(The Mammalian MPF)
activity during G2 prevents a cell from
rereplicating DNA that has already
been replicated earlier in the cell
cycle.
Cell and Molecular Biology concepts and experiments 6 edition

3- Licensing: Positive control of Replication
• An ORC protein complex has been
described as a “molecular landing
pad” because of its role in binding the
proteins required in subsequent steps.
• The Mcm ( licensing factor )proteins
are loaded onto the replication origin
at a late stage of mitosis, or soon
thereafter.
• Studies indicate that the Mcm2–
Mcm7 proteins are capable of
associating into a ring-shaped
complex that possesses helicase
activity.

3- Geminin : Negative control of
replication
• Geminin was identified as an inhibitor of DNA
replication and substrate of the anaphase
promoting complex (APC)
• Geminin is absent during G1 phase and
accumulates through S, G2 phase and M
phases of the cell cycle.
• At the start of the S-phase until late mitosis,
geminin inhibits the replication factor Cdt1,
preventing the assembly of the pre- replicative
complex.
• Geminin therefore ensures that one and only
one round of replication occurs during each cell
cycle.
sciencedirect.com

Molecular Pharmacology
Check points defects as
anticancer target:
1. Spindle defects: (by using
anti-microtubule drugs)
2. growth conditions: (by using
anti proliferative factors e.g.
DNA damage, ionizing
radiation and UV)
3. Replication faults: (by using
anti-metabolites) .
4. Chromosome catenation: (by
using topoisomerase
inhibitors )

Antiploriflative Factors
• Growth factors regulate behavior of
the cells including expression of
genes required for growth and
development.
• Mutation that constitutively activate
proteins along this path way
contributes to the development of
many human tumors by deriving
cellular proliferation.
• proteins communicate by adding
phosphate groups to a neighboring
protein, which act as an (on) or (off)

Types of oncogenesis in growth
factor receptors

A. Increased growth factor production

B. Increased growth factor receptors

D. Mutant transcription factors

Chronic myeloid leukemia
• It is a cancer of the white blood cells.
• It is a form of leukemia characterized by
the increased and unregulated growth
of predominantly myeloid cells in the
bone marrow and the accumulation of
these cells in the blood.
• CML is now largely treated with
targeted drugs called tyrosine kinase
inhibitors (TKIs) which have led to
dramatically improved long term
survival rates since the introduction of
the first such agent in 2001.

Gleevec
• Imatinib marketed by Novartis as Gleevec.
• It is a tyrosine-kinase inhibitor used in the
treatment of multiple cancers, most
notably Philadelphia chromosome-positive
(Ph+) chronic myelogenous leukemia.
• Like all tyrosine-kinase inhibitors, imatinib
works by preventing a tyrosine kinase
enzyme, in this case BCR-Abl ,
from phosphorylating subsequent proteins
and initiating the signaling cascade
necessary for cancer development, thus
preventing the growth of cancer cells and
leading to their death by apoptosis.
Gleevec

Continue..
• Because the BCR-Abl tyrosine kinase enzyme exists only in cancer
cells and not in healthy cells, imatinib works as a form of targeted
therapy—only cancer cells are killed through the drug's action.
• In this regard, imatinib was one of the first cancer therapies to show
the potential for such targeted action, and is often cited as a
paradigm for research in cancer therapeutics.
• Its use is advised against in patients on strong CYP3A4 inhibitors such
as clarithromycin, chloramphenicol and ketoconazole due to its
reliance on CYP3A4 for metabolism.

Epidermal Growth Factors Receptors

Epidermal Growth Factors Receptors
• The identification of EGFR as
an oncogene has led to the
development of anticancer
therapeutics directed against
EGFR.
• Including
• Gefitinib , Erlotinib for lung
cancer.
• Cetuximab for colon cancer.

Cetuximab
• It is an epidermal growth factor
receptor (EGFR) inhibitor used for the
treatment of metastatic colorectal
cancer and head and neck cancer.
Cetuximab is a chimeric
(mouse/human) monoclonal
antibody given by intravenous
infusion
• The monoclonal antibodies block the
extracellular ligand binding domain.
With the binding site blocked, signal
molecules can no longer attach there
and activate the tyrosine kinase.

Gefitinib
• Gefitinib and Erlotinib directly
target the EGFR.
• Patients have been divided into
EGFR-positive and EGFR-
negative , based upon whether a
tissue test shows a mutation.
• EGFR-positive patients have
shown a 60% response rate,
which exceeds the response rate
for conventional chemotherapy.

These EGFRI have an acne-like rash as a
common side effect .
What is your evidence based suggestion
for the cause of this case ?

Natural EGFR inhibitors
Natural inhibitors include:
1. potato carboxypeptidase inhibitor
(PCI). It is included in EGF family.
• Structural similarities with these
factors can explain the antagonistic
effect of PCI.
2. Grandinin is an ellagitannin found
in oaks.
• It suppresses the phosphorylation of
the epidermal growth factor receptor
in human colon carcinoma cells.
Grandinin
potato

Vascular endothelial growth factor
(VEGF)
• It is a signal protein produced by cells that stimulates
vasculogenesis and angiogenesis.
• Serum concentration of VEGF is high in bronchial asthma and
diabetes mellitus.

Types of VEGF and their functions
1. VEGF-A
• Angiogenesis
– ↑ Migration of endothelial cells
– ↑ mitosis of endothelial cells
– creation of blood vessel lumen
– creates fenestrations
• Chemotactic for macrophages and granulocytes
• Vasodilation (indirectly by NO release)

Continue …
2. VEGF-B : Embryonic angiogenesis (myocardial tissue,
specifically)
3. VEGF-C : Lymphangiogenesis
4. VEGF-D : Needed for the development of lymphatic
vasculature surrounding lung bronchioles

Alkylating
antineoplastic agent

Do not mix between Thymine
and Thiamine !
Nitrogenous bases :
• Pyrimidines :
– Cytosine (DNA, RNA)
– Uracil (RNA)
– Thymine (DNA)
• Purines
– Adenine (DNA, RNA)
– Guanine (DNA, RNA)

Do you know synthetic
analogs for these pyrimidines ?

Preparing for nucleic acid synthesis
• DNA and RNA are nucleic
acids, function in storage
and transmission of genetic
information.
• They are constructed of
monomers called
nucleotides.
• These nucleotides fuel the
DNA replication, RNA
transcription, mutation
correction , damage and
repair mechanisms.

Alkylating antineoplastic agent
• They are originally derived
from mustard gas used in the
war.
• They have the ability to
alkylate many molecules,
including proteins , RNA and
DNA.
• Alkylating agents work at any
point in the cell cycle and
thus are known as cell cycle-
independent drugs.
US Army World War II Gas Identification Poster,
ca. 1941–1945

Continue …
• The alkylating molecules may
either bind twice to one
strand of DNA (intrastrand
crosslink), or , may bind once
to both strands (interstrand
crosslink).
• If the cell tries to replicate
crosslinked DNA during cell
division, or tries to repair it,
the DNA strands can break.
• This leads to a form of
programmed cell death called
apoptosis.
Insights into the cross-linking mechanism of azinomycin B with DNA bases from
hybrid QM/MM Computations

Continue …
• They impair cell function by forming covalent
bonds with the amino, carboxyl, sulfhydryl, and
phosphate groups in biologically important
molecules.
• The most common toxicity of Alkilating agents;
Sterility
And
Secondary malignancy

Why Purines and Pyrimidines are important ?
• The sequence of amino acids in a polypeptide was specified by
the sequence of nucleotides in the DNA of a gene.
• With the discovery of messenger RNA as an intermediate in the
flow of information from DNA to protein, attention turned to
the manner in which a sequence written in a ribonucleotide
“alphabet” might code for a sequence in an “alphabet”
consisting of amino acids.

Purines and Pyrimidines synthesis
• Nearly all organisms synthesize
purines and pyrimidines "de
novo“
• Many organisms also "salvage"
purines and pyrimidines from
diet and degradative pathways
• Ribose generates energy, but
purine and pyrimidine rings do
not
• Nucleotide synthesis pathways
are good targets for anti-
cancer/antibacterial strategies
http://www.d.umn.edu/~jfitzake/Lectures/DMED/Antineoplastics/DNASynthesisInhibitors/NucleotideBiochemistry.html

Conversion of Ribose-5-phosphate
to PRPP
•The pentose sugar is always a ribose, which may be reduced to
deoxyribose after nucleotide synthesis is complete.
•5-Phosphoribosyl-1-pyrophosphate (PRPP) is also involved in
synthesis of pyrimidine nucleotides, NAD+, and histidine biosynthesis.
• Inhibited by AMP, GMP, and IMP
• Requires 4 ATP molecules

Purine de novo
synthesis
• First step of purine synthesis is committed step
and rate limiting step.
•Intracellular concentrations of glutamine and
PRPP control the reaction rate.
• Dependence on Glutamine in
committed step (Glutamine PRPP
amidotransferase ) lead to use
Azaserine as anti tumor .
• Dependence on THF in two steps
means that methotrexate and
sulfonamides block purine
synthesis
• Humans cannot synthesize folic
acid , and must rely on external
sources of this vitamin. Therefore,
sulfa drugs do not interfere with
human purine synthesis.
•The end product is
Inosine- 5’- mono phosphate

Azaserine block purine
synthesis
• De novo purine synthesis begins
with the conversion of ribose-5-
phosphate to 5-phosphoribosyl-
1-pyrophosphate (PRPP), a reaction
catalyzed by PRPP synthetase
(PRPS).
• The first committed step in
purine synthesis is the formation of
5-phosphoribosylamine via the
enzyme Glutamine PRPP
amidotransferase
• Azaserine – Glutamine analog-
inhibits Glutamine PRPP
amidotransferase and act as Anti-
tumor.

Sulfonamides block Microorganism
purine synthesis
• Sulfonamides are structural
analogs of para-aminobenzoic
acid that competitively inhibit
bacterial synthesis of folic acid
• Because purine synthesis
requires tetrahydrofolate as
a coenzyme, the sulfa drugs
slow down this pathway in
bacteria.
• In methotrexate this hydrogen
group is (methyl group)

Folic acid inhibitor
• Sulfonamides competitively
inhibit ( Dihydropteroate
synthetase ) in
the synthesis of folic and,
thereby, decrease
the synthesis of nucleotides
needed for the replication
of DNA.
• Dihydrofolate reductase is
competitively inhibited by
methotrexate, a folic acid
analogue used to treat
psoriasis, rheumatoid
arthritis and neoplastic
diseases.

Drugs target folate synthesis
1. FOLATE BIOCHEMISTRY
(e.g., METHOTREXATE)
Tetrahydrofolate is synthesized by two
mechanisms:
• Conversion of folate to
dihydrofolate and dihydrofolate to
tetrahydrofolate is catalyzed by
dihydrofolate reductase (DHFR).
• Methyltetrahydrofolate from liver
stores is converted to
tetrahydrofolate, a reaction that
requires VITAMIN B12.
Two steps in the conversion of
5-phosphoribosylamine to IMP (purine
synthesis) use tetrahydrofolate as a
carbon donor.
Tetrahydrofolate is also involved in the
generation of dTMP from dUMP
(pyrimidine synthesis) – this reaction is
catalyzed by thymidylate synthase

Mycophenolate (cellcept)
• Mycophenolate is derived from the fungus Penicillum
stoloniferum.
• Mycophenolate mofetil is metabolized in the liver to its active
moiety mycophenolic acid. It inhibits inosine monophosphate
dehydrogenase, the enzyme that controls the rate of synthesis
of guanine monophosphate in the denovo pathway of purine
synthesis used in the proliferation of B and T lymphocytes;
downstream it interferes with leukocyte adhesion to
endothelial cells through inhibition of E- selectin , P- selectin ,
and intercellular adhesion molecule -1.

Mycophenolate (cellcept)
• High levels shut down de novo
purine synthesis is caused by
AMP, GMP.
• If both AMP and GMP are
present in adequate amounts,
the de novo pathway of purine
synthesis is turned off at the
amidotransferase step.
Mycophenolic acid

Purine synthesis
clinical importance
Gout:
During purines degradation ; Nucleotidases
and nucleosidases release ribose and
phosphates and leave free bases .
Xanthine oxidase and guanine deaminase
route everything to xanthine.
Xanthine oxidase converts xanthine to uric
acid .
Allopurinol , which inhibits Xanthine oxidase, is
a treatment of gout .
•Pentostatin inhibits adenosine deaminase-
a critical pathway in purine metabolism.

Why do cancer patients take
high doses of allopurinol?

crystal
deposition
hyperuricemia
protein binding
receptor
binding
cytokine
release
influx of PMN’s
crystals
engulfed
inflammation
Acute attacks are treated with colchicine
and indomethecin for 3 weeks.
Long-term treatment with allopurinol
reduces the amount of uric acid in
circulation.
PMN is critical component
of crystal-induced
inflammation

Drugs induced gout
kinetically !
Certain drugs can cause secondary gout due to actions on the
kidney that affect uric acid reabsorption , secretion, and
excretion.
• Diuretics, due to their actions on the kidney, can increase uric
acid reabsorption and lead to hyperuricemia and resultant
gout.
• High doses of aspirin ( >3 g/day) are uricosuric .
• Pyrazinamide, ethambutol, and niacin are other agents
associated with gout because they suppress uric acid
secretion.

Hydroxyurea as
anti cancer
• The drug hydroxyurea destroys the free radical required for enzymic activity
of ribonucleotide reductase, and thus inhibits the generation of substrates
for DNA synthesis.
• For reductase to continue to produce deoxyribonucleotides, the disulfide
bond created during the production of the 2'-deoxy carbon must be
reduced.
• Hydroxyurea has been used in the treatment of cancers such as chronic
myelogenous leukemia.

Drugs target conversion of ribonucleotides to
deoxyribonucleotides
4. CONVERSION OF RIBONUCLEOTIDES
TO DEOXYRIBONUCLEOTIDES
(e.g., HYDROXYUREA)
This reaction is catalyzed by
ribonucleotide reductase.
NOTE : Hydroxyurea, an antitumor drug,
is therapeutically useful in SICKLE CELL
ANEMIA because it increases circulating
levels of Hb F, which decreases RBC
sickling. This leads to decreased
frequency of painful crises and reduces
mortality.

To BE CoNTINUED…….
15 MIN. BREAK

Purine synthesis salvage
path way
• Salvage pathways collect hypoxanthine and guanine
and recombine them with PRPP to form nucleotides
in the HGPRT reaction.
(hypoxanthine guanine phosphoribosyl transferase)
• Two enzymes are involved:
adenine phosphoribosyl transferase (APRT) and
hypoxanthine-guanine phosphoribosyl transferase
(HGPRT)
• Both enzymes use PRPP as the source of the ribose
5-phosphate group.
• The release of pyrophosphate and its subsequent
hydrolysis by pyrophosphatase makes these
reactions irreversible
• Absence of HGPRT is cause of Lesch-Nyhan
syndrome.

Azathioprine
• Azathioprine, a mercaptopurine
analog of adenine and
hypoxanthine
• It is a prodrug that is converted
first to 6 mercaptupurine (6-MP)
which in turn can be converted to
6- mercaptopurine nucleotides
leading to an inhibition of denovo
purine synthesis.
• Three enzymes play major roles in
the metabolism of azathioprine
and mercaptopurine
1- xanthine oxidase (XO)
2- thiopurine methyltransferase
(TPMT)
3- purine pathway salvage enzyme
hypoxanthine–guanine
phosphoribosyltransferase
(HGPRT).

Azathioprine
• Azathioprine (AZA) is converted to
6-mercaptopurine (6-MP) in the liver via a
glutathione-dependent process
accelerated by glutathione-S-transferase.
• 6-MP undergoes further metabolism by
1. Xanthine oxidase (XO).
2. Thiopurine methyltransferase (TPMT).
3. Hypoxanthine guanine
phosphoribosyltransferse (HPRT) within
the red blood cells.
• In red blood cells it is subsequently
converted by TPMT to 6-methyl-
mercaptopurine ribonucleotides or by
Inosine-5′- monophosphate
dehydrogenase (IMPDH) to 6-thioguanine
nucleotides.
• Deficiency of TPMT activity leads to
accumulation of 6-thioguanine
nucleotides, which may cause bone
marrow toxicity.
http://en.wikipedia.org/wiki/File:AZA_metabolism.svg
IMPDH
RBC

Drugs target purine synthesis
…Summary …
3. PURINE SYNTHESIS (e.g., 6-MP, 6-TG)
De novo purine synthesis begins with the conversion
of ribose-5-phosphate to 5-phosphoribosyl-
1-pyrophosphate (PRPP), a reaction catalyzed by
PRPP synthetase (PRPS). The first committed step in
purine synthesis is the formation of
5-phosphoribosylamine via the enzyme glutamyl
amidotransferase (GPAT).
IMP and GMP can also be created by via the “salvage
pathway” whereby PRPP is combined with
hypoxanthine or guanine bases (including 6-MP and
6-TG) by the actions of hypoxanthine-guanine
phosphoribosyl transferase (HGPRT).
6-MP and 6-TG (and their naturally occurring
analogues) inhibit guanylyl kinase, preventing the
conversion of GMP to GDP and causing
"pseudofeedback inhibition" of PRPS, GPAT, HGPRT
and the 2 steps that lead to the formation of XMP
and AMP from IMP.
One route for the degradation of purine nucleotides
(and 6-MP and 6-TG) occurs via conversion of IMP to
uric acid. Two steps in that process, conversion of
hypoxanthine to xanthine and xanthine to uric acid,
are catalyzed by the enzyme xanthine oxidase. This
enzyme is inhibited by ALLOPURINOL.

Pyrimidine synthesis
• Thymidylate Synthetase
The enzyme use N5,N10-methylene
tetrahydrofolate (CH3-THF)
as “a methyl donor”.
• This form of folate derives its carbon
from serine:
• Serine + THF ----> Glycine + CH3-THF
• 5-Fluorouracil is a pyrimidine analog
which is converted metabolically to its
toxic form, fluorodeoxyuridylate
(F-d UMP).
• As cells metabolically activate the
drug it acts as a "suicide" inhibitor of
Thymidylate Synthetase.

Drugs target pyrimidine synthesis
..Summary ..
2. PYRIMIDINE SYNTHESIS
(e.g., 5-FLUOROURACIL)
The rate limiting step in DNA
synthesis is the conversion of UMP to
TMP, which is catalyzed by
thymidylate synthase.
Conversion of UMP to UDP is
catalyzed by pyrimidine
monophosphate kinase; this reaction
is important in the development of
resistance to 5-FU.
One step in the degradation of
thymidine nucleotides is catalyzed by
dihydropyrimidine dehydrogenase;
an inherited deficiency of this enzyme
leads to greatly increased sensitivity
to 5-FU.

Regulation of DNA synthesis

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