mercaptopurine
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Genetic Basis of Variable Drug Response to MercaptopurineTRANSCRIPT
Genetic Basis of variable drug response to Mercaptopurine With a focus on a Single Nucleotide Polymorphism on the TPMT gene
Semester- 5
Kavya Anantha Narayanan Manipal University, Dubai
Department of Biotechnology
Mercaptopurine
Mercaptopurine, chemically known as l,7-dihydro-6H-purine-6-
thione monohydrate, is an analogue of two purine bases,
adenine and hypoxanthine. Mercaptopurine, and the closely
related immunosuppressant, azathioprine, are purine
antagonists and members of a group of chemotherapy drugs
called anti-metabolites.
It interferes with biochemical processes involving endogenous
purines, which are widely found in structures of DNA, RNA
and certain co-enzymes. Both Mercaptopurine and
Azathioprine have cytotoxic and immunosuppressive properties
that are useful in inhibiting protein, DNA and RNA synthesis.
Without these building blocks, a cell would stop growing, and
die.[Springer]
Also known by its trade name, PURINETHOL, mercaptopurine
is available in tablet form for oral administration. Each tablet
contains 50 mg mercaptopurine with inactive ingredients such
as corn and potato starch, lactose, magnesium stearate, and
stearic acid.
Figure 1- Chemical Structure of Mercaptopurine
Mercaptopurine was initially evaluated for the treatment of
leukaemia in the early 1950s [Burchenal et.al.1953]. Its
potential as an immunosuppressant was demonstrated by
Schwartz at al. (1958), who showed that in laboratory
animals injected with antigen, antibody response was
prevented by injection of Mercaptopurine.[1]
Today, mercaptopurine is indicated and used in maintenance
therapy of acute lymphocytic, lymphoblastic leukemia as part
of a combination regimen. Unlabelled uses include Non-
Hodgkin lymphoma, Crohn disease, ulcerative colitis.
Objective
Despite the widespread use of these compounds for over three
decades, precise details of their molecular basis of action in the
human body is still insufficient. Also, mutated genes/SNPs
associated with proteins involved in the metabolism or
transport of purine antagonists such as mercaptopurine would
cause an alteration in activity of the drug. The objective of this
study is to examine why some specific gene mutations cause
people to respond differently to Mercaptopurine.
Materials and Methods:
The modeling of proteins for comparative structural analysis
was made possible by the use of a molecular visualization
software called UCSF Chimera. The input files for this
visualization tool were obtained from the RCBS Protein Data
Bank.
Mercaptopurine itself has no anti-cancer activity- it is a
prodrug that requires extensive intestinal and hepatic
metabolism to be activated, after oral administration.
Mercaptopurine also undergoes extensive first-pass metabolism,
and the contributions of the three metabolic pathways to
individual variations in its metabolism have been the subject of
many pharmacogenetic studies. These include the Thiopurine
methyltransferase, xanthine oxidase and Hypoxanthine-guanine
phosphoribosyltransferase pathways.[1]
Figure 2-Pathway depicting metabolism and interaction of Mercaptopurine with related pathways. [Zaza
Gianluigi, Cheok Meyling, Krynetskaia Natalia, Thorn Caroline, Stocco Gabriele, Hebert Joan M, McLeod
Howard, Weinshilboum Richard M, Relling Mary V, Evans William E, Klein Teri E, Altman Russ B.
"Thiopurine pathway" Pharmacogenetics and genomics (2010).]
Thiopurine Pathway
The Thiopurine Pathway involves direct interaction of the
products of, and indirect influence by as many as 32 genes,
namely ABCC4, ABCC5, ADA, ADK, AHCY, AOX1, CBS, DHFR,
GART, GMPS, GSTA1, GSTA2, GSTM1, HPRT1, IMPDH1, ITPA,
MTHFD1, MTHFR, MTHFS, MTR, MTRR, NT5E, PPAT, PRPS1,
RAC1, SHMT1, SLC28A2, SLC28A3, SLC29A1, SLC29A2, TPMT,
TYMS, and four among these genes, MTHFR, TPMT, and two
related locally acting solute carrier proteins, SLC19A1 and
SLCO1B1, showed clinical annotations of varied responses with
Mercaptopurine.
More information on the proteins and their functions are given
in the table below.
Table 1- Genes and their functional proteins associated with altered activity of Mercaptopurine[3]
Gene coding for Protein that has
variable response to Mercaptopurine
The Protein that this gene codes for
How this protein interacts with Mercaptopurine
TPMT Thiopurine
S-methyltransferase enzyme
Methylation (metabolism) of mercaptopurine
SLC19A1 Solute carrier family 19
(folate transporter), member 1
Transporter of folate and is involved in the regulation of intracellular
concentrations of folate
MTHFR Methylenetetrahydrofolate
reductase enzyme
Simultaneously catalyzes formation of methionine from homocysteine, and
conversion of methyl-THF to THF*
SLCO1B1 Solute carrier organic anion transporter family member
1B1
Mediates the Na-independent uptake of many endogenous compounds and
drugs**
*THF is the form that folate is used as in biosynthetic pathways, including that of pyramidine nucleotide synthesis
**endogenous compounds: bilirubin, 17-beta-glucuronosyl estradiol and leukotriene C4; drugs: statins, bromosulfophthalein and rifampin
How do genes affect drug-response (to Mercaptopurine)?
Majority of the Clinical Annotiations for Mercaptopurine
corresponded with the well-studied TPMT gene, whose gene
product codes for a protein called Thiopurine S-
methyltransferase.
Thiopurines are pro-drugs, and they require extensive
metabolism, to exert their cytotoxic effects. This protein,
thiopurine methyltransferase, methylates these thiopurine
compounds such as 6-mercaptopurine and 6-thioguanine.
{In this reaction, S-adenosyl-L-methionine (produced from
reaction between methionine and ATP), is converted to S-
adenosyl-L-homocysteine, which in turn is hydrolyzed to
homocysteine i.e-the enzyme employs S-adenosyl-L-methionine
as the S-methyl donor, and S-adenosyl-L-homocysteine as the
by-product.}
In addition, 6MP is unique in that it can also be converted via
TPMT into a methyl-thioinosine 5-prime monophosphate
(MeTIMP), a metabolite that inhibits de novo purine synthesis
and likely contributes to the cytotoxic effect of 6MP.[4]
Genetic polymorphisms affecting enzymatic activity of TPMT
are often accompanied by a variation in sensitivity and
toxicity to such drugs within individuals. Defects in the TPMT
gene leads to a decrease in methylation (hence reduced
inactivation) of 6-MP. This tends to be the cause of enhanced
bone marrow toxicity, myelosuppression, leukopenia and
infection in patients with genetic polymorphisms on TPMT.
Identifying the Mutations:
Mutations in the genes and their functional proteins studied
above (in Table 1) may result in a altered activity of
Mercaptopurine due to its influence in the Thiopurine Pathway.
Table 2-Mutation in the alleles corresponding to genes under study, and their effects.[3]
Generic Name of the Drug
Gene coding for
Protein that has Variable
Response to the Drug
rsID of an identified
allele
Allele change
in Exon/
Intron/ Other?
Codon Sequence
Change (DNA)
Codon Sequence
Change (mRNA)
Amino Acid Sequence
Change +Translation
Effect
Mer
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TPMT rs1800462 Exon GCA ⇒ CCA GCA ⇒ CCA A [Ala] ⇒ P [Pro]
(Ala80Pro)
Change from a slightly less polar amino
acid residue to a contrasting, non-polar
amino acid
SLC19A1 rs1051266 Exon CAC ⇒ CGC CAC ⇒ CGC H [His] ⇒
R [Arg] (His27Arg)
Change from a basic, polar amino acid residue to a
different basic, polar amino acid residue
MTHFR rs1801131 Exon GAA ⇒ GCA GAA ⇒ GCA E [Glu] ⇒
A [Ala] (Glu429Ala)
Change from an acidic, polar
amino acid to a neutral, non-polar amino acid residue
SLCO1B1 rs11045879 Intron N/A N/A N/A N/A
The (G238C) SNP in the TPMT gene:
Altered TPMT activity predominantly results from single
nucleotide polymorphisms (SNPs). The mutant allele under
study, called TPMT*2 is defined by a single nucleotide
transversion (G238C) in the reading frame, as shown in Table 2
[GCA->CCA], followed by an amino acid substitution at
codon 80 [Ala80Pro]. [5]
Table 3- Hypothetical Symptoms of TPMT*2 mutant allele on Mercaptopurine
Allelic Configuration and Genotype
Hypothesized Symptoms corresponding with this Genotype**
Homozygous wild-type
CC
(example-
TPMT*1/*1)
May not be at increased risk for life-threatening myelosuppression.
Decreased inactivation of Mercaptopurine. Increased risk for toxicity with Mercaptopurine drugs as
compared to patients with TT genotype
Heterozygous mutant
CT
(example-
TPMT*1/*2)
Increased risk for life-threatening myelosuppression as compared to patients with the CC genotype. Decreased inactivation of Mercaptopurine.
Increased risk for toxicity with Mercaptopurine as compared to patients with TT genotype
Homozygous mutant
TT
(example-
TPMT*2/*2)
High risk for life-threatening myelosuppression as compared to patients with the CC or CT genotype.
Increased inactivation of Mercaptopurine. Decreased (but still prominent) risk for toxicity with Mercaptopurine compared to patients with CT or CC
genotype.
**Other genetic & clinical factors may also influence a patient's risk for toxicity. These symptoms were
hypothesized based on clinical annotations from patients of diverse race, and genetic characteristics.
In addition to the difference in activity of TPMT, some individuals with
a heterozygous genotype(CT) exhibit high activity whereas some
homozygous wild type subjects(CC) exhibit an intermediate
phenotype. Such discrepancies are due to the fact that the SNPs
discussed so far are not the only factors regulating catalytic activity.
How the [Ala80Pro] amino acid change matters?
Alanine is, except for glycine, the simplest amino acid
chemically, with a single methyl group for a side chain. Proline,
on the other hand, is a unique ‘imino acid’, that unlike primary
amino acids, has its side chain curving back from the alpha
carbon to bond to the amine nitrogen, forming a Cyclic
Structure as shown in the comparison table below. When
incorporated into a protein, proline therefore lacks an amide
proton-and the backbone near a proline thus tends to be
inflexible, prevent formation of certain secondary structures
(like alpha helices).
Table 4-A Comparison between Properties of an Alanine and Proline Residue
Structures:
Property Alanine Proline
Side chain flexibility: Limited Restricted
Interaction modes: Van der Waals Van der Waals
Potential side chain H-
bonds 0 0
Residue molecular weight 71 97
Isoelectric point 6 6.3
Hydrophobicity 0.806 0.678
Standard codon(s)
GCN
CCN
Biochemical Characteristics: Nonpolar Nonpolar
Methylene Methylene
Aliphatic Imino
Nonessential Nonessential
Variations:
As shown in the preceding section, these prominent changes in
biochemical properties of the wild type (Ala) and mutant (Pro)
amino acid residue, depending on the resultant conformational
changes, may cause an alteration in the catalytic activity of the
mutant protein/enzyme molecule. For the purpose of
understanding the conformational changes between the wild-type
and mutant Thiopurine methyltransferase protein, both the
structures were modeled using a modeling software program ,
UCSF Chimera, and illustrated below.
This observation was also experimentally observed in a study
conducted by Evans et. al., that assessed an inactivating
mutation (TPMT*2) in the human TPMT gene providing an
insight into inherited polymorphism in drug metabolism.
Figure 3A- Illustrated molecular model of the
Wild-type TPMT (Chain A: PDB ID: 3BGD)
with all Ala residues highlighted in Red, and
the wild-type Alanine residue highlighted and
labeled in Magenta.
Figure 4B- Illustrated molecular model of the
mutant TPMT*2 (Chain A, PDB ID: 2BZG) with
all Proline residues highlighted in magenta, and
the mutant Proline residue highlighted and
labeled in Red.
The experiment involved the use of a yeast (heterologous)
expression system, in which this mutation led to a 100-fold
reduction in TPMT catalytic activity specifically related to the
wild-type cDNA, despite comparable levels of mRNA for the
same being expressed in the system.[6]
This serves as conclusive proof of the underlying effect of this
TPMT*2 mutation on the catalytic activity of Thiopurine
methyltransferase. The study suggests that the mutation results
in a 100-fold reduction in activity. If the same would
observation can be duplicated in a human trial, this would
suggest that the activity of Mercaptopurine if administered (at
standard doses) would exert a higher cytotoxic effect.
Conclusion:
Genetic studies over the past few decades have showed that
polymorphism at the TPMT gene plays a major role in serious,
life-threatening myelosuppression which is a dose-related
toxicity of thiopurine drugs, TPMT polymorphism and activity
is one of the best models for the translation of
pharmacogenomic information to guide patient therapeutics
and also an effective method to personalize thiopurine therapy
in patients with disorders pertaining to toxicity, Inflammatory
Bowel Disorder(IBD) and other related disorders.
References:
[1] “The clinical Pharmacology of 6-mercaptopurine”-L.Leonard. A
special edition in Journal of Clinical Pharmacology (Springer, 1992)
[2] Zaza Gianluigi, Cheok Meyling, Krynetskaia Natalia, Thorn
Caroline, Stocco Gabriele, Hebert Joan M, McLeod Howard,
Weinshilboum Richard M, Relling Mary V, Evans William E, Klein
Teri E, Altman Russ B. "Thiopurine pathway" Pharmacogenetics and
genomics (2010).
[4](Vogt et al., 1993; Krynetski et al., 1995; Coulthard and
Hogarth, 2005)
[5] E Y Krynetski, J D Schuetz, A J Galpin, C H Pui, M V Relling, W
E Evans. “A single point mutation leading to loss of catalytic
activity in human thiopurine S-methyltransferase.” Proc Natl Acad
Sci U S A. 1995. PMCID: PMC42614
[6] K.H. Katsanos,1,2 Séverine Vermeire,1 Karolien Claes,1 Nele
Van Schuerbeek,1 Gert Van Assche,1P. Rutgeerts,1 E.V. Tsianos2.
“Thio-Purine Methyl Transferase Gene Single Nucleotide
Polymorphisms in Inflammatory Bowel Disease. (Annals of
Gastroenterology, 19(1):18-20 2006)
Bibliography:
[3] PharmGKB [www.pharmgkb.org; Clinical-PGx, Pharma-PGx,
Clinical Annotations]
[7] dbSNP Online SNP Database [http://www.ncbi.nlm.nih.gov/SNP]
[8] RCSB-Protein Databank [http://www.rcsb.org/pdb]