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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top

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ne

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]