Drug Targets and Receptor Polymorphisms

• Drug targets include receptors, enzymes, ion channels, and transporters, with a focus on receptors, enzymes, and ion channels.

Receptor Polymorphisms

• Polymorphisms in receptors and related proteins typically affect receptor function or expression levels, unlike metabolic enzyme polymorphisms that can cause a complete absence of activity.

G-Protein Coupled Receptors

• Polymorphisms can affect receptor or G-protein coupling in G-protein coupled receptors.
• These receptors have 7 transmembrane proteins.
• The linker region between transmembrane proteins 5 and 6 is important for coupling to G-proteins.

Studying Receptor Gene Polymorphisms

• Studying phenotypes is more difficult than with pharmacokinetic polymorphisms like CYP genes.
• Polymorphisms discovered by gene sequencing do not indicate functional importance, and are more useful when paired with knockout mouse studies.
• Knockout mouse studies provide clues about functional importance for behaviour effects.

Approaches to Studying Receptor Polymorphisms

• For coding sequence polymorphisms, variant function (response to agonist or antagonist) is compared with wild-type by expressing the variant in a cell line.
• Reporter gene studies, often using luciferase, are used for upstream polymorphisms.
• Luciferase is a sensitive assay using a luminometer but requires cell manipulation.
• Alkaline phosphatase reporter can be used instead of luciferase, with the product released into the growth medium, but is less sensitive.

Adrenoreceptors

• Adrenoreceptors are important drug targets.
• Beta 1 and 2 receptors increase cAMP when stimulated, unlike alpha receptors.

B2-Adrenergic Receptor

• The B2-adrenergic receptor is encoded by the ADRB2 gene, which contains introns and has a 1239 bp coding region.
• ADRB2 is highly polymorphic, especially in the coding sequence.
• Nine known non-synonymous polymorphisms exist in coding regions, resulting in four amino acid substitutions: Arg16Gly, Gln27Glu, Val34Met, and Thr164Ile.
  • Arg16Gly is a common SNP where the Gly16 variant enhances down-regulation after agonist binding compared to Arg16
• A single polymorphism exists in the leader peptide: Arg19Cys (at amino acid 16).
• Seven upstream polymorphisms are known.
• The Arg16 form of ADRB2 is associated with increased agonist-induced down-regulation and poorer response to inhaled short-acting beta agonists in asthmatic patients.
• Codon 16 affects short-acting agonists, but not clinically important as current treatments use long-acting agonists.
• Studies on B-adrenergic receptor antagonists for heart failure and hypertension have mostly failed to detect response differences related to codon 16 or 27 of ADRB2 genotype.
• The polymorphism site at amino acid 16 is the ligand binding site, outside the cell.
• G-protein coupling is unaffected.
• The arginine to glycine amino acid change is inside the 7-transmembrane domain.

Functional Effect of B2 Adrenergic Receptor Polymorphisms

• Change in FEV versus salbutamol was twice as high with 2/2 haplotype as with 4/4.
• Polymorphism in haplotype 6 has a marked effect.
• Individuals with mild asthma, where only homozygous were studied, haplotype 4 gives a better response to the placebo, specific to salbutamol.
• There is no difference in the response of long-acting agonists with these haplotypes.

In Vitro Effect of Different Haplotypes on Expression

• Lower protein expression of receptor is measured with the 4 haplotype.
• Individuals with mild asthma see a better response from placebo's in the 4 haplotype compared to the 2, only when homozygous.
• No difference will result in long-acting agonists, so genotyping is not carried out.

Connie et al Study, 2002

• Identified 13 SNPs in a continuous region of the 5' upstream and coding sequence of B2AR in humans.
• Showed that unique interactions of multiple SNPs within a haplotype ultimately affect the biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

B1 Adrenoreceptor Polymorphisms

• B1 receptors are the main type of adrenoreceptor in the heart.
• The ADRB1 gene has a common non-synonymous polymorphism leading to Gly389Arg substitution at codon 389.
• This is the main target for B blockers (e.g., atenolol).
• The Gly variant has a frequency of 0.26, and the Arg variant has a frequency of 0.74.
• The arginine form shows enhanced Gs protein interaction and increased adenylyl cyclase activation.
• Transfection studies demonstrated slightly higher basal expression levels for the arginine form
  • The arginine form shows 3-fold higher activity when stimulated with isoproterenol.

Johnson et al Study

• Patients with better responses had darker bars pure with Arg in the haplotype compared to glycine forms.

Vitamin-K Epoxide Reductase

• VKORC1 is the target enzyme for coumarin anticoagulants, including warfarin.
• VKORC1 was sequenced in 2004.
• Warfarin-resistant patients require more than 10 mg per day, due to rare mutations.
• Normal dosing is 5 mg/day, but each patient should be titrated to measure clotting.
• VKORC1 and CYP2C9 account for up to 40% of inter-individual variation in coumarin anticoagulant dose, with age, body surface area, and interfering concomitant medications accounting for an additional 15-20% of variation.
• Genotyping for CYP2C9 and VKORC1 may be helpful in settling warfarin dosing.
• Warfarin is only useful if the vitamin K epoxide can be measured.

Vitamin K Cycle

• The carboxylase enzyme needs vitamin K as a co-factor.
• Vitamin K epoxide is recycled by VKOR to produce vitamin KH2, which starts the cycle again.
• VKOR is present in the endoplasmic reticulum membrane, next to the GABA carboxylase.

VKORC1 Polymorphisms

• The VKORC1 is a small gene with only 3 exons.
• Two changes are almost always inherited together in Caucasian Europeans:
  • G-1639A (in the upstream region)
  • C1173T (in the first intron)

Reider et al Study, 2005

• Displays hepatic VKOR levels according to VKORC1 haplotype.
• The A variant has G-1639A and C1173T, while the B variant (wild type) has high mRNA expression for VKOR.
• Confirms the reason for lower doses of warfarin needed with G-1639A and C1173T, making less VKORC1 gene in the wildtype form.

VKORC1 Genotypes in Warfarin Patients

• A variant (-1639) is low expression.
• G variant (-1639) is high expression with AG intermediate.
• Most of the population has AG (heterozygotes).
• GG requires a higher dosage of warfarin.
• CYP2A9 metabolizes warfarin.

Warfarin Resistance

• Patients do not respond to warfarin or need unusually high doses (>10 mg/day).
• Some patients showing resistance have mutations in the coding region of VKORC1.
• These mutations affect warfarin binding but not vitamin K epoxide reduction.

VKORC1 Protein Structure

• The VKORC1 is present in the cytosol.
• D36Y & V66M are the rarest polymorphisms seen in the VKORC1 gene, located in the cytoplasmic loop (CL).
• D36Y is more common in Egypt, Israel, and Ethiopia (4 to 10%).
• Screening prior to treatment is not considered useful due to the rarity of these polymorphisms.

CYP2C9 and VKORC1 Genotype Effect on Warfarin Dose

• A study by Sconce et al. in 2005 showed:
• The wild-type for CYP2C9 (11) in the presence of G in the upstream region is associated with an increased dose requirement for warfarin.
• Significant increase with AA to GG (2 changes in the upstream region in the intron).
• 12 reduction in CYP2C9 activity globally decreases the dose requirement of warfarin.

Regression Equation

• CYP2C9/VKORC1 genotype, age, and height account for approximately 55% of warfarin dose variability.
• Equation quantitates effect of each factor and also dose prediction.
• Warfarin is being prescribed less commonly due to the availability of direct-acting anti-coagulants (DOACs).

HMG-CoA Reductase

• HMG-CoA reductase converts HMG-CoA into mevalonic acid, which is the rate-limiting step for cholesterol biosynthesis.
• Statins target the enzyme to lower plasma cholesterol levels.
• Two polymorphisms in the non-coding region of HMGCR, the gene encoding HMG-CoA reductase, are associated with a decreased response to statin treatment.
• The polymorphism is more common in African Americans (7%) than White Americans in the US population.
• The low haplotype frequency means that statin response can be studied on heterozygotes, as homozygous mutants are rare.
• The original study reported a smaller reduction in both total cholesterol and LDL in response to Pravastatin treatment.

Cystic Fibrosis Treatment

• Patients have a defect in the CFTR gene, which codes for an ion channel involved in Cl- transport.
  • The most common deficit is F508del (single amino acid [phenylalanine] deleted)
  • Protein is then not inserted into the cell membrane, abolishing activity
  • F508del deficits are screened in newborns
  • G551D is involved in approximately 5% of cases
  • Channel reaches the cell surface but does not transport Cl- due to a gating defect
  • The channel no longer responds to the ligand
• Ivacaftor targets the G551D gating defect, taking the place of the ligand and triggering a response in the receptor.

Ivacaftor Effect and Other Drugs

• Ivacaftor reduces sweat chloride levels, improving lung function (FEV1.0) by 11% and decreasing pulmonary exacerbations by 55%
• Additional drugs have been licensed in the UK, with others in the pipeline.
  • Orkambi targets the common F508del mutation
  • Orkambi is ivacaftor plus lumacaftor, which helps with CFTR movement to the plasma membrane
  • Symkevi (ivacaftor, tezacaftor, and elexacaftor) also targets F508del, more efficiently than Orkambi

Drug targets include receptors, enzymes, ion channels, and transporters. Receptor polymorphisms affect receptor function/expression. Studying receptor gene polymorphisms is more difficult than pharmacokinetic polymorphisms.