Liver plays an important function in the metabolism of a big number of drugs and toxins. Hepatic drug metabolism involves various procedures, loosely classified as Phase 1 (functionalization) and Phase 2 (conjugation). Glucuronidation, catalyzed by UDP-glucuronosyl transferase (UGTs), plays a cardinal role in the Phase 2 metabolism of a big number of drugs, as well as many endogenous substrates like bilirubin, steroids, etc., by increasing hydrophilicity and clearance. UGTs are various enzymes in terms of broad, yet overlapping substrate specificity, presence of numerous isoforms, genetic polymorphisms, etc.
Biological systems are recognized to be stereoselective in nature. A big number of drugs, about 50% of all marketed drugs, exist as either single enantiomers or racemates. Therefore, stereoselective behavior of drugs plays an important role in drug action, as well as absorption, distribution, metabolism, and elimination (ADME).
The purpose of this project is to identify the enantio-selectivity of drugs towards glucuronidation by UGT and also to determine whether enantio-selectivity is linked to specific isoforms of UGT. Furthermore, predicting the enantiomeric behavior of drugs may also help in rationalizing in-silico modeling of drug metabolism and thereby predicting metabolism of new chemical entities (NCEs).
Introduction
Biotransformation and elimination of drugs from the body involve several different metabolic pathways. These metabolic pathways are broadly classified into Phase 1 (functionalization) and Phase 2 (conjugation) drug metabolism.
The Phase 1 metabolic pathway involves transforming the drug into a more polar functionality through various reactions like oxidation, reduction, hydrolysis, isomerization, and so on, depending on the chemical nature of the drug. These reactions are catalyzed by enzymes such as cytochrome P450, NADPH-cytochrome P450 reductase, acetylcholinesterase, etc.
The product of Phase 1 drug metabolism may then act as a substrate for Phase 2 metabolism. This stage consists of conjugation of the drug substrate with endogenous ligands, leading to increased mutual opposition, hydrophilicity, and thereby elimination of the drug from the body through bile or urine. Conjugation reactions include glucuronidation, glycosidation, sulfation, methylation, etc. These reactions are catalyzed by UDP-glucuronosyltransferase, UDP-glycosyltransferase, sulfotransferase, methyltransferase, respectively. Among these, glucuronidation is the most prevalent conjugation reaction in the body.
Glucuronidation
Glucuronidation is the most common reaction in Phase 2 drug metabolism. This conjugation reaction, which is catalyzed by UDP-glucuronosyl transferase, forms about 35% of all drugs metabolized by conjugation. This is mainly due to the abundance in living systems of UDP-glucuronic acid, the co-factor for the reaction, as well as the permeant nature of the enzyme, UDP-glucuronosyl transferases (UGTs).
The process of glucuronidation involves:
- Formation of co-factor (UDP-glucuronic acid)
- Conjugation of UDP-glucuronic acid with substrate
The formation of co-factor (UDP-glucuronic acid):
This consists of a two-step process:
- Formation of UDP-glucose: Glucose-1-phosphate is present in high concentrations in almost all cells of the body. The first step of glucuronidation is related to glycogen synthesis through the common intermediate, UDP-glucose. The formation of UDP-glucose occurs by addition of a Uridine 5′-diphosphate (UDP), a pyrophosphate base in cells, to a molecule of Glucose-1-phosphate. The reaction is catalyzed by UDP-glucose pyrophosphorylase enzyme.
- Dehydrogenation of UDP-glucose to UDP-glucuronic acid: The above step is followed by dehydrogenation of UDP-glucose to UDP-glucuronic acid, catalyzed by the enzyme UDP-glucose dehydrogenase, in the presence of NAD+ co-factor.
Conjugation of the substrate with UDP-glucuronic acid:
The conjugation reaction involves the transfer of one α-D-glucuronic acid moiety from the co-substrate UDP-glucuronic acid (UDPGA), which acts as an energy-rich intermediate, to form the glucuronide conjugate of the drug molecule. The reaction is catalyzed by UDP-glucuronosyl transferase (UGT) enzyme.
The reaction is found to be a bimolecular nucleophilic substitution (SN2), whereby the C1-C of glucuronic acid, which is in α-configuration, during its reaction with the substrate inverts to form a β-D-glucuronide. The glucuronide formed is excreted via urine or bile, depending on the chemical nature and molecular weight of the conjugate.
The full reaction is summarized below:
Pharmacological relevance
Based on the functional group of the substrate molecule, the following types of glucuronide conjugates may be formed: O-Glucuronide, N-glucuronide, S-Glucuronide, and C-glucuronide.
O-Glucuronides are formed from phenols, alcohols, and carboxylic acids. They are chiefly excreted into bile and may undergo enterohepatic circulation. Examples of drugs that form O-glucuronides include morphine, chloramphenicol, salicylic acid, and clofibrate.
N-glucuronides are formed by the reaction of UDP-glucuronic acid (UDPGA) with aminoalkanes and amides, among others. Examples of drugs that form N-glucuronides include sulfanilamide, cyproheptidine, and dapsone.
S-Glucuronides are formed by the chemical reaction of thiol groups with UDPGA in the presence of UDP-glucuronosyl transferase. Examples of drugs that form S-glucuronides include disulfiram and 2-mercaptobenzothiazole.
C-glucuronides are an uncommon metabolic tract that occurs due to the direct attachment of UDPGA to the C skeleton of drugs. An example of a drug that forms C-glucuronides is sulfinpyrazone.
UDP-glucuronosyl transferase (UGT) enzymes are present in human beings and most other mammals. The enzyme is located in many tissues of the body, mostly in the liver but also in the kidney, lungs, small bowel, spleen, adrenal glands, and skin, to a lesser extent. Inside the cell, UGTs are bound to the membranes of endoplasmic reticulum. Most of the Phase 1 metabolic enzymes, including cytochrome P450s, are located in the endoplasmic reticulum. Therefore, the endoplasmic reticulum is regarded as an ideal site for UGT enzymes, as it facilitates glucuronide junction of Phase 1 substrates.
UDP-glucuronosyl transferase enzyme does not contain a prosthetic group. The monomeric molecular weight of the enzyme is found to be between 50-60 kilo Daltons. The protein sequence of the enzyme shows little variation between each individual form. A full-length crystal structure of UGTs is yet to be resolved, although the crystal structure of the binding sphere for UDP-glucuronic acid in human UGT2B7 has been published (by Miley et al., 2007).
In addition to being a major enzyme involved in Phase 2 drug metabolism, UGT enzymes play a number of other roles in the body. Many endogenous compounds, such as bilirubin, steroid hormones (e.g. tetraiodothyronine, liothyronine), and catechols (derived from catecholamine metabolism), act as substrates for UGT enzymes.
All these compounds are potentially dangerous if accumulated in the body. Lack of UGT enzyme results in hyperbilirubinemia. Familial diseases like Gilbert’s syndrome and Crigler-Najjar’s syndrome are associated with familial polymorphisms of the UGT gene. Apart from the metabolism of endogenous toxins, the enzyme also catalyzes glucuronidation of various exogenous chemicals and helps in the body’s defense mechanism against toxic substances.
Multiple forms
Assorted signifiers of UDP-glucuronosyl transferase (UGT) enzymes have been identified with the aid of studies based on purification, characterization of enzymes, molecular cloning, DNA sequencing, etc. About 50 vertebrate UGTs have been identified, among which 19 are found in humans.
UGT enzymes are divided into families and sub-families based on the similarity of their amino acid sequences. Two enzymes are in the same family if the similarity of their amino acid sequences is more than 50%, and will be grouped into the same subfamily if the similarity is greater than 60%.
Terminology
Divergent evolution and sequence similarity form the basis of the terminology of UGT enzymes. The name of the enzyme consists of 4 parts:
- Root Symbol: The root symbol ‘UGT’ stands for UDP-glucuronosyl transferase.
- Family: It is denoted by an Arabic figure, e.g. 1, 2, etc.
- Sub-family: Designated by an uppercase alphabet.
- Individual Form: An Arabic number is used for the unique designation of the individual signifier of the enzyme.
For example, UGT2B4, UGT1A6.
Mammalian UGTs are divided into four families: UGT1, UGT2, UGT3, and UGT8. Among these, only UGT1 and UGT2 catalyze the junction of glucuronide and are therefore discussed further.
The UGT1A family of enzymes is found to contain 9 functional proteins and is coded for by a single gene complex located at chromosome 2q37. The genes coding for this enzyme have common coding DNAs 2-5 (part of the gene which codes for the carboxyl endpoint of the enzyme) and a variable coding DNA 1. The first coding DNA is responsible for coding the N-terminal domain of the protein, and this explains why the enzymes are substrate-specific despite having a common C-terminal.
The UGT2 enzyme family, particularly UGT2B, plays a critical role in the metabolism of xenobiotics and endogenous ligands. Genes coding for UGT2 family enzymes are situated on chromosome 4q13. In the case of the UGT2B subfamily, protein sequences at the C-terminal give rise to the UDP-glucuronic acid binding domain, as well as aiding in the anchoring of the protein to the membrane of the endoplasmic reticulum.
The UGT2A subfamily is less studied and does not play a significant role in systemic metabolism. UGT2A1 is present in olfactory epithelial tissues and to a lesser extent in cells of the brain and lungs. UGT2A2 is in the liver and small bowel, while UGT2A3 is in the small bowel, liver, and adipose tissue.
Tissue specificity
The assorted forms of UGT enzymes show tissue specificity in adult males. The majority of these enzymes occur predominantly in the liver, (e.g., UGT 1A1, 1A4, 1A6, 2B7, etc.), while some others are found in various extrahepatic sites. An example is UGT1A10, which is present in the cells of all areas of the gastrointestinal tract and hence accounts for its broad range of substrate specificity, from phenol molecules to steroids.
Substrate specificity
UGTs show a broad, yet overlapping, range of specificity towards drugs and endogenous ligands. For example, glucuronidation of bilirubin is preferred by UGT1A1 and that of morphine by UGT2B7.
Interindividual variations
Several genetic polymorphisms in UGTs may lead to variations between individuals in the ability to glucuronidate drugs and endogenous substrates. Mutations in genes coding for UGT1 enzyme family have been identified as the cause of familial hyperbilirubinemia, characterized by jaundice due to high levels of unconjugated bilirubin in the body.
Furthermore, several genetic diseases – Gilbert’s syndrome and Crigler-Najjar’s syndrome – may occur due to mutations in genes coding for the UGT1A1 isoform.
Enzyme kinetics
The study of enzyme dynamics helps to understand the catalytic mechanism of the enzyme, the function played by the enzyme in metabolism, as well as the rate and activity of the enzyme. The Michaelis-Menten equation is used to describe enzyme-substrate interaction and is given by:
E + S ↔ ES → E + P
where E = Enzyme, S = Substrate, P = Product.
The Michaelis constant Km is given by:
Km = (k2 + k-1) / k1
The Michaelis constant Km is an index of the affinity of substrate for the enzyme as well as the rate of enzyme activity. The dynamics of drug metabolism can also be defined using the Michaelis-Menten equation and may be plotted in a graph of the rate of reaction (velocity) V. concentration of substrate. Although not all enzyme-substrate reactions are best described by this equation, a typical model of Michaelis-Menten plot is shown below: Here Vmax is the maximal speed of enzyme action. Vmax / Km is an index of the catalytic efficiency of the enzyme.
Stereoisomerism
Molecules having the same composition of atoms and sequence of covalent bonds but differ in their three-dimensional arrangement of atoms in space are known as stereoisomers. Stereoisomers are classified into geometrical (cis/trans) isomers, enantiomers, and diastereoisomers. Stereoisomers that are mirror images of each other and hence are not superimposable are called enantiomers. They differ from each other only by one chiral center. Isomers that are not mirror images are diastereoisomers. They may contain more than one chiral center.
While geometrical and diastereoisomers are chemically different molecules, enantiomers have identical chemical and physical properties, except for the way in which they rotate plane-polarized light. Enantiomers are of great significance in therapeutics as all biological systems represent a chiral environment. Hence, drug action as well as disposition (absorption, distribution, metabolism, and elimination) may differ between enantiomers.
Drugs as enantiomorphs
As discussed above, the pharmacokinetic and pharmacodynamic properties may change for each single enantiomer. In 1992, the United States Food and Drug Administration (US FDA) published a policy for the development of new stereoisomeric drugs. Approximately 50% of all marketed drugs are found to be racemates. Although many drugs can be safely administered as racemates, some others show better efficacy and fewer side effects when administered as a single enantiomer. For example, cardiac toxicity of the local anesthetic agent Levobupivacaine is chiefly associated with the R-enantiomer.
Furthermore, some drugs undergo chiral inversion inside the body to the other enantiomer (e.g. Ibuprofen: Non-steroidal anti-inflammatory agent), and some others undergo racemization after disposal. This is of particular concern, especially if one of the enantiomers is toxic. Hence, evaluating drugs for their stereochemistry is gaining importance.
Some examples of single enantiomeric drugs that have gained importance, compared to their racemate counterparts, are given below due to their improved pharmacodynamic- pharmacokinetic profiles:
L-dopa:
The use of levo-dihydroxyphenylalanine instead of racemic dihydroxyphenylalanine has resulted in a reduction in dosage and adverse effects (nausea, vomiting, anorexia, agranulocytosis).
Esomeprazole:
This proton-pump inhibitor, which is the S-enantiomer of Omeprazole, has shown a lower first-pass effect and higher plasma half-life compared to the R-enantiomer, thus maintaining the intragastric pH above 4 for a longer duration. The S-enantiomer also showed a decrease in the variability of the response between patients.
Levofloxacin:
It is a Quinolone antibiotic. As there are little differences in the disposition between enantiomers of this drug, a single S-enantiomer is preferred.
R-salbutamol (levalbuterol):
The S-enantiomer has shown an increased hyperreactivity of air passages, sensitivity to allergens, and some decrease in bronchodilator authority. While R-Salbutamol gives a significantly higher bronchodilator effect and lesser side effects.
R, r-methylphenidate:
This drug is found to be tenfold more potent than its S-enantiomer when used to treat attention deficit hyperactivity. The presystemic metabolism and disposition of the drug are enantioselective in nature. Furthermore, the R-enantiomer shows rapid onset of action and decreased adverse effects.
Aim of the project
This project aims to determine the rates of glucuronidation of enantiomeric pairs, of a wide range of drugs, to identify differences in metabolism between enantiomers of a drug and also to find out whether enantioselectivity is related to a particular family of UDP-glucuronosyltransferase (UGT) enzyme. The experiment may be done by in-vitro incubations of human recombinant UGTs or human liver microsomes with the selected substrates, followed by analysis using liquid chromatography (HPLC) equipped with a mass spectrometer for detection.
Laboratory analysis of enantiomers is usually done using any one of the following two methods:
- Chiral Chromatography, which make usage of a chiral column or chiral nomadic stage to divide the enantiomorphs.
- Derivatization of the analyte using a chiral derivative, followed by separation of the resulting diastereoisomers using a standard, achiral chromatographic method.
But in the instance of separation of drug conjugates, the analytical procedure is comparatively simple, as the glucuronide conjugates behave merely like derivatised diastereomers and hence may be separated by conventional liquid chromatography.
Many late-phase failures in drug development procedures are due to the inability to predict the pharmacokinetic properties of new chemical entities (NCE) before obtaining data from clinical tests. Hence, in-vitro approaches like computational (in-silico) modeling of drug metabolism are gaining acceptance in recent times.
Many approaches, such as 2D-Quantitative Structure Metabolism Relationship (2D-QSMR), 3D-Quantitative Structure Metabolism Relationship (3D-QSMR), pharmacophore identification, as well as non-linear pattern recognition techniques, are being studied to model drug-metabolizing enzymes. Although prognostic models for the metabolism of drugs by the Phase 1-metabolizing enzyme, Cytochrome P450, are widely accepted, the development of effective models for UDP-glucuronosyl transferases (UGTs)-catalyzed Phase 2 metabolism has received much less attention.
The versatility of this group of metabolic enzymes, in terms of broad but overlapping substrate specificity, drug-drug interactions, genetic polymorphisms, as well as the presence of a large number of isoforms, is some of the challenges facing the development of predictable models for UGTs. Furthermore, apart from a few catalytically relevant amino acids, the full X-ray crystal structure of the UGT enzyme is not yet elucidated.
Depending on the parameters being modeled (e.g. Km, Vmax, etc.), a number of physico-chemical and molecular forms, such as molecular size, shape, lipophilicity, H bonding, etc., are required to model molecular recognition of substrates and contact action by UGTs. Apart from this, the study of the electronic nature of the nucleophile and pKa is also important. Since chirality plays an important role in determining the metabolic behavior of drugs, design tools may be developed that address the issue of chirality. While 2D-descriptors will only predict molecular connectivity, 3D models predicting the enantiomeric properties of enzyme-substrate interactions might significantly improve the future of drug development procedures.
Conclusion
In conclusion, many biological systems represent a chiral environment. Therefore, evaluating the enantioselectivity of drug-metabolizing enzymes plays a significant role in predicting the pharmacokinetic behavior of drugs. The present project aims to identify the enantioselectivity of drugs in UDP-glucuronosyl transferase (UGT) metabolism, which is an important Phase 2 (conjugation) process of drug metabolism. Furthermore, knowledge of the enantiomeric behavior may assist in the development of 3D-Quantitative Structure Metabolism Relationship (3D-QSMR) models for predicting drug metabolism.
