Preliminary communication

Enzymatic thiolysis of azathioprine $\text{in vitro}$

Gastrointestinal disorders (GITs) are diseases that mainly affect the gastrointestinal tract and are the most common problems that need medical attention. Thanks to advancements in diagnostic and therapeutic technology, we are now better able to identify and manage the effects of diseases. GIT disorders include gastroesophageal reflux disease (GERD), diarrhea, inflammatory bowel disease (IBD), colorectal cancer (CRC), irritable bowel syndrome (IBS), and liver transplantation. Some illnesses cause symptoms even when the GI system appears to be in good health. The majority of digestive illnesses are treatable or preventable. GERD is a widespread clinical illness that affects millions of individuals worldwide, and in recent years, there has been a substantial rise in the prevalence of GERD. IBDs are a class of autoimmune disorders that are characterized by inflammation of the small and large intestines and occur when the body's immune system attacks the digestive system components. In individuals with acute liver failure and end-stage liver disease, a life-saving procedure is liver transplantation. IBS affects 9%–23% of people worldwide, and the affected individuals account for about 12% of patients in primary care. Age-related increases in colorectal cancer incidence make it the third most common cancer in the Western Hemisphere. In this chapter, our main focus is to study the clinical characteristics, pharmacological treatment, pharmacological consideration of GERD, IBD, IBS, CRC, and liver transplantation. The medications that can be used to treat a variety of disorders are clearly described in this chapter.

The phase II or conjugative enzymes of drug metabolism catalyze the combination of a functional group in a drug molecule or its phase I metabolite with a small, water-soluble, endogenous molecule. Products of most phase II enzyme-catalyzed reactions are more water-soluble than the starting drug or drug metabolite and many are anions at physiological pH, making them good substrates for elimination from the body aided by transporter proteins. The properties and chemical reactivity of the functional group or groups in the drug or drug metabolite will determine which phase II reactions occur. Functional groups may be nucleophilic, such as hydroxyl groups, carboxylic acids, amines, or electrophilic such as epoxides, α,β-unsaturated ketones or alkyl halides. The major phase II enzymes are UDP-glucuronosyltransferases (UGTs), PAPS-sulfotransferases (SULTs) and glutathione transferases (GSTs). Also important, but with a more limited range of substrates, are N-acetyltransferases (NATs), the enzymes that catalyze formation of amino acid conjugates of carboxylic acids and methyltransferases (MTs). In people and animals, a major site of phase II metabolism is the liver, though other tissues, such as kidney and intestine, also express these enzymes. The rate of formation of phase II metabolites depends upon the amount of enzyme and drug substrate present, the affinity of the drug substrate for the enzyme and the availability of the endogenous co-substrate.

Azathioprine (AZA) is a thiopurine prodrug which is widely used in patients with inflammatory bowel disease (IBD). However, the use is limited in one–third of patients because of adverse drug reactions (ADRs) or a lack of clinical response. It has been considered that the polymorphic enzyme thiopurine S–methyltransferase (TPMT) plays an important role in the in vivo process of AZA and the occurrence of its myelotoxicity. Glutathione S–transferase (GST) mutation is another pharmacogenetic polymorphism which is probably involved in AZA metabolism and tolerance. The aim of this study was to investigate the association among GST polymorphism, enzyme activity and AZA–related ADRs in Chinese Han patients with IBD.

We found that the patients who became neutropenic had a significantly higher GSTs activity when compared with of the patients who did not develop ADRs (analysis of variance, P < 0.001). There was also a significant underrepresentation of GSTP1*–₁₀₅V allele among patients developing ADRs (odds ratio [OR] = 0.125, 95% confidence interval [CI] = 0.022–0.709, P = 0.0012).

The patients with higher GST activity constituted a pharmacogenetic high risk group for leucopenia during AZA treatment. GST–P1 Ile105/Ile105 genotype appeared to be a promising marker indicating predisposition to AZA–related ADRs.

The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adverse side effects. The prodrug is activated by glutathione conjugation and release of 6-mercaptopurine, a reaction most efficiently catalyzed by glutathione transferase (GST) A2-2. Among five genotypes of GST A2-2, the variant A2*E has threefold–fourfold higher catalytic efficiency with azathioprine, suggesting that the expression of A2*E could boost 6-mercaptopurine release and adverse side effects in treated patients.

Structure–activity studies of the GST A2-2 variants and homologous alpha class GSTs were made to delineate the determinants of high catalytic efficiency compared to other alpha class GSTs. Engineered chimeras identified GST peptide segments of importance, and replacing the corresponding regions in low-activity GSTs by these short segments produced chimeras with higher azathioprine activity. By contrast, H-site mutagenesis led to decreased azathioprine activity when active-site positions 208 and 213 in these favored segments were mutagenized. Alternative substitutions indicated that hydrophobic residues were favored.

A pertinent question is whether variant A2*E represents the highest azathioprine activity achievable within the GST structural framework. This issue was addressed by mutagenesis of H-site residues assumed to interact with the substrate based on molecular modeling. The mutants with notably enhanced activities had small or polar residues in the mutated positions. The most active mutant L107G/L108D/F222H displayed a 70-fold enhanced catalytic efficiency with azathioprine. The determination of its structure by X-ray crystallography showed an expanded H-site, suggesting improved accommodation of the transition state for catalysis.

Hepatotoxicity due to immunosuppressive drugs is a rare cause of DILI, but when these reactions do occur, they run the gamut of clinical laboratory and histopathological presentations. As some immunosuppressive drugs are used less now than previously (like corticosteroids and azathioprine) and some not at all (like daclizumab that is no longer marketed), new agents are being produced at a fast rate. In the complicated setting in which these agents are usually used, many other causes of liver dysfunction are possible and indeed probable; therefore patients must be evaluated thoroughly and alternative diagnosis/diagnoses made before attributing the liver injury to immunosuppressive DILI. Sometimes there are characteristic histopathological abnormalities that provide a diagnosis but, even in these cases, concomitant causes of liver injury should still be sought. As with DILI in other situations, there is a great need for more information about patient monitoring, early detection, noninvasive tests, and, most of all, laboratory methods to identify DILI specifically and not simply by exclusion. The complicated diseases for which these drugs are prescribed and the increasing ingenuity of these agents continue to provide both a challenge and a fascination to physicians caring for these patients.

A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.