Carboxylesterase 2 : A Key Enzyme in Drug and Prodrug Metabolism

Carboxylesterases (CESs) play important roles in the metabolism of prodrugs containing ester bonds, which are often introduced to improve bioavailability and drug efficacy. CES1 and CES2 are major isozymes encoded by genes in the CES multigene family in humans, and their characteristics such as tissue distribution and substrate specificity have been extensively studied over the past few decades. This mini review briefly summarizes the current knowledge on CES2 focusing on its roles in the metabolism of anticancer prodrugs. We also discuss the regulatory mechanism of CES2 expression, which is often dysregulated in cancer cells. The development of CES2 inhibitors and methods to evaluate CES2 activity in biological samples are also discussed.


Tissue distribution and substrate preference of carboxylesterases
Carboxylesterases (CESs) are members of the serine hydrolase superfamily and catalyze the hydrolysis of a variety of endogenous and exogenous substrates, including esters, thioesters, carbamates, and amides 1) . CESs are classified into five groups based on the homology of their amino acid sequences 2) . In humans, two major isozymes CES1 and CES2 play important roles in drug metabolism. CES1 is highly expressed in the liver and distributed in macrophages as well as adipose, lung, and other tissues 3)-5) . In the liver, CES2 expression is lower compared with that of CES1. In contrast, in the small intestine and colon, CES2 is highly expressed whereas CES1 expression is low 6) 7) .
The difference in substrate preference of CES1 and CES2 appears to be related to the relative sizes of the alcohol and acyl substituents. For example, CES1 preferably hydrolyzes drugs with an acyl substituent larger than the alcohol substituent, such as oseltamivir, methylphenidate, and simvastatin. In contrast, drugs with an alcohol substituent larger than their acyl moiety, such as azilsartan, tenofovir disoproxil, and prasugrel, are the preferred substrates for hydrolysis by CES2 8) 9) . These substrates include irinotecan [camptothecin-11 (CPT-11)], an anticancer prodrug converted by CES2 to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) 7) 9) (Figure-1). Irinotecan has been used in the treatment of a wide spectrum of cancers, and irinotecan-containing regimens such as FOLFIRI and FOLFOXIRI are standard chemotherapy regimens for advanced colorectal cancer 10) . In addition, CES2 also hydrolyzes and activates other anticancer prodrugs such as capecitabine and LY2334737 (the prodrug of gemcitabine) 11) 12) . Therefore, understanding the tissue distribution and the regulatory mechanism of CES2 expression is critical with regards to the use of these anticancer prodrugs.

Regulatory mechanism of CES2 expression
Recent studies have reported that activation of p53, a tumor suppressor protein encoded by the TP53 gene, upregulates CES2 expression in colon carcinoma cell lines expressing functional p53 13)-15) . It is known that approximately 50% of colorectal tumors harbor missense mutations in TP53. Based on these premises, we investigated whether CES2 expression was decreased in colorectal cancer with a TP53 mutation 15) . First, we found that the expression and activity of CES2 were significantly decreased in colorectal cancer tissue compared to its adjacent normal tissue. However, there was no clear relationship between TP53 gene status and the decrease in CES2 expression and activity. Even in colon cancer with functional p53, CES2 was downregulated. We also found that the expression of p21, the downstream target of p53, was significantly reduced in cancer tissue independently of TP53 gene status. In addition, CES2 expression strongly correlated with p21 expression in the colon, even in the samples with nonfunctional p53. It has been reported that p21 expression can be upregulated by various stress signals independently of p53 16) . Taken together, these results suggest that the regulation of CES2 expression in colon cancer is complicated and mechanisms other than p53 are likely to be involved.

CES2 inhibitor
One of the most common side effects of taking irinotecan is severe diarrhea 17)-19) . This is likely, at least in part, because of the high expression level of CES2 in the intestine and the consequent intestinal conversion of this prodrug. Therefore, CES2 inhibitors that can prevent the local conversion of this prodrug may have therapeutic potential. In addition, CES2 may also be involved in the metabolism of endogenous substrates, such as lipids 20) 21) . Thus, CES2 inhibitors could be useful tools to reveal the roles of CES2 that are currently unknown.
In the past few decades, many compounds have been reported to possess inhibitory activity against CES2 1) . However, most of them are reversible inhibitors with poor potency and selectivity. A recent study discovered that sofosbuvir, an anti-hepatitis C virus (HCV) drug, is a potent and covalent CES2 inhibitor 22) . This study was performed based on the evidence that coadministration of sofosbuvir and anti-HIV drugs such as tenofovir disoproxil cause severe liver or kidney toxicity, which suggests a potential drugdrug interaction 23)-27) . These regimens are often used to treat patients coinfected with HCV and HIV. Anti-HIV drugs such as tenofovir disoproxil contain ester and/or amide bonds that could be hydrolyzed by CES. Indeed, tenofovir disoproxil was converted to its hydrolytic metabolite tenofovir when incubated with lysates from CES2-transfected cells, and sofosbuvir inhibited this conversion 22) . Further studies using biochemical and chemical biological techniques, such as activity-based protein profiling (ABPP), should be performed to fully characterize the specificity and efficacy of this drug in vitro and in vivo.

Methods to measure CES2 activity
Several methods to measure the enzymatic The anticancer prodrug irinotecan [camptothecin-11 (CPT-11)] contains an ester bond that is hydrolyzed by CES2 to produce its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). activity of CES2 have been reported. Such methods include an in vitro enzyme assay using CPT-11 as a substrate and monitoring the production of SN-38 by liquid chromatography coupled with mass spectrometry 28) . In addition to these in vitro methods, several chemical biological methods using fluorescent probes that enable CES2 activity monitoring in living cells and tissues have been recently developed 29) 30) . Another chemical biology technique referred to as ABPP can be also used to evaluate CES2 activity 15) 20) 31) . ABPP utilizes fluorophosphonate probes that can covalently label serine hydrolases in an activity-dependent manner 32) (Figure-2). The probe-bound serine hydrolases can be separated by SDS-PAGE and their activity can be visualized by fluorescence imager ( Figure-2A, B) or they can be affinity-purified and analyzed by proteomics (Figure-2C). Since ABPP simultaneously visualizes and detects multiple serine hydrolase activities, this method is especially useful for the identification of dysregulated serine hydrolase activities in pathophysiological conditions ( Figure-2A) and off-targets of serine hydrolase inhibitors (Figure-2B). Indeed, using this method, we successfully measured CES2 activities of human colorectal tissues and showed that not only CES2 expression but also its activity was downregulated in human colorectal cancer 15) . Therefore, ABPP and other chemical biological techniques are useful for evaluating CES activities in complex biological matrices.

Conclusions
Despite decades of extensive research on CES2, there are a number of questions that remain unanswered. The decrease in CES2 expression has been reported in many types of cancers, which could be partially explained by recent findings regarding the regulation of CES2 expression by a tumor suppressor p53. However, our recent study showed that the reduction of CES2 expression in human colorectal cancer was independent of TP53 genotypes 15) . Therefore, CES2 expression appears to be regulated by a variety of mechanisms. The endogenous substrates and products of CES2 are also poorly understood, which is mainly due to lack of suitable animal models. There are six CES genes in humans, whereas mice have twenty CES genes, which makes mouse/human ortholog assignment challenging 33) . Further development of tools, such as a transgenic mouse expressing a human CES2 gene and highly potent and selective in vivo active CES2 inhibitors, would be required to answer these remaining questions regarding CES2.