Carboxylesterase Activities and Protein Expression in Rabbit and Pig Ocular Tissues.

Anam Hammid,John K Fallon,Ari Tolonen,Paavo Honkakoski,Arto Urtti,Giulia Salluce,Philip C Smith,Toni Lassila,Achim Sauer

doi:10.1021/acs.molpharmaceut.0c01154

Abstract

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, via hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (d-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Highlights

Hydrolysis plays a vital role in the metabolism or activation of many clinicaldrugs, such as cancer therapeutics, opioids, and angiotensin-converting enzyme inhibitors, as well as in lipid hydrolysis and the elimination of potentially toxic xenobiotics.[1−3] Among many hydrolytic enzymes, carboxylesterases (CES, E.C. 3.1.1.1) are a group of serine hydrolases that cleave esters, amides, thioesters, and carbamates in numerous tissues including liver, kidney, intestine, placenta, heart, brain, and eye.[4−8] So far, six human, about 20 rodent [including eight Ces[1] (Ces1a−h), eight Ces[2] (Ces2a−h), two Ces[3] (Ces3a−b), and one each of Ces[4] and Ces[5] isoforms],9,10 three pig, and four rabbit CES proteins have been annotated in the Uniprot database
nitrophenyl acetate (NPA) is often used to measure the activities of various hydrolytic enzymes including CESs, Arylacetamide deacetylase (AADAC), acetylcholinesterase, and arylesterase in multiple species.[2,22,23,61]
Mg protein for NPA hydrolysis among most tissue homogenates, while the lowest activity was observed in RPE (17.5 ± 2.7 nmol/min/mg) and the highest in the choroid (81.6 ± 6.8 nmol/min/mg)

Summary

Introduction

Hydrolysis plays a vital role in the metabolism or activation of many clinical (pro)drugs, such as cancer therapeutics, opioids, and angiotensin-converting enzyme inhibitors, as well as in lipid hydrolysis and the elimination of potentially toxic xenobiotics.[1−3] Among many hydrolytic enzymes, carboxylesterases (CES, E.C. 3.1.1.1) are a group of serine hydrolases that cleave esters, amides, thioesters, and carbamates in numerous tissues including liver, kidney, intestine, placenta, heart, brain, and eye.[4−8] So far, six human, about 20 rodent [including eight Ces[1] (Ces1a−h), eight Ces[2] (Ces2a−h), two Ces[3] (Ces3a−b), and one each of Ces[4] and Ces[5] isoforms],9,10 three pig, and four rabbit CES proteins have been annotated in the Uniprot database (https://www.uniprot.org/uniprot/?query= CES&sort=score). CES1 is the major hepatic CES isoform but is present in the lungs, heart, and adipose tissue, while CES2 is quite abundant in the intestine and expressed in the kidney, liver, adrenal, and stomach.[9] The poorly studied CES3 isoform is expressed in the colon, brain, and at lower levels in the liver It has much lower activity than CES1 or CES2 but may participate in irinotecan hydrolysis and in lipolysis in adipocytes.[16,19,22−24] The metabolic roles of CES4 and CES5 are unknown, but they are expressed in neuronal cells.[25−27] CES6 is present in human and mouse cerebellums and is speculated to take part in detoxification within the neural system.[28] Arylacetamide deacetylase (AADAC)[16] is another serine hydrolase and has recently emerged as an important drug-metabolizing enzyme with a substrate specificity overlapping with CESs. It is known to prefer substrates with the smallest acyl moieties (Table 1).[29] The expression of human, rat, mouse, cynomolgus monkey, and dog AADAC has been found in the liver, kidney, and intestine.[27,29]

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