Original article
Effect of buffer components and carrier solvents on in vitro activity of recombinant human carboxylesterases

https://doi.org/10.1016/j.vascn.2007.11.003Get rights and content

Abstract

Introduction

The effects of buffer and substrate solvent conditions on in vitro activity of carboxylesterases (CE) have not been previously described. Therefore, it is unknown if the many different assay conditions used by various laboratories have a substantial impact on the activity of CE enzymes.

Methods

Three human CEs were expressed and purified, and the hydrolysis of 4-nitrophenyl butyrate was measured to assess enzyme activity. Four buffers (HEPES, potassium phosphate, sodium phosphate, and Tris) were evaluated for their effects on enzyme activity at concentrations ranging from 5 to 900 mM, as well as phosphate buffered saline. Five commonly used substrate-carrier solvents (acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol) ranging from 0.25 to 6% were also assessed for their effect on enzyme activity.

Results

The clearances for the CEs in HEPES, potassium phosphate, sodium phosphate, and Tris up to 100 mM were similar to the CE clearances obtained with phosphate buffered saline. Higher buffer concentrations resulted in differential activity of the CEs. All three CEs tolerated the substrate solvents up to 2% as indicated by little effect of solvent on catalytic activity. At substrate solvent concentrations above 2% the CE activities were found to gradually decrease. In general, CES3 displayed substantially lower activity than CES1 and CES2.

Discussion

In conclusion, any of the buffers examined up to 100 mM resulted in clearance values similar to that of phosphate buffered saline for the hydrolysis of 4-nitrophenyl butyrate by the human CEs. With regard to the substrate solvents tested, acetone, acetonitrile, or dimethyl sulfoxide appear to be well tolerated by the CEs up to 2% of the total reaction volume.

Introduction

While the carboxylesterases (CEs) are important for xenobiotic detoxification and prodrug activation (Satoh & Hosokawa, 2006), these enzymes have not been studied as extensively as other Phase I enzymes, such as the cytochrome P450s. However, interest in the CE family has increased in recent years, due in part to the sequencing of the genomes of many species and continued pursuits at understanding the structural elements of their active sites involved in hydrolysis (Bencharit et al., 2006) and substrate selectivity (Imai, 2006). The CEs are a multigene family of serine hydrolase enzymes found in organisms ranging from bacteria to mammals (Redinbo & Potter, 2005). These enzymes hydrolyze compounds containing ester, amide, or thioester linkages. In mammals, the substrates are both endogenous (i.e., acyl-glycerols and acyl-CoA esters) and exogenous (i.e., cocaine and heroin).

Interest in the CEs range from the design of herbicides with selective toxicity (Gershater, Sharples, Edwards, 2006) to the potential treatment for drug overdose, addiction, and chemical warfare, as well as prodrug therapy (Redinbo & Potter, 2005). It has been proposed that an engineered CE could be injected to reduce cocaine exposure quickly, while a CE inhibitor could be administered to decrease the conversion of heroin to its active metabolite, morphine (Redinbo & Potter, 2005). CEs are required for the activation of a number of prodrugs, such as irinotecan (CPT-11), an anti-cancer agent, which is converted to its active metabolite SN-38 (Humerickhouse, Lohrbach, Li, Bosron, Dolan, 2000). On the other hand, some drugs, such as aspirin and clopidogrel (Tang et al., 2006), are inactivated by hydrolysis. Thus, a thorough understanding of these enzymes could assist endeavors ranging from agricultural to medicinal pursuits.

To understand fully the CE enzymes, a routine and robust in vitro system to measure catalytic activity would provide significant insight into the interactions between the enzymes and their substrates. While the CEs are typically found bound to the endoplasmic reticulum (Satoh & Hosokawa, 2006), in some species a soluble form is also known to circulate in the plasma (Li et al., 2005). When a membrane-bound form of human CES1 is expressed as a soluble form, there does not appear to be an alteration in the activity of the enzyme (Scott, Chacko, Maxwell, Schlager, Lanclos, 1999). An example of the benefit of an in vitro system to elucidate the full kinetics between a substrate and CE is that of prasugrel with human CES2 (Williams et al., 2007). Unique kinetics are observed with the purified CES2 enzyme at high substrate concentrations that are not observed in vivo. Therefore, significant benefits exist for conducting in vitro assays to further knowledge of the pharmacological and potential toxicological significance.

Each mammalian species may have several CE forms that are expressed throughout the organism, with each form having some tissue-specific expression. In humans, three forms of CEs (CES1, CES2, and CES3) have been identified and two other genes (CAUXIN — carboxylesterase-like urinary excreted protein and AADAC-arylacetamide deacetylase) have been recently added as potential CEs (Satoh & Hosokawa, 2006). Two of the forms (CES1 and CES2) have undergone extensive investigation, and both have their highest mRNA expression in the liver (Satoh et al., 2002). Extrahepatic mRNA expression of CES1 has been detected in the stomach, testis, kidney, spleen, and colon, in decreasing order (Satoh et al., 2002). On the other hand, extrahepatic CES2 mRNA has also been identified in the colon, small intestine, and heart, in decreasing order. While CES3 was initially cloned from the brain (Mori, Hosokawa, Ogasawara, Tsukada, Chiba, 1999), its mRNA is expressed in the liver (Quinney et al., 2005), but its protein expression has not been shown since an antibody does not yet exist.

To date, there has not been a report systematically determining the incubation conditions that yield the highest intrinsic clearance when conducting in vitro metabolism experiments with the human CE forms. In the literature, an inconsistent use of incubation conditions is found, including 50 mM sodium phosphate (Böttcher, Brüsehaber, Doderer, Bornscheuer, 2007); 90 mM KH2PO4 and 40 mM KCl; 50 mM NaH2PO4 (Pindel et al., 1997); 50 mM HEPES (Scott et al., 1999); and 20 mM Tris (Nishi et al., 2006). In theory, these enzymes only require an aqueous environment in which to function (Satoh & Hosokawa, 2006). Thus, the current study considers the impact of four major variables of in vitro assay conditions, reaction buffer, substrate solvent, and the relative concentrations of each, that affect substrate hydrolysis in vitro by the human CEs.

Section snippets

Expression and purification of recombinant protein

CES1 cDNA was obtained by PCR from human liver cDNA (cat. no. 639307; BD Biosciences Clontech; Mountain View, CA) using PfuTurbo DNA polymerase (cat. no. 600250; Stratagene; La Jolla, CA) with a 5' primer (CGAGAACTGTCGCCCTTCCACGAT) and 3' primer (CAGACAATTCCCCAGCCATGGTAAGATG). Supplemental Fig. 1 contains the cDNA sequence obtained for CES1. The cDNAs for CES2 and CES3 were purchased from OpenBiosystem Co. (cat. no. MHS1011-7508842; Huntsville, AL) and Invitrogen (cat. no. 6145696; Carlsbad,

Results and discussion

Since the goal of this work was to arrive at robust assay conditions to routinely determine the catalytic activity of the CEs, the use of a single probe substrate is consistent with the recommendation made by PhRMA for several Phase I and Phase II enzymes (Bjornsson et al., 2003). Of the probe substrates used for the CEs, two common probe substrates of multiple forms are 4-nitrophenyl acetate (Li et al., 2005, Ross et al., 2006) and 4-nitrophenyl butyrate (4-NPB) (Soni et al., 2004, Ross et

Acknowledgments

The authors thank Barbara Ring, Michael Mohutsky, and Kevin Guo (Eli Lilly and Company) for their thoughtful discussion of the work, Jingqi Bao and Doreen Gillespie (Eli Lilly and Company) for their technical assistance, and Henry Strobel (University of Texas Health Science Center at Houston) for his editorial assistance. All funding was provided by Eli Lilly and Company.

References (22)

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