Elsevier

Biochemical Pharmacology

Volume 85, Issue 3, 1 February 2013, Pages 439-447
Biochemical Pharmacology

Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs

https://doi.org/10.1016/j.bcp.2012.11.026Get rights and content

Abstract

Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses.

Introduction

Obesity is probably the most important health issue and associated with a wide range of health conditions such as cardiovascular diseases, type II diabetes and certain types of cancers [1], [2], [3]. In Great Britain, obesity crisis has risen 10 times during the past decade [4]. In the United States (US), the direct cost of obesity is estimated as many as 10% of all medical cost [5]. Among the US population, more than 30% are considered obese and almost 70% are overweight. The prevalence of obesity is slightly lower in men than women, but the prevalence in men has shown a clear trend of increase during the past decade. Importantly, obesity is no longer an issue of adults only, and the prevalence has reached an all-time high in children and adolescents [6], and more than 10% of these populations are considered obese or overweight. Overweight children have much higher chance of becoming obese adults.

Obesity and overweight are caused by a combination of excessive food intake and lack of exercise. Clearly, changes in life styles probably represent the most effective approach to lose weight [7]. Intervention with medication has been increasingly used [8]. Orlistat is widely used for this purpose and marketed under prescription and over-the-counter [9]. The mechanism of action is to inhibit pancreatic and functionally related lipases. Pancreatic lipases are normally secreted into the lumen of the small intestine and hydrolyze triglycerides [11]. Lipids are absorbed upon hydrolysis, and inhibition of the lipases reduces the hydrolysis, thus decreasing the fat intake. In addition to weight loss, orlistat has been shown to improve conditions such as hypertension and type II diabetes [12]. On the other hand, orlistat has been implicated with severe liver toxicity [13].

Lipases, like carboxylesterases (CESs), structurally belong to the α/β fold hydrolase superfamily [10]. While CESs hydrolyze drugs and other xenobiotics, some CESs hydrolyze lipids as well [14], [15]. In the human genome, seven CES genes exist with one being a pseudogene [16], [17]. Nonetheless, only three human CESs are catalytically characterized: CES1, CES2 and CES3 [17]. CES3 is much weaker enzyme in terms of metabolizing common drugs. In a proteomic study, CES1 was identified as one of the top ten most abundant proteins in the adult liver [18]. More importantly, CES1 but not CES2 has been shown to hydrolyze neutral lipids [15]. In contrast, CES2 preferably hydrolyzes bulky molecules such as the anticancer agents: irinotecan [19] and pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) [20], [21].

This study was performed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. To test this hypothesis, recombinant CESs from human, mouse and rat were incubated with orlistat at various concentrations (1–100 nM) and the hydrolytic activity was determined. While orlistat inhibited all CESs tested, the relative potency varied markedly. Contrary to the hypothesis, CES2 was much more sensitive than CES1. Among all CESs, human CES2 and mouse ces2c were inhibited to the most extent and represent two of the most sensitive targets of orlistat. The inhibition was irreversible and occurred intracellularly. Orlistat inhibited the activation of PPD and irinotecan, thus decreasing its cell killing activity.

Section snippets

Chemicals and supplies

Cycloheximide (CHX), p-nitrophenylacetate, 1-naphthylacetate, orlistat and Hanks balanced salt solution were from Sigma (St. Louis, MO). Dulbecco's modified eagle medium (DMEM) and high fidelity Platinum Taq DNA polymerase were from Life Technologies (Carlsbad, CA). The antibody against glyceradehyde-3-phosphate dehydrogenase (GAPDH) was from Abcam (Cambridge, UK). The goat anti-rabbit IgG conjugated with horseradish peroxidase was from Pierce (Rockford, IL). Nitrocellulose membranes were from

Orlistat is a potent inhibitor of human and mouse but not rat CESs

Orlistat targets gastrointestinal lipases with high selectivity [29]. However, clinical observations suggest that this drug acts on other targets that may confer beneficial or adverse effects [13]. In this study, we made an effort to determine whether orlistat inhibits CESs, a class of enzymes that are structurally related to lipases. Pooled liver microsomes from humans, mice or rats were incubated with orlistat at various concentrations (0–1000 nM) and tested for hydrolytic activity. As shown

Discussion

For more than a decade, orlistat has been a major anti-obesity drug [9]. In addition to weight loss, orlistat is shown to improve metabolic syndrome in general [12]. In other cases, orlistat has been linked to certain organ toxicity as well [13]. The molecular targets responsible for the organ toxicity remain to be determined. In this study, we have characterized a novel target of orlistat: CES2, a major enzyme in drug metabolism and detoxication of xenobiotics [21]. Inhibition of CES2 by

Conflicts of interest

The authors indicate no potential conflict of interest.

Acknowledgements

This work was supported by NIH grants R01GM61988 and R01ES07965 to BY and R21CA143549-01A1 to THK. Further support to THK came from the Colorado Center for Drug Discovery.

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