Substrate Report for: Orlistat

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 alpha/beta 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 1nM 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.

Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-A-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

Orlistat (Xenical) is a lipase inhibitor developed by Roche for the treatment of obesity, which has been approved in New Zealand [288631], and several countries in Latin America and the Far East. Roche expects to introduce Xenical in its first markets later this year [287131] Analysts predict that it will be a $$1 billion drug for Roche [247775]. In May 1998, the US FDA issued a letter of approval for orlistat but final approval is still subject to certain conditions, including submission of follow-up safety data from the ongoing clinical trials and agreement on final labeling. Roche expects that this will be achieved by the first quarter of 1999 [287131]. In March 1998, the FDA's Endocrinologic and Metabolic Drugs Advisory Committee tied, 5 to 5, on recommending approval of orlistat [281197]. The EC's Committee on Proprietary Medicinal Products has given positive opinions to orlistat submitted under the centralized approvals procedure [283245]. It was also noticed that further clinical trials would be needed to rule out any links between breast cancer and the drug. In phase III studies, nine female patients taking orlistat developed breast neoplasms compared to one patient in the placebo group. After the 18-month follow-up, Roche found that two more women treated with 120 mg tid, and one more on placebo, developed breast cancer. Roche has estimated the relative risk of acquiring breast cancer through orlistat therapy to be 5.9-fold greater than through placebo, or 3.6-fold greater, including post-treatment follow-up; this risk has been shown to be statistically significant [282157]. In August 1997, Roche withdrew its NDA in order to gain more time to submit further analyses of available data to secure approval. The FDA reported that the data submitted supports orlistat's efficacy, but asked Roche to gather further information on breast cancer cases observed in clinical trials [260694]. The NDA for orlistat was resubmitted by Roche in November 1997 [269450].

Tetrahydrolipstatin (THL, 1a) has been shown to inhibit both mammalian and bacterial alpha/beta hydrolases. In the case of bacterial systems, THL is a known inhibitor of several Mycobacterium tuberculosis hydrolases involved in mycomembrane biosynthesis. Herein we report a highly efficient eight-step asymmetric synthesis of THL using a route that allows modification of the THL alpha-chain substituent to afford compounds 1a through 1e. The key transformation in the synthesis was use of a (TPP)CrCl/Co(2)(CO)(8)-catalyzed regioselective and stereospecific carbonylation on an advanced epoxide intermediate to yield a trans-beta-lactone. These compounds are modest inhibitors of Ag85A and Ag85C, two alpha/beta hydrolases of M. tuberculosis involved in the biosynthesis of the mycomembrane. Among these compounds, 10d showed the highest inhibitory effect on Ag85A (34 +/- 22 microM) and Ag85C (66 +/- 8 microM), and its X-ray structure was solved in complex with Ag85C to 2.5 A resolution. In contrast, compound 1e exhibited the best-in-class MICs of 50 microM (25 microg/mL) and 16 microM (8.4 microg/mL) against M. smegmatis and M. tuberculosis H37Ra, respectively, using a microtiter assay plate. Combination of 1e with 13 well-established antibiotics synergistically enhanced the potency of few of these antibiotics in M. smegmatis and M. tuberculosis H37Ra. Compound 1e applied at concentrations 4-fold lower than its MIC enhanced the MIC of the synergistic antibiotic by 2-256-fold. In addition to observing synergy with first-line drugs, rifamycin and isoniazid, the MIC of vancomycin against M. tuberculosis H37Ra was 65 microg/mL; however, the MIC was lowered to 0.25 microg/mL in the presence of 2.1 microg/mL 1e demonstrating the potential of targeting mycobacterial hydrolases involved in mycomembrane and peptidoglycan biosynthesis.

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 alpha/beta 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 1nM 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.

Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-A-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

Orlistat (Xenical) is a lipase inhibitor developed by Roche for the treatment of obesity, which has been approved in New Zealand [288631], and several countries in Latin America and the Far East. Roche expects to introduce Xenical in its first markets later this year [287131] Analysts predict that it will be a $$1 billion drug for Roche [247775]. In May 1998, the US FDA issued a letter of approval for orlistat but final approval is still subject to certain conditions, including submission of follow-up safety data from the ongoing clinical trials and agreement on final labeling. Roche expects that this will be achieved by the first quarter of 1999 [287131]. In March 1998, the FDA's Endocrinologic and Metabolic Drugs Advisory Committee tied, 5 to 5, on recommending approval of orlistat [281197]. The EC's Committee on Proprietary Medicinal Products has given positive opinions to orlistat submitted under the centralized approvals procedure [283245]. It was also noticed that further clinical trials would be needed to rule out any links between breast cancer and the drug. In phase III studies, nine female patients taking orlistat developed breast neoplasms compared to one patient in the placebo group. After the 18-month follow-up, Roche found that two more women treated with 120 mg tid, and one more on placebo, developed breast cancer. Roche has estimated the relative risk of acquiring breast cancer through orlistat therapy to be 5.9-fold greater than through placebo, or 3.6-fold greater, including post-treatment follow-up; this risk has been shown to be statistically significant [282157]. In August 1997, Roche withdrew its NDA in order to gain more time to submit further analyses of available data to secure approval. The FDA reported that the data submitted supports orlistat's efficacy, but asked Roche to gather further information on breast cancer cases observed in clinical trials [260694]. The NDA for orlistat was resubmitted by Roche in November 1997 [269450].