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CELLULAR AND MOLECULAR
Endocrine Research, Lilly Research Laboratories, Division of Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana
Received July 12, 2004; accepted September 8, 2004.
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Abstract |
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(CPT-1) is a key regulator of the oxidation of long-chain fatty acids in skeletal muscle and therefore a potential therapeutic target for diseases associated with defects in lipid metabolism such as obesity and type 2 diabetes. C75 [4-methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic acid] is an 
-methylene-butyrolactone that has been characterized as both an inhibitor of fatty acid synthase and more recently, an activator of CPT-1 (Thupari et al., 2002
). Using human CPT-1 expressed in the yeast Pichia pastoris, we demonstrate that C75 can activate the skeletal muscle isoform of CPT-1 and overcome inactivation of the enzyme by malonyl CoA, an important physiological repressor of CPT-1, and the malonyl CoA mimetic Ro25-0187 [{5-[2-(naphthalen-2-yloxy)-ethoxy]-thiophen-2-yl}-oxo-acetic acid]. We also show that C75 can activate CPT-1 in intact hepatocytes to levels similar to those achieved with inhibition of acetyl-CoA carboxylase, the enzyme that produces malonyl CoA. Finally, we demonstrate that concentrations of C75 sufficient for activation of CPT-1 do not displace bound malonyl CoA. We conclude that CPT-1 is an activator of human CPT-1 and other CPT-1 isoforms but that it does not activate CPT-1 through antagonism of malonyl CoA binding.
). This reaction is a principal regulatory step in the metabolism of acyl CoAs because trans-esterification to acyl-carnitine is required for their entry into mitochondria and subsequent oxidation (McGarry and Brown, 1997; Zammit, 1999; Eaton et al., 2001). CPT-1 resides in the outer mitochondrial membrane, and mapping studies suggest it contains two transmembrane regions and a short intramitochondrial loop. A large C-terminal catalytic domain and a short N-terminal domain face the cytosol (Fraser et al., 1997). There are two known isoforms of CPT-1. CPT-1 (88 KDa, rat) is the predominant isoform in skeletal muscle, heart, adipose, and testis, whereas CPT-1 (88 KDa, rat) predominates elsewhere (McGarry and Brown, 1997). Both CPT-1 isoforms are regulated by the binding of malonyl CoA, the product of acetyl CoA carboxylase, to a high-affinity binding site (McGarry et al., 1978; McGarry and Brown, 1997; Zammit, 1999). Removal of the short N-terminal domain, or selected mutagenesis within it, disrupts malonyl CoA inhibition of CPT-1 (Cohen et al., 1998; Swanson et al., 1998; Jackson et al., 2000; Shi et al., 2000).
In human and rodent skeletal muscle, malonyl CoA levels, activity, and therefore, presumably, CPT-1 correlate very strongly with utilization of long-chain acyl CoAs as fuel (Bavenholm et al., 2000; Chien et al., 2000). Low levels of lipid oxidation are associated with and predictive for obesity in both humans and rodents (Zurlo et al., 1990; Pagliassotti et al., 1997), and decreased skeletal muscle CPT-1 activity has been demonstrated in obese patient populations (Simoneau et al., 1999; Kim et al., 2000). In rodents, lowering skeletal muscle malonyl CoA levels through genetic deletion or pharmacological inhibition of acetyl CoA carboxylase results in increased lipid oxidation, decreased adipose mass, and protection from diet-induced obesity and diabetes (Abu-Elheiga et al., 2001, 2003; Harwood et al., 2003). In rodents, inhibition of CPT-1 with the catalytic site inhibitor etomoxir results in accumulation of fat mass and pronounced insulin resistance (Dobbins et al., 2001). Together, these studies suggest that mitigating the inhibition of CPT-1 by malonyl CoA may have beneficial effects in obese patients.
The characterization of C75, an -methylene-butyrolactone inhibitor of fatty acid synthase (Kuhajda et al., 2000), as a CPT-1 activator was of considerable interest because of the potential for agents that enhance CPT-1 activity to treat obesity (Thupari et al., 2002). Treatment of rodents by either intraperitoneal or intracerebroventricular administration of C75 causes pronounced anorexia and loss of fat mass (Loftus et al., 2000). This anorectic effect of C75 has been proposed to be due to inhibition of fatty acid synthase in the central nervous system (Loftus et al., 2000). Thupari and colleagues also observed that C75 treatment caused increased energy expenditure and weight loss beyond that seen in pair-fed controls. They proposed that the additional weight loss seen with C75 was due to the direct effects on CPT-1 in the periphery. In support of this, it was shown in semipermeabilized cells that C75 increased the activity of CPT-1 several-fold, with a concomitant increase in palmitate oxidation (Thupari et al., 2002). C75 was also reported to increase CPT-1 activity in the presence of inhibitory concentrations of malonyl CoA (Thupari et al., 2002). In an attempt to understand mechanism of CPT-1 activation by C75, we have used human CPT-1 expressed in Pichia pastoris to measure CPT-1 activity and malonyl CoA binding in the presence of C75.
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Materials and Methods |
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was obtained from Dr. G. Hofle (Gesellschaft fur Biotechnologische Forschung, Baunschweig, Germany). C75 was purchased from Alexis Corporation (Lausen, Switzerland); L-[methyl-3H]carnitine hydrochloride and [14C]malonyl CoA were purchased from Amersham Biosciences Inc. (Piscataway, NJ). Structures of compounds are shown in Fig. 1. Dulbecco's modified Eagle's medium and Hanks' balanced salt solution (HBSS) were from Invitrogen (Carlsbad, CA). Ready Solve HP scintillation cocktail was from Beckman Coulter (Fullerton, CA).
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Expression of Human CPT-1 in P. pastoris. The full-length cDNA of human CPT-1 was generated by PCR using human skeletal muscle Marathon-Ready cDNA (BD Biosciences Clontech, Palo Alto, CA) as a template. The primers used for PCR amplification were the following: forward, 5'-AGGCTCGAGAATAATGTCTGCGGAAGCTCACCAGGCCGTGG-3'; and reverse, 5'-AGGGCGGCCGCGCTGTAGGCCTTGGGAACTTGG-3'. The resultant PCR product was ligated into the XhoI/NotI sites of expression vector pPICZ B (Invitrogen). The identity of human CPT-1 construct was confirmed by DNA sequencing. The plasmid was linearized with the restriction enzyme SstI and transformed to P. pastoris strain GS115 by electroporation with a Gene Pulser II system (Bio-Rad, Hercules, CA). Ten zeocin-resistant colonies were chosen for characterization using the CPT-1 activity assay. One clone, pPICZB-hCPT-1-N8, which showed a high level of enzymatic activity, was used for subsequent experiments. P. pastoris strain GS115 transformed with vector pPICZ B was used as a negative control.
Preparation of P. pastoris Mitochondria. A single colony of hCPT1-N8 was inoculated in buffered minimal glycerol medium with 1.34% yeast nitrogen base without amino acids/1% glycerol/100 mM potassium phosphate, pH 6.0/4 x 10–5% biotin/0.004% histidine and grown with shaking overnight at 30°C. The yeast cells were pelleted, the supernatant was removed, and the pellet was resuspended to an OD600 = 1.0 in buffered minimal methanol medium (same as buffered minimal glycerol medium but with 0.5% methanol and without glycerol) for protein induction.
Highly enriched yeast mitochondria were prepared as described previously (Glick and Pon, 1995; Jackson et al., 1999) with some modifications. Yeast cells were harvested and washed once with distilled water. The yeast pellet was resuspended in 1.2 M sorbitol and 20 mM potassium phosphate buffer, pH 7.4, (2 ml/g of cells) containing 1 mg/ml Zymolase 20T (Seikagaku Corporation, Tokyo, Japan) and incubated at 30°C for 1 h. The yeast spheroplasts were collected by centrifugation at 2000g for 5 min and mixed with an equal volume of acid-washed glass beads (0.5 mm; Sigma-Aldrich). The yeast spheroplasts were broken by several cycles of vortex mixing and incubation on ice. The yeast mitochondria were extracted by adding 0.5 volumes of buffer (50 mM sodium phosphate, pH 7.4, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM EDTA, and 5% glycerol), vortex mixing, and centrifuging at 2000g for 5 min. The supernatants containing the mitochondria were either assayed immediately or stored at –80°C.
Preparation of Rat Heart Mitochondria. Rat housing and handling protocols were approved by an institutional animal care committee and are in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Rats were anesthetized with an i.p. injection (6 mg/100 g of body weight) of pentobarbital sodium and killed by cervical dislocation. Hearts were quickly removed and placed in ice-cold sucrose buffer (250 mM sucrose, 0.5 mM EDTA, 10 mM Hepes, 0.5% BSA, and Roche Complete Protease Inhibitor, pH 7.0). Hearts were finely minced with a scalpel and homogenized in a 15-ml glass Dounce homogenizer (three hearts per 10 ml of buffer). The homogenate was centrifuged at 1200g for 5 min at 4°C, and the supernatant was removed. The homogenate was resuspended with an additional 10 ml of buffer and centrifuged again at 1200g for 5 min. The supernatants were pooled and centrifuged in a Beckman SW-40 at 8000 rpm for 20 min at 4°C to pellet mitochondria.
CPT Activity Assay. CPT activity was assayed radiochemically with either L-[methyl-3H]carnitine hydrochloride or L-[methyl-14C]carnitine hydrochloride in the direction of palmitoylcarnitine formation (Bremer et al., 1985). The assay buffer contains 50 mM KCl, 5 mM Tris-HCl, 1 mM Mg2Cl, 0.1% fatty acid-free BSA, 1 mM KCN, 0.1 mM ATP, and 0.5 µM palmitoylCoA, pH 6.0. Five micrograms of mitochondria and 0.1 µM[3H]carnitine were used in a total reaction volume of 150 µl. The reaction was carried out at room temperature for 10 min. The reaction mixtures were applied to a PVDF filtration plate (Millipore, Bedford, MA) after adding 50 µl of 5 N HCl to terminate the reaction. The filtration plate was washed three times with a total of 600 µl distilled water. The radioactive palmitoylcarnitine was counted with a Microbeta counter (PerkinElmer Life and Analytical Sciences, Boston, MA) after drying plate at 60°C and adding Optiphase Supermix scintillation cocktail (PerkinElmer Life and Analytical Sciences). Filtration capture of labeled palmitoylcarnitine gave similar results to those obtained by butanol extraction (McGarry et al., 1978; data not shown) but allowed higher assay throughput. For determination of KM of carnitine, 10 mM cold carnitine was mixed with [3H]carnitine, and the final concentration ranged from 1 to 500 µM.
Palmitoylcarnitine Synthesis Assay. Palmitoylcarnitine formation was also analyzed in human hepatocellular carcinoma cells (Hep G2). Hep G2 cells were seeded in 96-well cell culture plates at a density of 4 x 104 cell/well and cultivated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C with 5% CO2 for 24 h. Fifty microliters of compound in HBSS was added to triplicate wells after removing the culture media and washing once with HBSS. L-[Methyl-3H]carnitine hydrochloride or L-[methyl-14C]carnitine hydrochloride was also added at final concentration of 0.1 µM. After 2-h incubation, the reaction mixtures were removed, and the cells were washed with phosphate-buffered saline. The cells were lysed by adding 200 µl of distilled water. The cell lysates were either applied to a PVDF filtration plate or analyzed by thin layer chromatography (data not shown). The filtration procedures are same as the CPT activity assay. Dimethyl sulfoxide was used as vehicle control.
Malonyl CoA Binding. Malonyl CoA binding was measured essentially as described previously (Shi et al., 1998, 1999). Briefly, isolated mitochondria from transfected and untransfected yeast were suspended in 0.350 ml of ice-cold binding buffer consisting of 72 mM sorbitol, 60 mM KCl, 25 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, and 1.3 mg/ml fatty acid-free BSA (pH 6.8). The mitochondria were added to 0.1 ml of [14C]malonyl CoA (final concentration 350 nM) and 0.1 ml of compound from 30 nM to 100 µM. The suspension was incubated on ice for 30 min with periodic mixing followed by a 30-min spin at 20,000g at 4°C. Buffer was decanted, and the resulting pellet was carefully washed with 0.5 ml of ice-cold binding buffer and centrifuged again as described above. After decanting the 0.5 ml of buffer, the pellet was resuspended in 0.3 ml of 2% SDS, and the entire contents were electrotransferred to 10 ml of ReadySolve HP scintillation cocktail for radioactive counting.
Statistical Analysis. Data from CPT activity assays are presented as means ± standard error of the mean from multiple determinations. Data were analyzed using PRISM 3.0 (GraphPad Software Inc., San Diego, CA). Data from binding assays were analyzed using GraphPad PRISM 3.0 using nonlinear regression for sigmoidal dose response with variable slope. Background was removed from all points by subtracting nonspecific binding (100 µM unlabeled malonyl CoA), and percentage of binding was calculated as percentage of maximum radioligand binding (no drug added).
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) because it provided us with a convenient source of human CPT-1, and it is free of CPT-2 activity. Human CPT-1 was cloned and expressed essentially as described previously (Zhu et al., 1997). Intact mitochondria isolated from Untransformed P. pastoris were used as source of CPT-1 for enzymatic studies. A time course of CPT-1 activity shows linear product formation up to the 10-min time point used for measuring compound activity (Fig. 2A). Untransformed yeast mitochondria showed negligible activity. The KM values for the substrates carnitine and palmitoyl CoA were 42.5 and 0.59 µM, respectively (Fig. 2, B and C). The reaction was inhibited by malonyl CoA (IC50 = 0.58 µM) and Ro25-0187 (IC50 = 7.9 µM), a CPT-1 inhibitor whose binding has been mapped to a high-affinity malonyl CoA binding site (Fig. 2D) (Cook et al., 1994; Kashfi et al., 1994; Anderson, 1998).
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Activation of CPT-1 in Isolated Mitochondria. To determine the effect of C75 on the activity of CPT-1 expressed in yeast, we measured the formation of [3H]palmitoylcarnitine in the presence of increasing amounts of the compound. C75 caused a dose-dependent increase in hCPT-1 activity in isolated mitochondria, with a maximum increase of 165% of control (Fig. 3A). Similar activation of CPT-1 was seen with isolated mitochondria from untransfected HepG2 hepatocytes (Fig. 3B). C75 activation of the enzyme in yeast mitochondria was also able to overcome inhibition caused by malonyl CoA and Ro25-0187 (Fig. 3A).
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Activation of CPT-1 in Intact Cells. We next measured CPT-1 activity in intact HepG2 cells in the presence of C75 and two well characterized isoform nonselective inhibitors of acetyl CoA carboxylase. Activity was measured by incubating cells with [3H]carnitine and test compounds, lysing the cells by hypotonic shock and capturing labeled palmitoylcarnitine on a PVDF 96-well filter plate. The assay showed a linear increase palmitoylcarnitine production up to the 2-h time point at which compound effects were measured (Fig. 4A). C75 (5 µM) caused an increase in signal, which was suppressed by the CPT-1 inhibitor etomoxir (Fig. 4B) (Anderson, 1998). A similar C75-dependent increase in palmitoylcarnitine was measured from cell extract using thin layer chromatography (data not shown). C75 caused a similar increase in cellular CPT-1 activity to the natural product ACC inhibitor soraphen (Gerth et al., 1994; Gubler and Mizhrahi, 2003), although C75 is much less potent. The ACC inhibitor CP-640186 (Harwood et al., 2003) also increased CPT-1 activity in this system but not to the extent seen with C75 or soraphen.
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Malonyl CoA Binding. It has been suggested that C75 may act at the malonyl CoA regulatory binding site on CPT-1 as a malonyl CoA mimetic (Thupari et al., 2002). To test this hypothesis, we studied the ability of C75 and Ro25-0187 to displace bound malonyl CoA from human CPT-1 and rat heart CPT-1. Specific [14C]malonyl CoA binding to transfected yeast mitochondria was shown by suppression of binding in the presence of 100 µM unlabeled malonyl CoA and by the lack of binding to mitochondria from yeast transfected with vector alone (Fig. 5A). Saturation binding, performed under conditions that resulted in less than 10% of ligand bound, was not affected by the presence of 20 or 40 µM C75 (Fig. 5A). Competition binding studies using 350 nM [14C]malonyl CoA showed displacement by cold malonyl CoA and Ro25-0187, but not C75 (Fig. 5B). Even 100 µM C75, a concentration well above that needed for activation of CPT-1 in isolated mitochondria and intact cells (Figs. 3, A and B, and 4, B and C), showed no displacement of labeled ligand. Similarly, competition binding studies using mitochondria isolated from rat heart, which expresses CPT-1 predominantly (McGarry and Brown, 1997), showed strong suppression of [14C]malonyl CoA binding in the presence of unlabeled malonyl CoA and Ro25-0187, but not C75 (Fig. 5C).
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Discussion |
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). Using the well established P. pastoris expression system for CPT-1 (Zhu et al., 1997), we have found that C75 can activate the human skeletal muscle isoform of CPT-1 (hCPT-1) in a dose-dependent manner. Activation of hCPT-1 is of particular relevance because skeletal muscle is a major site of lipid utilization, and CPT-1 is therefore a potentially attractive therapeutic target for obesity associated with abnormalities in lipid oxidation. Although we have not analyzed CPT-1 activity in a highly purified system, our results show that C75 can activate human CPT-1 in a yeast membrane that does not contain an endogenous CPT-I/II system. Therefore, it is likely that C75 acts directly on CPT-1 and not on other protein components in the mitochondrial membrane.
In the presence of C75, CPT-1 activity was suppressed by both malonyl CoA and Ro25-0187, a CPT-1 inhibitor that also binds to the malonyl CoA regulatory site (Cook et al., 1994; Kashfi et al., 1994; Anderson, 1998). However, CPT-1 in the presence of either inhibitor still exhibited a dose-dependent increase in activity with C75. Although C75 increases the overall activity of CPT-1, it leaves some measure of regulation by malonyl CoA intact. Nevertheless, the maximum fold-increase in CPT-1 activity was approximately the same in the presence and absence of malonyl CoA. It is also noteworthy that it seems to require somewhat higher concentrations of C75 to achieve maximal CPT-1 activation in the presence of malonyl CoA (Fig. 3A). The cause of this apparent shift in C75 potency is unclear, but we speculate that it may be due to conformation changes in CPT-1, caused by malonyl CoA binding, that influence C75's ability activate the enzyme.
In intact HepG2 hepatocytes, C75 was also able to activate CPT-1, as measured by synthesis of palmitoylcarnitine. This is consistent with previous studies that used digitonin-permeabilized cell systems to assess the effects of C75 on CPT-1 (Thupari et al., 2002). Our studies also showed that C75 enhances CPT-1 activity in intact cells to levels similar to those obtained with two nonisoform selective inhibitors of acetyl CoA carboxylase (Gubler and Mizhrahi, 2003; Harwood et al., 2003). These compounds, soraphen and CP-640186, have been shown to activate CPT-1-mediated lipid oxidation through lowering cellular levels of malonyl CoA, the product of ACC, and thus derepression of CPT-1 (Gubler and Mizhrahi, 2003; Harwood et al., 2003). It is particularly interesting that C75 activates CPT-1 to a similar extent in intact cells as ACC inhibitors that have been shown to increase lipid oxidation rates and cause loss of fat mass in vivo. This suggests that direct pharmacological activation of CPT-1 has the potential to increase lipid oxidation rates to therapeutically meaningful levels.
Displacement binding studies with hCPT-1 in yeast mitochondria demonstrated that concentrations of C75 that markedly activated the enzyme did not displace bound malonyl CoA (Fig. 5). In contrast, Ro25-0187 was able to displace malonyl CoA, a finding that supports earlier studies that used limited proteolysis and dual inhibitor analysis to map Ro25-0187's site of action to a high-affinity malonyl CoA binding site on CPT-1 (Cook et al., 1994; Kashfi et al., 1994). It has been hypothesized that C75 may act as a malonyl CoA mimetic and activate CPT-1 by competing with malonyl CoA for a regulatory binding site (Thupari et al., 2002). This hypothesis was based in part on the work of Cook and colleagues who suggested that a dicarbonyl motif found in a number of small molecules that inhibit CPT-1, such as malonyl, succinyl CoA, and Ro25-0187, is required for binding to a high-affinity site on the enzyme (Cook et al., 1994; Kashfi et al., 1994). Free CoA was shown to bind to a separate lower affinity site (Cook et al., 1994). Our studies show that despite the presence of a similar carbonyl motif on C75, it does not compete for malonyl CoA binding (Fig. 5). Ro25-0187 does compete for malonyl CoA binding and acts functionally the same as malonyl CoA to inhibit CPT-1 activity (Fig. 2D).
CPT-1 activity and inhibition by malonyl CoA have been shown to be highly sensitive to the composition of the enzyme's membrane environment. Mapping studies suggest that CPT-1 resides in the outer mitochondrial membrane with the catalytic domain and a short N-terminal sequence facing the cytosol. Between the cytosolic domains are two transmembrane regions and a short intramitochondrial loop (Fraser et al., 1997; Zammit, 1999). Malonyl CoA binding and inhibition of both CPT-1 and CPT-1 can be disrupted by deletions or point mutations in both the short N terminus and the C-terminal catalytic domain 1 (Cohen et al., 1998; Swanson et al., 1998; Jackson et al., 2000; Shi et al., 2000; Napal et al., 2003). Interestingly, Zammit and colleagues have shown that deletions within the N-terminal region can actually increase sensitivity of CPT-1 to inhibition by malonyl CoA (Jackson et al., 2000). It has also been shown that the changes in the fluidity of the CPT-1 membrane environment can affect not only malonyl CoA sensitivity but also basal enzymatic activity in the absence of malonyl CoA (Kolodziej and Zammit, 1990; McGarry and Brown, 2000). Together, these data point to the importance of CPT-1's interaction with the mitochondrial membrane for its activity and regulation. We speculate that C75 may activate CPT-1 by somehow altering the enzyme's interaction with its transmembrane environment. Because C75 has a very hydrophobic "tail" it may bind transmembrane portions of CPT-1 or insert into the lipid bilayer around the enzyme. Alternatively, C75 may interact with an uncharacterized regulatory domain on CPT-1 that is distinct from the malonyl CoA binding site. Future studies will address the questions of how C75 activates CPT-1 and what effects it has on the enzyme's kinetic properties.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: CPT-1, carnitine palmitoyltransferase-1; C75, 4-methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic acid; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; hCPT, human carnitine palmitoyltransferase; ACC, acetyl-coenzyme-A carboxylase; Ro25-0187, {5-[2-(naphthalen-2-yloxy)-ethoxy]-thiophen-2-yl}-oxo-acetic acid; CP-640186, anthracen-9-yl-[3-(morpholine-4-carbonyl)-[1,4']bipiperidinyl-1'-yl]-methanone.
Address correspondence to: Dr. Craig Hammond, Lilly Research Laboratories, Lilly Corporate Center, Drop Code 0304, Indianapolis, IN 46285. E-mail: hammond_craig{at}lilly.com
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