Mechanistic Characterization of Long Residence Time Inhibitors of

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Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2) Brandon Pabst, Kentaro Futatsugi, Qifang Li, and Kay Ahn Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01096 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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Biochemistry

Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

Brandon Pabst,† Kentaro Futatsugi,‡ Qifang Li,‡ and Kay Ahn*,†,§ Pfizer Worldwide Research and Development, †Cardiovascular and Metabolic Diseases Research Unit, ‡Medicinal Chemistry, 1 Portland Street, Cambridge, Massachusetts 02139.

*Corresponding author: Cardiovascular and Metabolic Diseases Research Unit, Pfizer Worldwide Research and Development, 1 Portland Street, Cambridge, Massachusetts 02139. Email: [email protected]. §Present

address: Molecular and Cellular Pharmacology, Janssen Research and Development,

1400 McKean Road, Spring House, Pennsylvania 19477. E-mail: [email protected].

1

ACS Paragon Plus Environment

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ABSTRACT Diacylglycerol acyltransferase2 (DGAT2) catalyzes the final step in triacylglycerol (TAG) synthesis. Genetic knockdown or pharmacological inhibition of DGAT2 leads to reduction in very-low-density lipoprotein (VLDL) TAG secretion and hepatic lipid levels in rodents, indicating DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia and hepatic steatosis. We have previously described potent and selective imidazopyridine DGAT2 inhibitors with high oral bioavailability. However, the detailed mechanism of DGAT2 inhibition has not been reported. Herein, we describe imidazopyridines represented by (PF06424439, 1) and (2) as long residence time inhibitors of DGAT2. We demonstrate that 1 and 2 are slowly reversible, time-dependent inhibitors, which inhibit DGAT2 in a noncompetitive mode with respect to the acyl-CoA substrate. Detailed kinetic analysis demonstrated that 1 and 2 inhibit DGAT2 in a two-step binding mechanism, in which the initial enzyme-inhibitor complex (EI) undergoes an isomerization step resulting in a much higher affinity complex (EI*) with overall inhibition constants (Ki* values) of 16.7 and 16.0 nM for 1 and 2, respectively. The EI* complex dissociates with dissociation half-lives of 1.2 and 1.0 hr for 1 and 2, respectively. A binding assay utilizing [125I]-labeled imidazopyridine demonstrated that imidazopyridine binding to DGAT2 mutant enzymes, H161A and H163A, dramatically decreased to 11-17% of that of the WT enzyme, indicating that these residues are critical for imidazopyridines to bind to DGAT2. Taken together, imidazopyridines may thus represent a promising lead series for the development of DGAT2 inhibitors that display an unprecedented combination of potency, selectivity, and in vivo efficacy. 2


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Biochemistry

ABBREVIATIONS DGAT, diacylglycerol acyltransferase; DAG, diacylglycerol; DMSO, dimethyl sulfoxide; MAFP, methyl arachidonyl fluorophosphonate; SD, standard deviation; TAG, triacylglycerol; VLDL, very-low-density lipoprotein

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INTRODUCTION The synthesis of triacylglycerol (triglyceride, TAG) serves critical physiological functions, such as intestinal dietary fat absorption and intracellular storage of energy in mammals. However, excess accumulation of TAG in adipose tissue leads to obesity and, in nonadipose tissues, is associated with various pathological conditions such as nonalcoholic steatohepatitis, insulin resistance, and cardiovascular diseases.(1) Diacylglycerol acyltransferase (DGAT) enzymes catalyze the final step in triacylglycerol (TAG) synthesis by transferring the acyl group from acyl-CoA to diacylglycerol.(2) In mammals, two structurally unrelated DGAT enzymes, DGAT1 and DGAT2, have been characterized. DGAT enzymes belong to different gene families that do not share sequence homology.(3, 4) Both DGAT1 and DGAT2 are integral membrane enzymes with potentially multiple transmembrane domains embedded in the membrane bilayer.(5, 6) In humans, DGAT1 is highly expressed in the small intestine and plays a main role in fat absorption.(3, 7) DGAT2 is highly expressed in liver and adipose tissues.(4) It has been demonstrated that DGAT1 and DGAT2 can compensate each other for TAG synthesis, but TAG synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes TAG destined to very-low-density lipoprotein (VLDL) assembly.(8) Mice lacking DGAT1 (Dgat1-/- mice) are viable, have modest reductions in tissue TAGs, and are resistant to diet-induced obesity.(9, 10) In contrast, mice lacking DGAT2 (Dgat2-/- mice) have severe (~95%) reductions in whole body TAGs and die shortly after birth.(11) Due to the lethality of Dgat2-/mice, much of the preclinical data on DGAT2 function is derived from studies using antisense oligonucleotides (ASO) and overexpression.(12-15) In preclinical rodent models, knockdown of DGAT2 by ASO treatment has been shown to lead to reduction in hepatic lipids (diacylglycerol and TAG), hepatic VLDL TAG secretion, and plasma cholesterol, as well as protection against 4


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Biochemistry

diet-induced hepatic steatosis and insulin resistance.(12-14) More recently, pharmacological inhibition of DGAT2 has been shown to recapitulate in vivo efficacy similar to those demonstrated by ASO-mediated knockdown of hepatic DGAT2.(16-18) These data indicate that DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia, hepatic steatosis, and metabolic syndrome. Several DGAT2 inhibitors displaying modest in vitro potency have been reported.(16-24) However, there are only very few literature reports on DGAT2 inhibitors which display in vivo efficacy.(16-18, 24) Herein, we describe a highly reproducible and HTS-compatible DGAT2 assay, where it was critical to inhibit endogenous acyl-CoA hydrolyzing enzyme(s) in the membrane fraction from insect cells commonly used as a source of DGAT2 enzyme. For our search for novel DGAT2 inhibitors, we carried out a high-throughput screening (HTS) of the Pfizer compound library and identified a new chemical series of agents that share an imidazopyridine scaffold. Improvements made to potency and physical/chemical properties engendered the DGAT2 inhibitors 1-3 shown in Figure 1. We have previously reported the imidazopyridines DGAT2 inhibitors that are highly potent and selective with excellent pharmacokinetic properties.(16) In the present study, we report a detailed characterization on the mechanism of DGAT2 inhibition and inhibition kinetics of 1-3 utilizing enzyme kinetic and direct binding methods. We demonstrate that 1 and 2 are reversible, time-dependent inhibitors of DGAT2 with long residence time while 3, despite its structural similarity to 1 and 2, is a fully reversible inhibitor of DGAT2. We also show binding of 1-3 to DGAT2 by establishing a radioligand binding assay utilizing [125I]imidazopyridine as a radioactive ligand with potency similar to inhibition kinetic constants, which verified the binding of 1-3 to DGAT2. 5


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Our findings thus promote imidazopyridines as a promising leading series for developing reversible time-dependent inhibitors of DGAT2 with long residence time that display an unprecedented combination of potency, selectivity, oral bioavailability, and in vivo efficacy. Furthermore, our study demonstrates the importance of mechanism of inhibition studies during structure-activity relationship (SAR) process. This study illustrates that relatively minor modifications in compound series could potentially change the mechanism of inhibition and SAR could be misguided as IC50 values typically utilized under standard assay conditions during SAR does not represent true potency when mechanism of inhibition changes.

Cl

N

O N

N

N

N N

N

Cl

N

O

O

N N

N

N H

N

N

N

N

N

N H

1

2

N H

3

Figure 1. Structures of imidazopyridine DGAT2 inhibitors. MATERIALS AND METHODS Materials. Bac-to-Bac baculovirus expression system with pFastBac1 vector and Sf900II media were from Life Technologies (Grand Island, NY). Wave Bioreactor System 20/50P wave bags were purchased from GE Healthcare (Piscataway, NJ). MgCl2, Dimethyl sulfoxide (DMSO), acetone, fatty acid free bovine serum albumin (BSA), 2-[4-(2-hydroxyethyl)piperazin1-yl]ethanesulfonic acid (Hepes), 2-Amino-2-hydroxymethyl-propane-1,3-diol hydrochloride (Tris-HCl), phosphate buffered saline (PBS), sucrose, and ethylenediaminetetraacetic acid (EDTA)

were

purchased

from

Sigma

Aldrich

6


(St.

Louis,

MO).

3-[(3-

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Biochemistry

cholamidopropyl)dimethylammonio]-1-propanesulfonate

(CHAPS),

methyl

arachidonyl

fluorophosphonate (MAFP), and complete protease inhibitor tablets were obtained from Calbiochem (Gibbstown, NJ), Cayman Chemical (Ann Arbor, MI), and Roche Diagnostics (Indianapolis, IN), respectively. MicroScint-E, Top Seal-A covers, 384-well white Polyplates, and custom-synthesized [1-14C]decanoyl-CoA (50 mCi/mmol) were from Perkin Elmer (Waltham, MA). 1,2-didecanoyl-sn-glycerol and H3PO4 were purchased from Avanti Polar Lipids (Alabaster, AL) and J.T. Baker (Phillipsburg, NJ), respectively. All reagents were of the highest quality commercially available. Compounds 1 and 2 were synthesized as reported in our previous study.(16) Compound 1 (PF-06424439) is commercially available from Sigma Aldrich (catalog number PZ0233). Procedures for the synthesis of compounds 3, 4 , and [125I]4 are described in Supporting Information.

Generation of DGAT2 and Mutant Constructs. Constructs for human diacylglycerol acyltransferase 2 (DGAT2) wild-type and mutants were generated with N-terminal FLAG tag. For the FLAG -tagged DGAT2 constructs, the cDNAs for DGAT2 were custom-synthesized at Genscript (Piscataway, NJ) and cloned into the pFastBac1 vector to generate an N-terminally FLAG-tagged pFastBac1-FLAG-DGAT2 (amino acids 1-388) construct. All DGAT2 constructs were confirmed by sequencing in both directions. DGAT2 Expression and Preparation of the Detergent-Solubilized Membrane Fraction. Recombinant baculovirus for the FLAG-tagged DGAT2 construct was generated in SF9 insect cells using Bac-to-Bac baculovirus expression system according to the manufacturer’s protocol. For the expression of DGAT2, SF9 cells (20 L) grown in Sf900II media were infected with DGAT2 baculovirus at a multiplicity of infection of 1 in a Wave Bioreactor System 20/50P wave bag. After 40 h of infection, the cells were then harvested by centrifugation at 5,000 x g. 7


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The cell pellets were washed by resuspending in phosphate buffered saline (PBS) and collected by centrifugation at 5,000 x g. The cell paste was flash frozen in liquid N2 and stored at -80 oC until needed. Detergent-solubilized membrane fraction was prepared and used as a source of DGAT2 enzyme. All operations below were at 4 oC unless otherwise noted. The cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 250 mM sucrose) including 1 mM EDTA and complete protease inhibitor cocktail at a ratio of 3 ml buffer per 1 g cell paste. The cells were lysed by dounce homogenizer. The cell debris was removed by centrifugation at 1,000 x g for 20 min, and the supernatant was centrifuged at 100,000 x g for 1 h. The resulting pellet was rinsed three times by filling ultracentrifuge tubes to the top with PBS before decanting. The washed pellet was resuspended with gentle stirring for 1 h in lysis buffer containing 8 mM CHAPS at a ratio of 1 mL buffer per 1 g of original cell paste and centrifuged again at 100,000 x g for 1 h. The resulting supernatant was aliquotted, flash frozen in liquid N2, and stored at -80 oC until use. DGAT2 activity assay. DGAT2 activity was determined by measuring the incorporation of the [1-14C]decanoyl moiety into triacylglycerol using [1-14C]decanoyl-CoA and 1,2didecanoyl-sn-glycerol. The radioactive triacylglycerol product generated was quantified following lipid extraction either by scintillation counting or by thin layer chromatography (TLC) separation followed by quantification as described below. DGAT2 reactions that were analyzed by TLC were carried out in 1.5 ml polypropylene tubes in a total reaction volume of 200 l. Reactions were stopped by the addition of 300 l CHCl3:MeOH (2:1, v/v) and extraction of lipids was achieved by vortexing the tubes for 1 min at the highest setting followed by centrifugation at 14,000 rpm for 1 min. The lower organic product layer (50 μL) was loaded onto the TLC plate (Whatman, LK6D silica gel plates, dried at 80 oC in a vacuum oven 8


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Biochemistry

overnight). Radiolabeled lipids were separated using a mixture of ethyl acetate:isopropyl alcohol:CHCl3:MeOH:0.25% KCl (100:100:100:40:36, v/v/v/v/v) as the first solvent system. Once the solvent front migrated 7 cm above the origin, plates were removed and dried under nitrogen. Samples were separated further using a mixture of hexane: diethyl ether: acetic acid (70:27:3, v/v/v) as the second solvent system. Plates were removed and dried under nitrogen after migration of the second solvent front to the top of the plate. The TLC plates were exposed for ~24 hr to a PhosphorImager screen (GE Healthcare, Piscataway, NJ). Bands were visualized and quantitated using PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using a standard curve generated from known amounts of [14C]-labeled standard. Determination of DGAT2 kinetic parameters. To determine the KM values for 1,2didecanoyl glycerol and decanoyl-CoA, all assay components, including 50 mM Hepes, pH 7.4, 10 mM MgCl2, 1 μM MAFP, 3% acetone, 1,2-didecanoyl glycerol, and decanoyl-CoA, in a volume of 160 μL were added to each 1.5 mL polypropylene tube. Assays to determine the KM for 1,2-didecanoyl glycerol contained 60 μM [1-14C]decanoyl-CoA (50 mCi/mmol) and 1.6 - 100 μM 1,2-didecanoyl glycerol. Assays to determine the KM for decanoyl-CoA contained 50 μM 1,2-didecanoyl glycerol and 0.2 - 300 μM [1-14C]decanoyl-CoA. Reactions performed in duplicates were initiated by the addition of 40 μL of DGAT2 (0.09 mg/ml, detergent-solubilized membrane fraction) in 50 mM Hepes, pH 7.4, 10 mM MgCl2, 1 μM MAFP (dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock). Reaction mixtures were vortexed for 30 s and incubated at room temperature (RT) for 30 min. Reactions were stopped by the addition of 50 μL of 1% H3PO4. Extraction of lipids was achieved by the addition 400 μL Microscint-E and tubes were vortexed for 1 min at the highest setting before a centrifugation at 16,000 x g for 1 min. The generated [14C]tridecanolyglycerol was quantitated; 9


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100 μL of the upper organic product layer was added to 5 mL optiphase scintillation fluid and read in a Wallac 1409 liquid scintillation counter (Perkin Elmer) for 1 min. The background activity was determined for each reaction condition in the absence of DGAT2 and was subtracted from each reaction. Under the assay conditions established, the product generated was in the linear portion of the time course under the initial velocity conditions. The KM values for decanoyl-CoA and 1,2-didecanoyl glycerol were determined by plotting the reaction rates (Vo, in M per second) as a function of substrate concentration ([S]) and fitting to the Michaelis– Menten equation 1,

V0 =

Vmax [S] (1)

Km + [S]

where Vmax (M per second) is the maximal rate of the reaction and KM is the concentration of substrate required to reach 1/2Vmax. The kcat value was calculated by dividing the Vmax by the concentration of DGAT2 in the reaction (300 pM). DGAT2 concentration from the membrane fraction was determined by Western blot analysis using the FLAG monoclonal antibody (Sigma Aldrich) and the known amount of purified FLAG-tagged bacterial alkaline phosphatase (Sigma Aldrich) as standards. Determination of IC50 values for DGAT2 inhibitors. For determination of IC50 values for DGAT2 inhibitors, the reactions were carried out in 384-well white Polyplates in a total volume of 20 μL. The final assay mixture contained 50 mM Hepes-NaOH, pH 7.4, 6 μM [114C]decanoyl-CoA,

25 μM 1,2-didecanoyl-sn-glycerol, 10 mM MgCl2, 100 nM methyl MAFP,

0.01% BSA, 5% DMSO, 2.5% acetone, and 0.1 μg of the detergent-solubilized DGAT2 10


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Biochemistry

membrane. To 1 μL of compounds dissolved in DMSO (or DMSO for controls) and spotted at the bottom of each well, 5 μL of 0.04% BSA was added and the mixture was kept at RT for 20 min. To this mixture, 10 μL of the detergent-solubilized DGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM MAFP was added. After this mixture was preincubated at RT for the indicated period of time, DGAT2 reactions were initiated by the addition of 4 μL of substrates containing 30 μM [1-14C]decanoylCoA and 125 μM 1,2-didecanoyl-sn-glycerol dissolved in 12.5% acetone. The reaction mixtures were incubated at RT for 40 min and the reactions were stopped by the addition of 5 μL of 1% H3PO4. After the addition of 45 μL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, CA). Plates were centrifuged at 2,000 x g for 1 min and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase. Various preparations of DGAT2 were carefully titrated so that the final DGAT2 concentrations were under the conditions where the time course of DGAT2 reaction was linear. Background activity obtained using 50 μM of (1R, 2R)-2-({3'-Fluoro-4'-[(6-fluoro-1, 3benzothiazol-2-yl)amino]-1,1'-biphenyl-4-yl}carbonyl)cyclopentanecarboxylic acid (US 20040224997, Example 26) for complete inhibition of DGAT2 was subtracted from all reactions. To determine IC50 values, the data were plotted as percentage of the control relative to the uninhibited reaction versus inhibitor concentration and fit to equation 2,

y=

100

(2)

z

1 + (x/IC50)

11


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where IC50 is the inhibitor concentration at 50% inhibition and z is the Hill slope (the slope of the curve at its inflection point). Determination of DGAT2 inhibition kinetic parameters. The DGAT2 reactions for determination of inhibition rate constants were carried out in 384-well plates using a procedure similar to that described above with minor modifications. Typical preincubation times with the indicated concentrations of inhibitors were 0, 2, 4, 8, 12, 15, 20, 25, 30, 35, 45, 60, 75, 90, 105, and 120 min utilizing approximately 10-fold greater DGAT2 enzyme concentration (1 μg of the detergent-solubilized DGAT2 membrane) compared to that used in IC50 value determinations above. Separate 384-well plates were used for different preincubation times. After initiating the enzyme reaction by adding substrates, the DGAT2 reaction was carried out at RT for 4 min. The concentrations of inhibitors were varied in 1.5-fold increments from 0.25 to 250 nM. Experimental conditions were carefully established so that there is no inhibition observed at inhibitor concentrations employed during the 4-min reaction time. E+I

E+I

k1 k2

k1 k2

EI

EI

Scheme 1

k3 k4

EI*

Scheme 2

For determination of inhibition rate constant kobs value at each inhibitor concentration, the data were plotted as percentage of control versus inhibitor concentration and fit to the pseudofirst-order decay equation 3, 12


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Biochemistry

yt = (y0 – y1) [exp(-kobst)] + y1

(3)

where yt is the measured percent of control after preincubation time t in min, and y0 and y1 are the percent of controls at preincubation time 0 and infinite times, respectively, yielding the first order rate constant for enzyme inactivation (kobs) at each inhibitor concentration. Each kobs value was corrected for autoinactivation of the enzyme by subtracting the uninhibited reaction. The corrected kobs values were then plotted versus inhibitor concentration ([I]) and fit to equation 4 where Kiapp is the apparent value of Ki for the initial enzyme-inhibitor complex.

kobs = k4 +

k3[I]

(4)

Kiapp + [I]

The true affinity of an inhibitor that conforms to a two-step enzyme isomerization mechanism (scheme 2) is defined by the dissociation constant (Ki*app) for the final high-affinity conformation of the enzyme-inhibitor complex given in equation 5. Equation 5 describes the relationship between the dissociation constant Kiapp for the initial EI complex and a second dissociation constant Ki*app for the second enzyme conformation in a two-step binding mechanism (scheme 2).

Ki*app

=

Kiapp k4

(5)

k3 + k4

Substituting equation 5 into 4, equation 4 can be recast to equation 6.

13


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1+ kobs = k4

[I] Ki*app

(6)

[I] 1+ Kiapp

Mode of inhibition by imidazopyridines towards decanoyl-CoA. Experiments to determine the mode of DGAT2 inhibition by imidazopyridine with respect to the decanoyl-CoA substrate were performed similar to those for determining inhibition rate constants above with minor modifications. DGAT2 reactions were performed in a final volume of 200 μL in polypropylene tubes. The assay mixture consisted of 50 mM Hepes-NaOH, pH 7.4, 10 mM MgCl2, 1 μM MAFP, 150 nM PF-439 (or DMSO for controls), and DGAT2 membrane (6 μg). After the mixture was preincubated at RT for the indicated period of time, DGAT2 reactions were initiated by the addition of 6 or 60 μM [1-14C]decanoyl-CoA in the presence of 100 μM 1,2-didecanoyl-sn-glycerol. The DGAT2 inactivation rates (kobs values) at various decanoylCoA concentrations were calculated same as above. Rapid dilution reversibility studies of the imidazopyridine DGAT2 inhibitors. A rapid dilution experiment was conducted to assess the reversibility of DGAT2 inhibitors. In a total volume of 100 L, 5 L of inhibitor dissolved in DMSO at concentrations of 20-fold greater than IC50 values (or DMSO for control) was mixed with 25 μL of 0.04% BSA. To this mixture, 70 μL of the DGAT2 membrane fraction (1.71 mg/mL) containing Hepes-NaOH, pH 7.4, MgCl2, and MAFP was added to final concentrations of 50 mM, 10 mM, and 100 nM, respectively. At the end of preincubation for 1 h, 3 L of the enzyme-inhibitor mixture was rapidly diluted 300-fold into the DGAT2 assay mixture containing substrates, 60 μM [114C]decanoyl-CoA

and 100 μM 1,2-didecanoyl-sn-glycerol. All other assay conditions were 14


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Biochemistry

identical to those described under determination of IC50 values besides substrate concentrations. The reaction mixture was incubated at room temperature for the indicated time and the reactions were stopped by addition of 300 μL of 1% H3PO4. Phase partitioning of substrate and product was achieved by the addition of 500 μL Microscint-E scintillation fluid and vortexing at the highest setting for 1 min followed by centrifugation at 16,000 x g for 1 min. DGAT2 activity was determined by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase; 100 μL of the organic layer was transferred to vials for counting. Optiphase Supermix (5 mL, Perkin Elmer) was added to the vials, which were counted for 1 min in a Wallac 1409 liquid scintillation counter (Perkin Elmer). Radioligand binding assay. A radioligand binding was developed using a [125I]-labeled inhibitor 4 ([125I]4). The dissociation constant of radioligand (Kd value) was measured in saturation binding experiments. For saturation binding, increasing concentrations of the radioligand [125I]4 (2,200 Ci/mmol) at concentrations varying by 1.5-fold between 0.20 and 130 nM (or DMSO for controls) was incubated in 96-well polypropylene BD Falcon plates in a total volume of 100 l containing 100 g of DGAT2 membrane in 50 mM Hepes, pH 7.4, 10 mM MgCl2, 1 M MAFP, and 10% DMSO. The resulting mixtures were incubated while being gently mixed in a shaker for 3 h at RT, filtered using a Perkin Elmer FilterMate Harvester with unifilter GC/F plates, and washed fifteen times with 200 l of ice-cold binding buffer (20 mM Hepes, pH 7.4). Filter plates were left to dry overnight at RT. After addition of 30 l optiphase scintillation fluid, plates were sealed and counted using a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). Non-specific binding was determined in parallel in the presence of 4 at 200 M, and was subtracted from total binding to determine specific binding. The data were plotted and fit to equation 7. 15


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y =

Bmax[I]

(7)

Kd + [I]

In competition experiments, 5 L of 6 nM [125I]4 in 100% DMSO and 5 L of the compound at varying concentrations dissolved in 100% DMSO were incubated in a total volume of 100 l containing 20 g DGAT-2 membrane under the same conditions described above. RESULTS The DGAT2 membrane expressed in SF9 cells contains carboxy thioesterase(s) that efficiently hydrolyze acyl-CoA substrate. We have utilized the DGAT2 membrane fraction prepared from Sf9 insect cell expression system as a source of DGAT2 enzyme, which has been widely used in the literature.(4, 16-18, 20, 21, 23, 25, 26) DGAT2 activity was determined by measuring the incorporation of the [1-14C]decanoyl moiety into triacylglycerol using [1-14C]decanoyl-CoA and 1,2-didecanoyl-sn-glycerol as substrates. After phase partitioning by the addition of a phase partition scintillation fluid (MicroScint-E, Perkin-Elmer), which serves as both a scintillation fluid and a phase partition agent, the generated product [14C]tridecanoylglycerol ([14C]TAG) was quantified from the upper organic phase.(27) An initial experiment comparing DGAT2 versus mock membranes (1.5 g for each), which was analyzed on TLC, showed that mock membrane generated significant levels of [14C]decanoic acid (1.26 M, 13% hydrolysis of [14C]decanoylCoA) (Fig. 2A, lane 1) while DGAT2 membrane generated 1.73 M [14C]TAG as well as 0.44 M [14C]decanoic acid (Fig. 2A, lane 6) under the DGAT2 assay conditions described under Methods except 10 M [14C]decanoyl-CoA and 30 M didecanoylglycerol were used as substrates in this assay. Since the background activity ([14C]decanoic acid generated by mock 16


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Biochemistry

membrane) is significant (~25% of [14C]TAG) in the DGAT2 reaction, this system would not allow an optimal DGAT2 assay window without separation of [14C]TAG from [14C]decanoic acid. We have speculated that this acyl-CoA hydrolytic enzyme activity is likely due to thioesterase(s) which belong(s) to the serine hydrolase family of enzymes. Therefore, we utilized a general serine hydrolase inhibitor, methyl arachidonyl fluorophosphonate (MAFP),(28) at 0.02 – 2 M to assess whether it can be used to inhibit endogenous thioesterase activity without inhibiting DGAT2 activity. Figure 2A shows that thioesterase activity in both mock and DGAT2 membranes was inhibited to near completion by MAFP at 20-100 nM (lanes 2, 3, 7 and 8). MAFP at 20-500 nM did not affect DGAT2 activity (Figure 2A, lanes 6-9) but partially inhibited DGA2 activity at 2 M (Figure 2A, lane 10). As shown in Figure 2B, denaturation of enzymes by addition of acid to the reaction mixture before initiating the reaction by addition of substrates did not yield [14C]decanoic acid for mock (lane 1) and DGAT2 (lane 6), confirming that the generation of [14C]decanoic acid is due to enzymatic activity, and not chemical hydrolysis of [14C]decanoyl-CoA under assay conditions. This thioesterase activity was inhibited to near completion by the presence of 100 nM MAFP in mock (Figure 2B, lane 3) and DGAT2 (Figure 2B, lane 8) compared to controls in the absence of MAFP (Figure 2B, lanes 2 and 7). [14C]Decanoic acid was generated both in the presence (Figure 2B, lanes 2 and 7) and absence (Figure 2B, lanes 4 and 9) of 1,2-didecanoyl-sn-glycerol (DAG) verifying that, as expected, the thioesterase activity does not require DAG substrate for its activity. Furthermore, inhibition of the thioesterase activity by 100 nM MAFP was not affected by the presence of DAG (Figure 2B, lanes 3 and 8) compared to controls in the absence of DAG (Figure 2B, lanes 5 and 10). A higher level of [14C]decanoic acid was generated by mock (lanes 2 and 4, 1.52 M) 17


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 51

compared to that by DGAT2 (lanes 7 and 9, 0.045 M) in the absence of MAFP in Figure 2B. This is most likely due to competition for [14C]decanoyl-CoA substrate between thioesterase and DGAT2 in the DGAT2 reactions while only thioesterase is utilizing [14C]decanoyl-CoA in the mock reactions. A. Mock 1

2

DGAT2

3

4

5

6

7

8

9

10 TAG Decanoic acid

B.

Mock MAFP

+

-

+

DAG

+

+

1

2

DGAT2 +

+

-

+

+

-

-

+

+

3

4

5

6

7

+

+

-

8

9

10

TAG

Decanoic acid

18


Page 19 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. Inhibition of endogenous acyl-CoA hydrolytic enzyme activity by methyl arachidonyl fluorophosphonate (MAFP). (A) DGAT2 reactions were carried out at RT for 40 min with 10 M [1-14C]decanoyl-CoA and 30 M 1,2-didecanoyl-sn-glycerol as described in the DGAT2 activity assay using Mock and DGAT2 membranes (1.5 g each). The results were analyzed by TLC using the procedure described under Materials and Methods. Lanes 1-5 (Mock) and 6-10 (DGAT2) contained 0, 0.02, 0.1, 0.5, 2 M MAFP, respectively, in DGAT2 reactions. Arrows indicate free fatty acid (decanoic acid) and TAG (tridecanoyl glycerol). (B). DGAT2 reactions were carried out same as in (A). Enzymes were denatured first by the addition of 50 L of 1% H3PO4 before addition of substrates for Mock (lane 1) and DGAT2 (lane 6). DGAT2 reactions were carried out in the absence or presence of MAFP (lanes 2 and 7 for mock and DGAT2, respectively, in the absence of MAFP; lanes 3 and 8 for mock and DGAT2, respectively, in the presence of MAFP). DGAT2 reactions were carried out without 1,2-didecanoyl-sn-glycerol substrate in the absence or presence of 100 nM MAFP (lanes 4 and 9 for Mock and DGAT2, respectively, in the absence MAFP; lanes 5 and 10 for mock and DGAT2, respectively, in the presence of MAFP). Taken together, inhibition of endogenous thioesterase activity in Sf9 insect cells by MAFP allowed us to utilize DGAT2 membranes prepared from insect cell expression and establish DGAT2 assays where the quantitation of [14C]TAG in the organic layer could be performed without separation steps as [14C]decanoic acid generated by endogenous thioesterase in the absence of MAFP also partitions to the organic phase. Importantly, inhibition of carboxy thioesterases by MAFP allowed characterization of DGAT2 under the conditions where one of its substrates decanoyl-CoA is not degraded by other enzymes during DGAT2 reactions (See below). DGAT2 enzyme kinetic parameters. To establish DGAT2 assay conditions for inhibitor screening and characterization, we first determined the KM and kcat values for didecanoylglycerol and decanoyl-CoA. Due to relatively low solubility of substrates, we determined that use of substrates with decanoyl (C10:0) groups for both acyl donor and acceptor yielded more reproducible assay than with those containing oleoyl (C18:1) group as acyl 19


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

donor/acceptor. The Michaelis-Menten plots for the determination of KM and kcat values for both decanoyl-CoA and 1,2-didecanoyl-sn-glycerol are shown in Figure 3A and 3B, respectively. Due to solubility limits of 1,2-didecanoyl-sn-glycerol, the highest concentration used was 100 M. The KM and kcat values for decanoyl-CoA in the presence of 100 M 1,2-didecanoyl-snglycerol were determined to be 4.17 ± 0.80 M and 4.52 ± 0.22 s-1, respectively. The KM and kcat values for 1,2-didecanoyl-sn-glycerol were determined to be ~222 ± 72 M and ~28.7 ± 6.9 s-1, respectively at a saturating decanoyl-CoA concentration (60 M); these are approximate values as the highest concentration used was below the KM value (100 M) for 1,2-didecanoyl-snglycerol. The DGAT2 catalytic efficiency (kcat/KM value) for decanoyl-CoA and 1,2-didecanoylsn-glycerol were 1.08 x 106 and ~1.29 x 105 M-1s-1, respectively. As described in Methods, kcat values were calculated using DGAT2 concentrations determined by using western blot analysis with purified Flag-tagged bacterial alkaline phosphatase as standards. Based on these kinetic parameters, for the inhibitor characterization studies, DGAT2 reactions were conducted with substrate concentrations at 6 M for decanoyl-CoA, which is near the KM value with an optimal assay window, and 25 M for 1,2-didecanoyl-sn-glycerol, which is below the KM value due to solubility limit but yields highly reproducible assay signal. DGAT2 reactions were routinely carried out in 384-well plates under these standard substrate concentrations as described in Materials and Methods. As shown in Figure 3C, DGAT2 reactions were linear for at least ~45 min and resulted in Z’ values of 0.67, 0.72, and 0.63 after the 20, 30, and 40 min reaction, respectively, yielding a robust assay.

20


Page 20 of 51

Page 21 of 51

A.

B. 0.20

[14C]Triacylglycerol (M/min)

[14C]Triacylglycerol (M/min)

0.10 0.08 0.06 0.04 0.02 0.00 0

20

40

60

80

0.16 0.12 0.08 0.04 0.00 0

100

[Decanoyl-CoA] (M)

20

40

60

80

100

[Didecanoylglycerol] (M)

C.

6000 5000

Signal (CPM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

4000 3000 2000 1000 0

10

20

30

40

50

Reaction Time (min)

Figure 3. Determination of DGAT2 enzyme kinetics parameters. Determination of DGAT2 KM and kcat values for decanoyl-CoA (A) and 1,2-didecanoyl-sn-glycerol (B). The reactions were carried out with 100-200 pM DGAT2 (0.5-1 g membrane) and the initial reaction rates were determined as described under Materials and Methods. Data are averages, and error bars represent the SD from two separate experiments. (A) The decanoyl-CoA concentrations were varied from 0.20 to 100 μM with 1,2-didecanoyl-sn-glycerol concentration at a maximum solubility limit of 100 μM. The initial rates were plotted as a function of decanoyl-CoA concentration, and the data were fit to equation 1. (B) The 1,2-didecanoyl-sn-glycerol concentrations were varied from 1.6 to 100 μM with a saturating decanoyl-CoA concentration of 60 μM. Due to solubility limitations of 1,2-didecanoyl-sn-glycerol, the highest concentration of 100 μM was used. The initial rates were plotted as a function of 1,2-didecanoyl-sn-glycerol 21


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

concentration, and the data were fit to equation 1. (C) The DGAT2 reaction were carried out in 384-well plates for the indicated period times at substrate concentrations of 6 and 25 μM for decanoyl-CoA and 1,2-didecanoyl-sn-glycerol, respectively, under the conditions described in Materials and Methods. Compounds 1 and 2 inhibit DGAT2 in a time-dependent manner in contrast to compound 3. We first determined IC50 values of three imidazopyridine inhibitors 1-3 under the standard DGAT2 IC50 assay conditions described under Material and Methods. Without preincubation, all three inhibitors displayed comparable potency in DGAT2 inhibition with IC50 values of 184, 194, and 245 nM for 1 and 2, and 3, respectively (Figure 4A-C and Table 1). As an initial step to characterize the mechanism of action by the imidazopyridine DGAT2 inhibitors 1-3, we assessed time-dependent inhibition by measuring IC50 values following variable preincubation times. DGAT2 was preincubated with inhibitors for 0, 10, 60, and 120 min before initiating the DGAT2 reaction by the addition of the DGAT2 substrates decanoyl-CoA and 1,2didecanoyl-sn-glycerol. Even though all three structurally related imidazopyridine inhibitors had similar potency without preincubation, they displayed marked differences in their timedependence. 1 and 2 exhibited dramatic enhancements in their potency as the preincubation time increased (Figure 4A and B). 1 inhibited DGAT2 approximately 3.0-, 8.5-, and 24-fold more potently with 10-, 60-, and 120-min preincubation time, respectively, compared to its potency with no preincubation time (Figure 4A and Table 1). A similar time-dependent inhibition was observed for 2 (Figure 4B and Table 1). In sharp contrast, 3 did not display time-dependent inhibition of DGAT2 as similar potency was obtained regardless of preincubation time despite its core structure similarity to 1 and 2 (Figure 4C and Table 1).

22


Page 22 of 51

Page 23 of 51

A.

B.

C.

100

% Inhibition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0 10 min 60 min 120 min

50

0 0.1

1

10

100

1000

0.1

10000

1

10

100

1000

10000

[Compound 2] (nM)

[Compound 1] (nM)

0.1

1

10

100

1000

10000

[Compound 3] (nM)

Figure 4. Time-dependent DGAT2 inhibition by imidazopyridine inhibitors. DGAT2 reactions were carried out with preincubation times of 0, 10, 60, and 120 min using the conditions described under “Determination of IC50 values for DGAT2 inhibitors” in Materials and Methods. The data were plotted as a percentage of inhibition versus inhibitor concentration and fit to equation 2 to determine the IC50 values for compound 1 (A), compound 2 (B), and compound 3 (C). The concentrations of inhibitors were varied 2-fold from 0.1 nM to 100 μM. Three separate experimental were performed with similar results, and one experiment was represented. Table 1. IC50 values of DGAT2 inhibitors with various preincubation times. Preincubation Time (min)

IC50 (nM) 1

2

3

0

184

194

245

10

60.9

70.0

323

60

21.7

11.4

260

120

7.66

7.51

313

DGAT2 reactions were carried out in 384-well plates as described under Materials and Methods. The concentrations of inhibitors were varied from 100 M to 0.0954 nM with a 2-fold serial dilution. IC50 values were determined as described in the Figure 4 legend. Reversibility of the imidazopyridine inhibitors (Rapid dilution). Time-dependent inhibition could be carried out by a reversible slow-binding or covalent, irreversible mechanism 23


Biochemistry

of inhibition. To assess whether 1 and 2 acted as reversible or irreversible inhibitors, a rapid dilution experiment was carried out. DGAT2 was incubated with inhibitors (or DMSO as a control) at concentrations approximately 20-fold higher than their IC50 values obtained. Under these conditions, DGAT2 is expected to be completely inhibited. After incubation for 60 min at room temperature, the mixture was rapidly diluted 300-fold with buffer containing a saturating concentration of [14C]decanoyl-CoA (60 M) and 100 M 1,2-didecanoyl-sn-glycerol and the recovery of DGAT2 activity was measured at various reaction times. As shown in Figure 5, DGAT2 inhibition by 3 was rapidly reversible and the recovered activity was indistinguishable from the control reaction preincubated with DMSO, which is expected from a fully reversible inhibitor, confirming no time-dependent inhibition observed above (Figure 4C). In contrast, DGAT2 preincubated with 1 and 2 recovered activity slowly; 60, 54, and 46% of the control activity by 1, and 66, 50, and 50 % of the control activity by 2, at 10, 20, and 40 min, respectively (Figure 5). These data combined with the time-dependent inhibition shown above indicate that 1 and 2 inhibit DGAT2 by a slowly reversible, time-dependent mode of inhibition with long residence time. In contrast, 3 is a fully reversible inhibitor of DGAT2. 10

[14C]Triacylglycerol (M)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8 6

DMSO 1 2 3

4 2 0 0

10

20

30

40

Incubation Time (min)

24


Page 24 of 51

Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. Reversibility of DGAT2 inhibition by imidazopyridine inhibitors. DGAT2 (6.8 g membrane) was preincubated for 60 min with inhibitors (or DMSO for controls) at concentrations approximately 20-fold greater than their IC50 values with 60-min preincubation shown in Table 1. Aliquots of the enzyme-inhibitor mixture were diluted 300-fold into the DGAT2 assay buffer containing saturating substrate concentrations (50 μM [1-14C]decanoylCoA and 100 μM 1,2-didecanoyl-sn-glycerol ), and recovery of DGAT2 activity was measured after incubation for 10, 20, and 40 min. Data are averages, and error bars represent the SD from duplicates. Determination of DGAT2 inhibition kinetic parameters for the imidazopyridine inhibitors. The above results indicated that compounds 1 and 2 possess measurable residence times of inhibition whereas 3 is rapidly reversible. To further characterize time-dependent inhibition of DGAT2 by the imidazopyridine inhibitors, the residual DGAT2 activity was measured after various preincubation times in contact with the indicated concentrations of inhibitors before initiating the reaction with DGAT2 substrates. To ensure the measurement of DGAT2 residual activity, assay conditions were modified where DGAT2 reaction time was reduced to 4 min. To achieve a sufficient assay signal with a 4-min reaction time, enzyme concentrations were increased by ~10-fold. As shown in Figure 6A, 3 showed similar DGAT2 inhibition regardless of the preincubation time as expected from a simple fully reversible inhibitor. In contrast, 1 and 2 exhibited a rapid decrease in DGAT2 activity with increasing preincubation time and the rate of this activity decrease increased with increasing inhibitor concentrations (Figure 6B and C). The data were fit to a pseudo-first-order decay equation 2 to determine kobs values (rate constants for DGAT2 inactivation) at various concentrations of 1 and 2. Plotting these kobs values as a function of inhibitor concentration revealed a straight line as shown in Figure 6D and E. These data indicate two slow binding modes of inhibition; either a single-step binding event, as in scheme 1, or a two-step binding mechanism (scheme 2) for which the first step is a simple equilibrium binding of inhibitor to enzyme to form a complex EI 25


Biochemistry

and the second step is an isomerization of the enzyme to form a higher affinity complex E*I where Kiapp >> Ki*app. To differentiate between these two binding modes, we have determined the IC50 values with no preincubation and very short reaction time (4 min). Under these conditions, the IC50 values are approximately equal to the Kiapp values, which are initial binding dissociation constants in scheme 2 and were obtained to be 3.80 and 1.71 M for 1 and 2, respectively. The corresponding IC50 values with 120 min preincubation, which represent steady state and approximately equal to the Ki*app values, were 11.8 and 8.9 nM for 1 and 2, respectively. This dramatic increase in potency with increasing preincubation times compared to that with no preincubation time observed with 1 and 2 above is consistent with a two-step binding mechanism (scheme 2) for which Kiapp is much greater than Ki*app. Under these circumstances in a two-step binding mechanism (scheme 2), equation 6 is thus reduced to equation 8. kobs = k4 1 +

[I] Ki*app

(8)

A.

B.

100

100 nM 250 nM 750 nM

C.

15 nM 22 nM 33 nM 74 nM 170 nM

100

80

% of Control Activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 51

100

17 nM 25 nM 37 nM 56 nM 83 nM 125 nM

60

50

40

50

20

0 0

20

40

60

80

100

Preincubation Time (min)

120

0 0

50

100

150


26


0 0

30

60

90


120

Page 27 of 51

D.

E.

0.18

0.12

0.12

0.08

kobs (min-1)

kobs (min-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0.06

0.00 0

0.04

100

200

[Compound 1] (nM)

300

0.00 0

50

100

150

[Compound 2] (nM)

Figure 6. Determination of inhibition kinetic parameters for the imidazopyridine inhibitors. After DGAT2 (1 μg of the detergent-solubilized DGAT2 membrane, 10-fold higher enzyme concentration than that in standard assay) was preincubated with inhibitors (or DMSO for controls) for the indicated times, the residual DGAT2 activity was measured by carrying out DGAT2 reactions (20 l) for 4 min as described in Materials and Methods. The data were plotted as percentage of control versus preincubation time as shown in (A), (B), and (C) for inhibitors 3, 1, and 2, respectively. The data in (B) and (C) were fit to the equation 3 yielding the first order rate constant for enzyme inactivation (kobs) at each inhibitor concentration. The kobs values were then plotted versus inhibitor concentration ([I]) and fit to equation 7 as shown in (D) and (E) for inhibitors 1 and 2, respectively. In this case, a plot of kobs as a function of [I] would yield a straight-line relationship where the y-intercept is the kinetic rate constant k4 and the slope is the ratio k4/Ki*app. Accordingly, from the linear fit of the data as shown in Figure 6D and E, the kinetic rate constant k4 (from the y-intercept) and the ratio k4/Ki*app (from the slope) were obtained. The kinetic constants k4 values determined for 1 and 2 were (1.64 ± 0.23) x 10-4 and (1.97 ± 0.30) x 10-4 s-1, respectively, which correspond to dissociation half-lives of 1.19 ± 0.17 and 0.991 ± 0.15 hr. The resulting Ki*app values for 1 and 2 were calculated to be 16.7 ± 0.38 and 16.0 ± 4.6 nM, respectively (Table 2). In a two-step model enzyme inhibition, since the dissociation rate constant (koff) of the enzyme-inhibitor complex is approximately equal to k4, residence time 27


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 51

(1/koff) was also calculated (Table 2). These data indicate 1 and 2 possess similar binding affinity and residence time for DGAT2. Due to the limited solubility of DGAT2 substrate 1,2didecanoyl-sn-glycerol, Ki*app values could not be converted to true Ki* values in the present study. Table 2. Inhibition Kinetic Parameters Compounds

Ki*app (nM)

t½ (hr)

Residence Time (hr)

1

16.7 ± 0.38

1.19 ± 0.17

1.69 ± 0.24

2

16.0 ± 4.6

0.991 ±0.15

1.41 ± 0.21

Half-life (t½) was derived from t½ = 0.693/k4. Residence time = 1/koff ; In a two-step model of enzyme inhibition (scheme 2) where k2

Mechanistic Characterization of Long Residence Time Inhibitors of

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