Discovery of a Selective Covalent Inhibitor of Lysophospholipase

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Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism Kay Ahn, Matthew F. Brown, Markus Boehm, Jessica Calloway, Ye Che, Jinshan M. Chen, Kimberly Fennell, Kieran F Geoghegan, Adam M. Gilbert, Jemy A. Gutierrez, Amit S. Kalgutkar, Adhiraj Lanba, Chris Limberakis, Thomas V Magee, Inish O'Doherty, Robert Oliver, Brandon Pabst, Jayvardhan Pandit, Kevin Parris, Jeffrey A. Pfefferkorn, Timothy P. Rolph, Rushi Patel, Brandon Schuff, Veerabahu Shanmugasundaram, Jeremy T. Starr, Alison H. Varghese, Nicholas B Vera, Cecile Vernochet, and Jiangli Yan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00266 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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ACS Chemical Biology

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Inhibition of LYPLAL1

O

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O

N

N

N

N

O N

OH

N Compound 11 LYPLAL1 IC50 = 0.006 µM CES1 IC50 > 50 µM

Glucose Production

Lipids species

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Key Words: LYPAL1, serine hydrolase, hepatic glucose production

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Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism Kay Ahn,¥,# Matthew F. Brown,§ Markus Boehm,§ Jessica Calloway,¥ Ye Che,§ Jinshan Chen,§ Kimberly F. Fennell,§ Kieran F. Geoghegan,§ Adam M. Gilbert,§ Jemy A. Gutierrez,¥ Amit S. Kalgutkar,£ Adhiraj Lanba,¥ Chris Limberakis,§ Thomas V. Magee,*,§ Inish O’Doherty,¥ Robert Oliver,§ Brandon Pabst,¥ Jayvardhan Pandit,§ Kevin Parris,§ Jeffrey A. Pfefferkorn,¥ Timothy P. Rolph,¥ Rushi Patel,¥ Brandon Schuff, § Veerabahu Shanmugasundaram,§ Jeremy T. Starr,§ Alison H. Varghese,§ Nicholas B. Vera,¥ Cecile Vernochet,*,¥ and Jiangli Yan§ ¥

Cardiovascular, Metabolic, and Endocrine Diseases (CVMED) Research Unit, §Worldwide Medicinal Chemistry, and £Pharmacokinetics, Dynamics, & Metabolism, Pfizer Inc, 610 Main Street, Cambridge, MA 02139 and Eastern Point Road, Groton, CT 06340, USA ABSTRACT: Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio and non-alcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

INTRODUCTION Serine hydrolases form a large and diverse class of enzymes in the human proteome, with more than 200 members divided almost equally between serine proteases and distinct metabolic serine hydrolases. Many enzymes in the metabolic group have yet to be assigned distinct functions,1 but the general druggability of the class is confirmed by approved medicines such as sitagliptin for diabetes,2 orlistat for weight loss,3 and rivaroxaban for thrombosis.4 Advances in the genomic analysis of populations have revealed many linkages between specific genetic variations and human disease. These results can suggest that a specific protein is a valid target for therapeutic intervention, but only occasionally is a selective inhibitor or activator readily available to test the hypothesis. A suitable agent need not have all the attributes to qualify as a fully-fledged drug, but its potency and selectivity against the target should be adequate to assess the value of the therapeutic strategy. Agents of this kind are called "chemical tools",5 and their expedited discovery and application represents an increasingly important strategy for target validation. The value of the approach is apparent. For example, Ogasawara et al. developed two selective, centrally active irreversible diacylglycerol lipase (DAGL) inhibitors which, when studied with a related control

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compound, assisted with uncovering the role of DAGL in neuroinflammation.6 Separately, Naydenov et al. showed that a brain penetrant ABHD6 blocker protected against PTZ (pentylenetetrazole)-induced epileptiform seizures.7 Recently, genome-wide association studies (GWAS) identified genetic variants in 19 loci near LYPLAL1, the gene that encodes the serine hydrolase lysophospholipase-like 1 (LYPLAL1).8-10 These variants were significantly associated with fat distribution, waist-to-hip ratio, and non-alcoholic fatty liver disease (NAFLD). Fasting serum triglyceride levels and insulin resistance were also associated with the major Gallele of LYPLAL1, adding to the interest of investigating the biological function of LYPLAL1 in metabolically relevant tissues such as adipose and liver. In obese humans, LYPLAL1 gene expression is upregulated in white adipose tissue, which contrasts with results from obese rodent models.11,12 As a further complication, overexpression or knockdown studies of LYPLAL1 in mouse adipocytes have not revealed a direct role for the enzyme in differentiation, triacylglycerol accumulation, or insulin signaling.13 Therefore, while the genetic associations suggest a potential role for LYPLAL1 in human metabolic disease, current knowledge of the biological function of LYPLAL1 remains limited and clarifying insights are required. Against this backdrop, we undertook an effort to develop a selective pharmacological tool that would enable evaluation of the functional role of LYPLAL1 in human and other metabolic systems.

RESULTS LYPLAL1 expression in human and rodent liver and protein turnover. As the LYPLAL1 risk variant is significantly associated with hepatic triglyceride (HTG) content,14 LYPLAL1 expression was initially investigated in human livers separately affected by a number of diseases. LYPLAL1 mRNA levels in homogenized tissue samples were analyzed by total RNA isolation with a Qiagen RNeasy Plus Mini Kit, followed by transcription into cDNA with the Bio-Rad iScript kit and PCR of LYPLAL1, TBP and PPIA transcript using ABI 7900HT (see Methods). LYPLAL1 expression was normalized to the TBP and PPIA signals (see Methods). Hepatocellular carcinoma (HCC) and fibrotic human liver expressed the highest levels of LYPLAL1 mRNA compared to normal or fatty liver (Figure 1A). LYPLAL1 expression also tended to be higher in steatotic liver than in a normal control, but this difference did not reach statistical significance. Livers from rodent models of various metabolic diseases were studied in the same way (Figure 1B). As in the human samples, hepatic LYPLAL1 expression was increased in mouse models of metabolic diseases, such as high fat-fed C57Bl6 mice, ob/ob (leptin-deficient) and db -/- (leptin receptor-deficient) mice compared to the respective control animals [chow diet fed (CD) or db +/-]. Detection of elevated expression levels of LYPLAL1 in human and rodent diseased liver samples reinforced the relevance of developing a chemical tool that would permit the biological function of LYPLAL1 to be clarified and could potentially validate LYPLAL1 as a therapeutic target.

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Figure 1. LYPLAL1 expression is elevated in A) diseased human liver and in B) rodent models of metabolic disease. C) Half-life of LYPLAL1 in overexpressing COS7 cells as determined by 35S pulse-chase analysis. Results are statistically significant using One Way Anova *p≤ 0.05, **p≤ 0.01 and ***p≤ 0.001.

LYPLAL1 Crystallization and LYPLAL1 Inhibitor Generation The precise activity and endogenous substrate of LYPLAL1 remain uncertain, but kinetic studies and the crystal structure imply that it is a carboxyester hydrolase that prefers short acyl chains to long ones.15 Pulse-chase labeling studies using 35S indicated that the half-life of protein turnover in COS7 cells overexpressing LYPLAL1 was 9.6 h (Figure 1C), suggesting that exposure to a selective covalent inhibitor could suppress its activity for long enough to allow its function to be uncovered. With this in mind, we utilized in-gel activity-based protein profiling (ABPP) with a fluorescent probe in search of novel leads.16 Our initial hit matter was derived from a regioisomer of N1-substituted piperidinyl-1,2,3-triazole ureas previously shown to be selective inhibitors of the serine hydrolase ABHD6 (Figure 2).17,18 Further gelbased screening against human recombinant LYPLAL1 and related serine hydrolase targets then led to the identification of N2-substituted triazole analogs with superior selectivity against LYPLAL1 (Table 1).

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This was a purely serendipitous finding resulting from the isolation and screening of minor N2substituted byproducts observed in the process of synthesizing the known ABHD6 inhibitors.

Figure 2. The N1-substituted triazole urea previously disclosed as an inhibitor of ABHD6, and the N2-substituted isomer discovered herein as an inhibition motif for LYPLAL1.

In addition to the beneficial effect of the triazole N2-substitution on potency against LYPLAL1, resolution of the stereoisomers of the 2-phenyl piperidine showed a 266-fold preference for the (R) (2) versus the (S) (1) configuration (Table 1). These leads, however, lacked the desired selectivity for LYPLAL1, as they inhibited human recombinant carboxyesterase CES1 at least ten times more potently than they inhibited LYPLAL1 (Table 1). Our primary screening strategy from this point forward was to test against human recombinant forms of LYPLAL1 and CES in parallel, with the CES1 subtype representing the CES family. Although kinact/Ki values are considered the most accurate measure of potency for irreversible inhibitors, IC50 values determined using a consistent enzyme:inhibitor incubation time of 30 min were used to rank order potencies under the assumption that kinact would be comparable across the compound set due to the structural similarity of the reactive core of this chemical series. Changes to the biaryl sidechain had minimal or no effect on the CES1 activity but did impact LYPLAL1 potency (Table 1). For example, replacement of the carboxylic acid of 2 with the dimethylsulfonamide of 3 gave a nearly 3-fold increase in potency. Modification of the 1,2,3-triazole to the 1,2,4-triazole (4) diminished activity against LYPLAL1 by 2-fold, but was adopted for routine use because it greatly simplified the synthesis and isolation of pure N-2 substituted analogs. Replacement of the terminus of 4 with a simple pyridyl (5) did not compromise LYPLAL1 potency, but the morpholinopyrimidine sidechain (6) was consistently the most potent and became the default biaryl sidechain appendage. Focusing our attention on removing CES activity, we obtained a crystal structure of covalently modified LYPLAL1 by soaking with the racemic mixture of 1 and 2. As expected, the biaryl sidechain was absent, leaving the (R)-2-phenylpiperidine-1-carbonyl moiety of 2 attached to the hydroxyl of the active site serine (Ser-124) (Figure 3).

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Figure 3. X-ray crystal structure (PDB Code: 5KRE) of LYPLAL1 covalently modified by 2 (2.0 Å). A) Active site serine (Ser-124) O-acylated by (R)-2-phenylpiperidine-1-carbonyl. Nitrate derived from the crystallization buffer makes an ionic interaction with the guanidinium of Arg-80. B) Flipped perspective with Van der Waals surface of the binding pocket and overlap with CES1 Leu-255 (yellow; PDB Code: 2DR0). C) Unbiased Fobs-Fcalc map calculated around the inhibitor binding site and contoured at 1.4σ.

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A unique feature of the LYPLAL1 active site relative to the CES family and other serine hydrolases is the presence of an arginyl residue (Arg-80). The X-ray crystal structure of the covalent adduct revealed the close proximity of Arg-80 to the piperidine ring of the acylating group, suggesting that a well-placed carboxylate functionality at its 3- or 4-position could improve LYPLAL1 potency (Figure S1). Consistent with this idea, the structure showed that a nitrate anion derived from the crystallization buffer was present in this region and interacted with the Arg-80 guanidinium group. The strongest argument for carboxylation of the piperidine was the lipophilic residue in the overlapping position of CES1, namely Leu-255 (Leu-262 and Ile-263 for CES2 and CES3, respectively), improving the odds that the carboxylate would render the compound incompatible with off-target enzymes. The (2R,4S)-2phenylpiperidine-4-carboxylic acid (7) was duly found to lack CES1 activity, but its LYPLAL1 potency was also significantly reduced relative to the parent 6. Not surprisingly, the (2S,4S)-2-phenylpiperidine-4carboxylic acid (8) was generally inactive, but the (2R,4R)-isomer (9) displayed very good separation between LYPLAL1 and CES1 inhibition while delivering quite potent inhibition of LYPLAL1. Finally, the (2S,3S)-isomer (10) was also impotent, but its antipode, the (2R,3R)-2-phenylpiperidine-3-carboxylic acid (11), was both exquisitely potent against LYPLAL1 (IC50 = 0.006 µM) and inactive versus CES1 (IC50 > 50 µM) and was by far the most promising analog for querying the LYPLAL1 mechanism further. The mass spectrum of LYPLAL1 treated with 11 showed a mass shift of +231 Da (allowing for small experimental error) consistent with the expected acylation adduct (Figure S2). Because the crystal structure of LYPLAL1 treated with 2 clearly showed the 2-phenyl on the piperidine on the outer edge of the binding pocket, we reasoned that a ‘clickable’ handle such as an alkyne could be installed at the 3position of the phenyl without compromising LYPLAL1 potency. Consistent with this expectation, the 3alkynyl analog (12) retained the LYPLAL1 and CES1 profiles of the parent 11. Selectivity against a sample of the broader human serine hydrolase proteome (>30 enzymes) was also tested using in-gel fluorescence with enzymes overexpressed from Cos7 cells (Table 2). Although the leaving group sidechain did have some impact on proteome selectivity, e.g. analogs 2 versus 6, the acids 11 and 12 were the most selective LYPLAL1 inhibitors. LYPLAL1 Activity, Target Engagement and Impact on Hepatic Glucose production. The LYPLAL1-directed alkyne probe (12) allowed us to assess levels of active LYPLAL1 in normal, fatty and human hepatocellular carcinoma (HCC) liver samples. After incubation of human liver lysates with 12 in vitro, labeled LYPLAL1 was visualized by copper-catalyzed azide-alkyne cycloaddition of TAMRA-azide followed by SDS-PAGE and fluorescent scanning of the gel. The bands observed matched the predicted molecular weight of LYPLAL1 (~26 kDa) and were eliminated by pre-treatment with 11 before addition of 12 (data not shown). Although quantification of the band intensities did not reach statistical significance due to the low number of human samples available, LYPLAL1 activity appeared to follow the mRNA transcript level, with a slight decrease in fatty liver and a slight elevation in HCC samples (Figure 4A). Furthermore, 12 was shown to be selective at ≤1 µM against endogenous FPrhodamine reactive serine hydrolases in human hepatic cells (HepG2) (Figure S3) and in liver and brain tissues from C57Bl/6J mice (Figure S4).

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To establish that 11 engaged LYPLAL1 in human cells and to further assess its selectivity, a SILAC experiment was performed in HepG2 cells using a pan-active serine hydrolase probe (FP-biotin) and quantitation by mass spectrometry (Figure 4B).19-21 HepG2 cells are a well-established human hepatic cell line. The results showed that treating the cells with 11 selectively blocked the ability of FP-biotin to capture LYPLAL1 when the cells were lysed. DMSO treatment of heavy and light HepG2 cells was used to normalize any changes in serine hydrolase expression between cells cultured in light and heavy SILAC media, giving the x-coordinate of each enzyme point in Figure 4B. The corresponding y-coordinates report the SILAC ratio for each serine hydrolase captured from a mixture of 11-treated light cells and DMSO-treated heavy cells. The exquisite selectivity of 11 at 50 nM for LYPLAL1 was evident in the almost fivefold enrichment of LYPLAL1 in material captured from DMSO-treated cells compared to 11treated cells, while the SILAC ratio for other serine hydrolases was essentially unaffected. As a further study of specificity, HepG2 cells were treated with the clickable compound 12 (50 nM) either without or with pretreatment by 50 nM nonclickable 11. Following cell lysis and click chemistry to introduce TAMRA to 12-modified proteins, the cell lysates were fractionated by SDSpolyacrylamide gel electrophoresis and TAMRA-bearing proteins were located by fluorescent imaging (Figure 4C). The sole specifically TAMRA-labeled band occurred in the 12-treated sample at close to 26 kDa, the mass of LYPLAL1, and proteomic analysis of this band unequivocally showed that LYPLAL1 was present in it. In a control analysis, LYPLAL1 was not detected in a section of the DMSO-treated lane cut at a region corresponding to about 80 kDa. A similar experiment conducted with rat hepatocytes (Figure S5) similarly confirmed co-localization of competing TAMRA incorporation with LYPLAL1, but indicated the presence of cross-reactivity with protein migrating at about 56 kDa. Proteomic analysis of the labeled region indicated that the labeled protein co-migrated with high levels of rat carboxylesterases. This result raises caution that the strong indications of LYPLAL1 specificity for 12 derived from experiments with human proteins and cells may not extend without exception to other species.

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Figure 4. A) LYPLAL1 activity in normal and diseased human liver assessed using the LYPLAL1-directed alkyne probe 12. B) LYPLAL1 selectivity of 11 in HepG2 cells. Each point represents a serine hydrolase captured by FP-biotin. X-values are SILAC ratios (heavy/light) reflecting relative quantities of enzyme captured from DMSO-treated heavy and light cells (control), and y-values indicate relative amounts captured when heavy cells received DMSO and light cells received 11 (50 nM). C) Selectivity of probe action in HepG2 cells assessed using (i) DMSO as a control; (ii) the LYPLAL1-directed alkyne probe 12 (50 nM) (lane 12); and (iii) 11 (50 nM) followed by 12 (50 nM) (lane 11,12). Proteins modified by 12 were click-coupled to TAMRA, fractionated by SDS-PAGE, and visualized by fluorescent imaging of the gel. Proteomic analysis indicated that LYPLAL1 was present in the fluorescent band at 26 kDa in lane 12. LYPLAL1_ACSChemBiol


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We also investigated the biological impact of LYPLAL1 inhibition by 11 on hepatic glucose production. As expected for a commonly used control of glucose output, AMP-activated protein kinase (AMPK) activation by 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed glucose production in human (Figure 5A), rat (Figure 5B) and mouse (Figure 5C) hepatocytes. In contrast, incubation with 11 (50 nM) led to a significant increase in glucose production by human (Figure 5A), rat (Figure 5B) and mouse (Figure 5C) hepatocytes by 12%, 20% and 27%, respectively, suggesting that the function of LYPLAL1 is consistent across these species. The mechanism underlying these effects remains to be elucidated but the results collectively suggest that LYPLAL1 plays a role in hepatic metabolism.

Figure 5. A) Effects of 100 µM AICAR or 50 nM 11 on glucose levels in human hepatocytes. B) Same as panel A in rat hepatocytes. C) Same as panel A in mouse hepatocytes. Results are statistically significant using One Way Anova *p≤ 0.05, **p≤ 0.01 and ***p≤ 0.001.

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CONCLUSION Although GWAS studies strongly indicate a link between variations in the gene encoding LYPLAL1 and aspects of human physiology connected to energy and the metabolism of lipids, the underlying enzymatic function of this metabolic serine hydrolase remains unclear. Here we have shown that the expression of LYPLAL1, and potentially its activity, are upregulated in fibrotic liver and HCC and tend to increase in human steatotic liver as well. Having discovered a highly potent and selective covalent inhibitor of LYPLAL1 (11) and its clickable analog (12), both devoid of CES and other off-target activities both in human cells and against an extensive catalog of human recombinant metabolic serine hydrolases, we demonstrated that LYPLAL1 inhibition has a significant impact on hepatic gluconeogenic functions in human and rodent hepatocytes. While we caution that the selectivity demonstrated in human systems cannot be assumed to be equivalent in other species, these results tend to suggest that LYPLAL1 inhibition would be detrimental rather than beneficial for the treatment of either NAFLD and/or diabetes. More generally, they exemplify how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of classical biochemical characterization.

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Table 1. Structures and IC50 values (µM) of key analogs against human recombinant LYPLAL1 and CES1 as determined by in-gel ABPP. The degree of fluorescent labeling was determined by measuring the integrated optical density of the bands between compound treated samples and DMSO controls. IC50 values were determined from half-log dose-response curves from three trials at each inhibitor concentration. Purified LYPLAL1 (10 nM final) was incubated at RT for 30 min with 1.0–10,000 nM inhibitor. Lysate from CES1 transiently expressed in COS7 (1.75 µg final) was incubated for 30 min at RT with 5.0–50,000 nM inhibitor. The data were plotted as percentage of effect versus inhibitor concentration and fit to the equation, y = 100/[1 + (x/IC50)z]. Cmpd 1

Structure

LYPLAL1 16

CES1 10% labeling was achieved. In a final reaction volume of 25 μL each serine hydrolase pool diluted in 50 mM Tris pH 7.5 (23 μL), was incubated for 30 min at room temperature with 1 μL DMSO or candidate inhibitor (10 μM, final) dissolved in 100% DMSO. To this mixture, 1 μL of freshly prepared 50 μM FP-rhodamine diluted in 25% DMSO was added (2 μM FP-Rh final). After 30 min, the reactions were quenched with 7.5 μL 4x SDS-PAGE loading buffer containing 300 mM DTT, boiled for 2 min at 80°C before 15 μL of sample was loaded per lane, separated by 10% Bis-Tris SDS-PAGE, and visualized in-gel using a flatbed fluorescence scanner (Typhoon Trio). The degree of fluorescent labeling was determined by measuring the integrated optical density of the bands between compound-treated and DMSO controls. All ABPP custom proteome gels can be found in Supporting Information. Selectivity of LYPLAL1 Alkyne Probe by Gel-Based ABPP with HepG2, C57Bl/6J Liver and Brain Lysates HepG2 cells were lysed in ice cold PBS (pH 7.5) using a point sonicator set at 30% amplitude for 15 s. The lysates were spun at 1000 x g for 15 min at 4 °C to remove cellular debris, the supernatant was removed (lysate) and protein concentration was determined by BCA assay kit. Preparation of C57Bl/6J tissue lysates can be found under Preparation of Liver and Brain Lysates. The final assay mixture contained 1x PBS (pH 7.5), 5% DMSO, and 2.0 mg/mL of either HepG2, C57Bl/6J liver or brain lysate. To 23 μL of lysate (2.17 mg/mL) diluted in PBS, 1 μL of LYPLAL1 alkyne probe (12) at 25x the desired final concentration dissolved in 100% DMSO or DMSO (control) was added and the mixture was incubated at room temperature for 30 min. To this mixture, 1 μL of freshly prepared 50 μM FP-rhodamine diluted in 25% DMSO was added (2 μM FP-Rh, final). The reactions were incubated for 30 min at room temperature and quenched with 7.5 μL of 4x SDS-PAGE loading buffer containing 300 mM DTT, and boiled for 2 min at 80 °C before 15 μL of sample was loaded per lane, separated by 10% Bis-Tris SDS-PAGE, and visualized Magee_Vernochet_LYPLAL1_ACSChemBiol ACS Paragon Plus Environment

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in-gel using a flatbed fluorescence scanner (Typhoon Trio). The degree of fluorescent labeling was determined by measuring the integrated optical density of the bands between compound-treated and DMSO controls. All ABPP proteome gels from HepG2, C57Bl/6J liver and brain lysates can be found in Supporting Information. LYPLAL1 Activity, Target Engagement and Impact on Hepatic Glucose production. Design of Serine Hydrolase Constructs All serine hydrolase constructs were designed with NheI and Kozak sequences on the 5'-end (GCTAGCACC), and stop codons and HindIII sequence on the 3'-end (TGATGAAGCTT). They were synthesized in the pUC57 vector (GenScript, Piscataway NJ) using UniProtKB sequences (accession numbers in Table 2) and subcloned into pcDNA3.1(-)zeo. Preparation of Liver and Brain Lysates All operations were performed at 4 oC unless otherwise noted. Tissues were homogenized in lysing matrix D tubes in PBS, pH 7.5 (1 mL PBS per 50 mg tissue) using an MP FastPrep-24 at speed setting 5.5 for 3 min. A low-speed spin (1000 x g, 10 min) was performed to remove cellular debris. The total protein concentration from the resulting supernatant was determined using a BCA protein assay kit (Pierce). Samples were aliquoted, flash frozen in liquid N2, and stored at −80 °C unel further use. Donor information and source of human liver samples can be found in Supporting Information. Assessment of Active LYPLAL1 Levels in Human Liver by Click Chemistry To investigate active levels of LYPLAL1 in human liver lysates, reactions were carried out in 1.5 mL microcentrifuge tubes in a total reaction volume of 125 μL. To 115 μL of 0.7 mg/mL human liver lysate diluted in PBS, pH 7.5, 10 μL of a LYPLAL1-specific alkyne probe (12) dissolved in 62.5% DMSO was added and the mixture was incubated at room temperature for 60 min (10 μM 12, 5% DMSO, final). To this mixture, 7.5 μL of freshly prepared click chemistry mix (2.5 μL 50 mM CuSO4, 2.5 μL 50 mM TCEP, 1.25 μL 10 mM TBTA in DMSO:TBA (1:4 v/v), 1.25 μL 10 mM TAMRA azide in DMSO) was added. The final concentration of components from the click chemistry mix in the reaction was 1 mM CuSO4, 1 mM TCEP, 0.05 mM TBTA, and 0.05 mM TAMRA azide. The reactions were incubated for 60 min at room temperature followed by the addition of 200 μL water and 375 μL of methanol:chloroform (4:1 v/v). Tubes were vortexed at the highest setting for 40 s before centrifugation at 18,000 x g at 4 °C for 5 min. The liquid above the resulting layer of protein precipitate was carefully removed. Excess TAMRA azide was washed from protein with the addition of 800 μL methanol followed by vortexing at the highest setting for 40 s and centrifugation at 18,000 x g at 4 oC for 5 min. Methanol was aspirated from the tubes and the aforementioned methanol wash step was repeated four additional times. Once methanol was removed after the final wash the resulting protein pellets were dried under a gentle stream of nitrogen before resuspension in 40 μL 2x SDS-PAGE loading buffer containing 300 mM DTT. Samples were boiled for 5 min at 80°C before 30 μL of sample (60 μg protein) was loaded per lane, separated by 10% Bis-Tris SDS-PAGE, and visualized in-gel using a flatbed fluorescence scanner (Typhoon Trio). Active levels of LYPLAL1 were determined by measuring the integrated optical density of the bands.

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Generation of SILAC HepG2 Cell Lines The heavy cells were generated after five passages in RPMI (Fisher Scientific, PI88421) with 10% (v/v) dialyzed FBS (Fisher Scientific, PI88440), 1% penicillin/streptomycin (Life Technologies, 10378-016), 50 mg/mL L-proline (Fisher Scientific, PI88430), 50 mg/mL [13C6,15N2]-L-lysine-2HCl, (Fisher Scientific, PI88209), 50 mg/mL [13C6,15N4]-L-arginine-HCl (Fisher Scientific, PI-89990). The light cells were generated over five passages in the same medium except that heavy lysine and arginine were replaced by 50 mg/mL L-lysine-2HCl (Fisher Scientific, PI-88209), 50 mg/mL L-arginine-HCl (Fisher Scientific, PI-89989). HepG2 cells were plated at 4.5 x 106 cells per T-75 flask and grown to 90% confluence over two days in 15 mL of heavy or light growth media. Once the cells had reached the desired confluence, the medium was removed and 12 mL of fresh medium was added. A 250 nM solution of 11 was made in 3 mL of OptiMEM I (Life Technologies, 31985-088) with 2.5% DMSO (v/v) with vigorous vortexing upon preparation. This probe solution was added to the flask of cells such that the final probe and DMSO concentrations were 50 nM and 0.5% (v/v) respectively and incubated for 5 h at 37°C. The cells were then washed twice with PBS pH 7.4 and lifted from the flask with cell dissociation buffer (Life Technologies, 13151-014), spun down at 1200 x g for 5 min at 4 °C and washed with ice-cold PBS before being lysed in 0.5 mL of ice-cold 50 mM Tris, pH 7.4, using a point sonicator. The lysate was centrifuged at 1000 x g for 15 min at 4°C to remove the cellular debris. Membrane preparations were made by centrifuging the cell lysate at 100,000 x g for 45 min at 4 ˚C. The membrane pellets were resolubilized in 250 µL cold 50 mM Tris pH 7.4 buffer using an insulin syringe (Fisher Scientific, 1482028) and dounced 40 times. Protein concentrations were measured by BCA assay (Fisher Scientific, PI-23222). The heavy and light soluble fractions were combined into two sample types. The first combined equal amounts of DMSO-treated heavy cell lysates and DMSO-treated light cell lysates, (n=3), the second combined equal amounts of DMSO treated heavy cell lysates and 11-treated light cell lysates, (n=3). The total amount of protein in the combined soluble preparations was 420 μg at 2 mg/mL concentration. The same combinations were made for the membrane fractions, yielding a total of 12 samples. The individual samples were reacted with 2.9 μL of FP-biotin (3 mM) for 2 h, vortexing at 45 min intervals. The reaction was then diluted to 500 μL with PBS pH 7.4 and the excess unreacted FP-biotin was removed in a NAP-5 column (GE Healthcare Life Sciences, 17-0853-02). The column was first equilibrated with 5 x 2 mL 50 mM Tris pH 7.4 before adding 500 μL of the FP-biotin reaction. The flowthrough was collected, and the column was washed with 1 mL mM Tris pH 7.4 combining the eluate with previous fraction. Seventy five microliters of 24% SDS (w/v) stock solution was added for a final concentration of 1.2%, and the samples were then heated at 90°C for 5 min. Samples were transferred to a 15 mL tube and diluted with PBS to a final 0.2% SDS concentration. Washed high capacity streptavidin resin (Fisher Scientific, PI-20359) was added to each of the samples and incubated for 30 min at room temp on an end-over-end mixer before being moved to 4 °C overnight. The next morning the tubes were rotated at room temperature for 2 h to dissolve the SDS. The beads were then spun down at 1400 x g for 3 min and the supernatant was removed. The beads were washed with 5 mL 0.2% SDS PBS on an end-over-end mixer for 10 min, followed by four separate 5 mL washes with PBS at 1 min each, and then three washes using 5 mL of water for 1 min each. The beads were spun down and the supernatant was removed between individual washes. The beads were transferred to 1.5 mL Eppendorf tubes with 1 mL water, spun down and the supernatant removed. Five hundred microliters of 6 M urea in PBS was added to the beads followed by 25 μL of 200 mM DTT (Sigma-Aldrich, 43819). The samples were heated at 65°C for 20 min, shaking Magee_Vernochet_LYPLAL1_ACSChemBiol ACS Paragon Plus Environment

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every 10 min. The samples were allowed to cool at 37°C and then 25 μL of 400 mM iodoacetamide (Sigma-Aldrich, I1149) was added. The samples were kept at 37˚C in the dark with agitation. The samples were diluted with 950 μL PBS, centrifuged and the supernatant removed. A premixed 200 μL solution made up of 2 M urea PBS, 1.1 μg trypsin, 2 μL 100 mM CaCl2 was added to each sample before incubating overnight at 37˚C with agitation. The peptides were harvested, desalted, dried down and stored -80°C until further analysis.

Liquid Chromatography-Mass Spectrometry Analysis of SILAC-treated Peptides Dried samples of tryptic digests were redissolved in 20 μL of 0.1% formic acid, and 7 μL portions were analyzed by LC-MS. A blank analysis was run between each pair of digest samples to minimize any effect of carry-over. Nano-LC was conducted at a flow rate of 300 nL/min with solvents: A, 0.1% formic acid; and B, 0.1% formic acid in acetonitrile. The gradient program was: 0-3 min, isocratic 2% B; 3-125 min, 270% B; 125-130 min, 70-90% B; 130-135 min, isocratic 90% B; 135-140 min, 90-2% B; 140-160 min, isocratic 2% B. Sample injection was conducted using a trapping method. The trapping column was a Waters ACQUITY UPLC PST C18 nanoACQUITY Trap 10K psi VV, 100Å, 5 µm packing particle, 180 µm x 20 mm [p/n 186006526], and the analytical HPLC column was a Waters ACQUITY UPLC PST C18 nanoACQUITY Column 10K psi, 130Å, 1.7 µm packing particle, 100 µm x 100 mm [p/n 186003546]. The HPLC was connected through an empty (i.e. no packing) ThermoFisher Scientific EASY-Spray interface to a ThermoFisher Scientific LTQ Orbitrap Elite mass spectrometer programmed to operate in top 10 mode with resolution set to 60,000 for the Full MS survey scan and MS/MS spectra collected following collision-induced dissociation in the ion trap. Dynamic exclusion was set to 1 with the exclusion duration and repeat duration values both set to 45 s and the exclusion list size set to the maximum value of 500. Lock mass values were set to 371.101233 and 445.120025 ([M+H]+ values for polysiloxanes), and masses of identified peptides generally agreed with theoretical values to within 1 ppm. Identification of peptides and determination of SILAC ratios were conducted using Proteome Discoverer 1.4 (Thermo Scientific) with Mascot (licensed from Matrix Science, Boston, MA) as the search engine. Searches were conducted against the UniProtHuman sequence database enriched with sequences for common contaminant proteins. Carbamidomethyl (C) was selected as a static modification, and dynamic modifications were Label: 13C(6)15N (R), Label: 13C(6)15N(2) (K), and Oxidation (M). Precursor mass tolerance was 10 ppm and fragment mass tolerance was 0.6 Da. The maximum allowed fold change in SILAC ratio was set to 20, giving a corresponding minimum permitted value of 0.05. Quantification labels for the Heavy channel were Arg10 (Label: 13C(6)15N(4)/+10.008 Da) and Lys8 (Label: 13C(6)15N(2)/+8.014 Da). The following options were selected: Use Only Unique Peptides, Replace Missing Quan Values With Minimum Intensity, and Use Single-Peak Quan Channels. Mass precision in the Event Detector was set to 2 ppm. Selectivity of LYPLAL1 Alkyne Probe by Click Chemistry in HepG2 Cells and Rat Hepatocytes On the day before compound treatment, rat hepatocytes and HepG2 cells were seeded into in DMEM containing 10% FBS at densities of 1.0 x 107 and 5 x 106 cells/T-75 flask, respectively. On the day of the experiment, cells were washed twice with warm PBS, pH 7.5, before the addition of 15 mL of DMEM Magee_Vernochet_LYPLAL1_ACSChemBiol ACS Paragon Plus Environment

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containing 1% DMSO in the presence or absence of 50 nM compound 11. After 2 h, the media were removed and the cells were washed twice with warm PBS. Fifteen milliliters of DMEM containing 1% DMSO in the presence or absence of 50 nM alkyne probe 12 was added to each flask and incubated for 4 h. Cells were harvested by washing the flask twice with warm PBS before using 5 mL of ice cold PBS and a cell scraper to lift cells from the plate. The cells were pelleted at 4°C in a refrigerated centrifuge and the supernatant was removed. Cells were lysed in ice cold PBS (pH 7.5) using a point sonicator set at 30% amplitude for 15 s. The lysates were spun at 1000 x g for 15 min at 4 °C to remove cellular debris, the supernatant was removed (lysate) and protein concentration was determined by BCA assay kit. Lysates were snap frozen and stored in -80 oC prior to use. To investigate the selectivity of the alkyne probe 12, reactions were carried out in 1.5 mL microcentrifuge tubes in a total reaction volume of 250 μL. To 235 μL of 2.13 mg/mL cell lysate diluted in PBS, pH 7.5, 15 μL of freshly prepared click chemistry mix (5 μL 50 mM CuSO4, 5 μL 50 mM TCEP, 2.5 μL 10 mM TBTA in DMSO:TBA (1:4 v/v), 2.5 μL 10 mM TAMRA azide in DMSO) was added. The final concentration of components from the click chemistry mix in the reaction was 1 mM CuSO4, 1 mM TCEP, 0.05 mM TBTA, and 0.05 mM TAMRA azide. The reactions were incubated for 60 min at room temperature, after which 200 μL of water and 375 μL of methanol:chloroform (4:1 v/v) were added to each. Tubes were vortexed at the highest setting for 40 s before centrifugation at 18,000 x g at 4 °C for 5 min. The liquid above the resulting layer of protein precipitate was carefully removed. Excess TAMRA azide was washed from protein with the addition of 800 μL methanol followed by vortexing at the highest setting for 40 s and centrifugation at 18,000 x g at 4 oC for 5 min. Methanol was aspirated from the tubes and the methanol wash step was repeated four additional times. Once methanol was removed after the final wash, the resulting protein pellets were dried under a gentle stream of nitrogen before being resuspended in 100 μL of 2x SDS-PAGE loading buffer containing 300 mM DTT. Samples were boiled for 5 min at 80°C before 20 μL of sample (100 μg protein) was loaded per lane, fractionated by 4-12% Bis-Tris SDS-PAGE, and visualized in-gel using a flatbed fluorescence scanner (Typhoon Trio). Lysates from cells treated with DMEM and 1% DMSO alone were used to evaluate non-specific TAMRA binding, while those lysates from cells pretreated with 11 before the addition of 12 were used to assess LYPLAL1 target engagement. Bands unique to treatment with 12 were excised for further proteomic analysis. Click chemistry gels specific to rat hepatocytes can be found in Supporting Information. Proteomic Analysis of Gel Sections TAMRA-containing fluorescent bands and nonfluorescent control pieces excised from the gel were washed twice with 50% acetonitrile before being dried in a centrifugal concentrator. They were next treated with 0.125 mL of 0.025 M NH4HCO3, 4.5 mM DTT, and incubated with shaking for 30 min at 50 o C. After being cooled to room temperature, the samples were treated with 0.02 mL of 0.1 M iodoacetamide, and incubated with shaking for 50 min. They were next treated with 0.5 µg (microgram) of trypsin and incubated with shaking at 37 oC overnight. Digests were collected, gel pieces were extracted with (i) 0.2 mL and (ii) 0.1 mL of 50% CH3CN, 5% TFA, extracts were combined with the corresponding digests, and the samples were reduced in volume to about 0.1 ml. They were then subjected to solid-phase extraction using StageTips, dried, redissolved in 0.02 ml of 0.1% formic acid, Magee_Vernochet_LYPLAL1_ACSChemBiol ACS Paragon Plus Environment

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and analyzed by nano LC-MS using a Waters Nano ACQUITY HPLC system and a Thermo Scientific Orbitrap Fusion mass spectrometer. Data were searched for protein identification using Mascot. Rat hepatocyte Isolation Livers of 9 to 12 week old rats were perfused using liver perfusion media Life Technologies 17701-038) and submitted to digestion using KRB buffer (Sigma-Aldrich K4002) to which 0.5 mM CaCl2, collagenase/elastase and DNase were freshly added (Worthington Biomedical LK003172-LK002067). Livers were removed and disrupted in serum free DMEM (0.25% BSA+Pen/Strep) by tearing. Cells in suspension were filtered through 40µM filters and centrifuged at 4°C at 50 x g for 5 min. Supernatant was removed, fresh serum free DMEM added (0.25% BSA+Pen/Strep) and cells were centrifuged at 4 °C at 50 x g for 5 min again until the solution was clear. Percoll solution was added (Fisher Scientific 45001-747) (45 mL Percoll + 5 mL 10X PBS and 25 mL serum free DMEM) and centrifugation was performed at 120 x g at 4°C for 5 min. Finally, cells were suspended in DMEM 25 mM glucose containing 5% FBS, 1 μM dexamethasone and 700 nM insulin. After 4 h, media was replaced with William’s E media containing 1 μM dexamethasone, ITS, 2 mM glutamine and Pen/Strep. Hepatic Glucose Production Human cryopreserved (Corning), freshly isolated rat hepatocytes and freshly isolated mouse hepatocytes (Biomedical Research Models Inc.) were plated onto rat tail type I collagen-coated plates in high glucose DMEM containing 5% heat-inactivated FBS, 1 µM dexamethasone, 4 µg/mL recombinant human insulin, 25 mM HEPES pH 7.4 and 1,000 units/mL of penicillin and 1,000 µg/mL of streptomycin. After cells had attached (4 h), the medium was replaced with serum and phenol red-free DMEM containing 5 mM glucose, 1 µM glycogen in presence or absence of LYPLAL1 inhibitor, 11. After 16-18 h incubation, cell medium was replaced with assay buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 1.2 mM magnesium sulfate, 1.2 mM monopotassium phosphate, 0.1% BSA, 10 mM HEPES presence or absence of LYPLAL1 inhibitor, 11. After 16-18 h incubation, cell medium was replaced with assay buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 1.2 mM magnesium sulfate, 1.2 mM monopotassium, 1.4 mM calcium chloride, 20 mM sodium bicarbonate) containing 1 µM glucagon and 11. After 1 h, media was replaced with stimulation medium (assay buffer + 1 µM glucagon + 10 mM lactate + 1 mM pyruvate +/- 50 nM 11 +/- 100 µM AICAR (Tocris) and incubated for either 4 h (human hepatocytes) or 8 h (rat hepatocytes) in 37oC/5% CO2 incubator. Glucose output was assessed enzymatically using Amplex Red Glucose/Glucose Oxidase assay kit (Life Technologies) according to manufacturer’s instructions, normalized to total protein content and expressed as % DMSO control. Data are the Mean +/- s.e.m. n=3 with p value calculated by one-way ANOVA with Dunnett’s multiple comparisons test.


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ASSOCIATED CONTENT Supporting Information Detailed supplemental methods, spectral data, and pharmacokinetic data are provided in Supporting Information.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] *Email: [email protected]

Present Address #

Janssen Research & Development

1400 McKean Road Spring House, PA 19477

ACKNOWLEDGEMENTS We would like to thank M. J. Kim for assistance in generating recombinant serine hydrolases, S. Perez and T. Turner for technical assistance and G. Tesz for biology input. We also thank D. Anderson and K. Farley for assistance with structure elucidation. Finally, we thank BioDuro-Shanghai and WuXi-Shanghai for assistance with synthesis.


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Discovery of a Selective Covalent Inhibitor of Lysophospholipase

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