JX06 Selectively Inhibits Pyruvate Dehydrogenase Kinase PDK1 by a Covalent Cysteine Modification

Published OnlineFirst October 19, 2015; DOI: 10.1158/0008-5472.CAN-15-1023 Cancer Therapeutics, Targets, and Chemical Biology Research

JX06 Selectively Inhibits Pyruvate Dehydrogenase Kinase PDK1 by a Covalent Cysteine Modiﬁcation Wenyi Sun1, Zuoquan Xie1, Yifu Liu2, Dan Zhao3, Zhixiang Wu4, Dadong Zhang1, Hao Lv1, Shuai Tang1, Nan Jin1, Hualiang Jiang3, Minjia Tan4, Jian Ding1, Cheng Luo3, Jian Li2, Min Huang1, and Meiyu Geng1

Abstract

Pyruvate dehydrogenase kinase PDK1 is a metabolic enzyme covalent modification at C240 induced conformational changes responsible for switching glucose metabolism from mitochon- at Arginine 286 through Van der Waals forces, thereby hindering drial oxidation to aerobic glycolysis in cancer cells, a general access of ATP to its binding pocket and in turn impairing PDK1 hallmark of malignancy termed the Warburg effect. Herein we enzymatic activity. Notably, cells with a higher dependency on report the identification of JX06 as a selective covalent inhibitor of glycolysis were more sensitive to PDK1 inhibition, reflecting a PDK1 in cells. JX06 forms a disulfide bond with the thiol group of metabolic shift that promoted cellular oxidative stress and apo- a conserved cysteine residue (C240) based on recognition of a ptosis. Our findings offer new mechanistic insights including hydrophobic pocket adjacent to the ATP pocket of the PDK1 how to therapeutically target PDK1 by covalently modifying the enzyme. Our investigations of JX06 mechanism suggested that C240 residue. Cancer Res; 75(22); 1–14. 2015 AACR.

Introduction piration and enhances aerobic glycolysis via phosphorylating and inactivating pyruvate dehydrogenase (PDH), a gate-keeping mito- Cancer cells feature a unique metabolic profile of high aerobic chondrial enzyme (6, 7). PDH catalyzes the oxidative decarbox- glycolysis, also known as "Warburg effect," which describes the ylation of pyruvate to acetyl-CoA, the only entry leading pyruvate phenomenon of the enhanced conversion of glucose into lactate into tricarboxylic acid cycle in mitochondria (8). Phosphorylation even in the presence of oxygen (1). Aerobic glycolysis confers a of PDH by PDK, which occurs in the PDH complex (PDC), significant growth advantage of cancer cells by supplying essential restricts the access of glycolytic products into the mitochondrial ATP production, generating precursors for biosynthesis, and pro- respiration. To date, four PDK isoforms (PDK1-4) have been viding reducing equivalents for antioxidant defense (2, 3). identified in human mitochondria with tissue-specific expression, Although Warburgeffect has been observed for decades, how cancer all of which participate in the phosphorylation of serine 293, the cells gain this unique metabolic profile remains unclear (4, 5). main regulatory site of PDH activity (9–11). Mitochondrial pyruvate dehydrogenase kinase (PDK) func- Recently, an increasingly recognized linkage between PDK1 tions as a molecular switch that diminishes mitochondrial res- and upstream oncogenic proteins has provided a glimpse into the molecular basis of the metabolic reprogramming in cancer cells. PDK1 is directly trans-activated by hypoxia-inducible transcrip- 1 Division of Antitumor Pharmacology, State Key Laboratory of Drug tion factor 1 (HIF-1) or the cooperation of dysregulated c-Myc and Research, Shanghai Institute of Materia Medica, Chinese Academy of – Sciences, Shanghai, China. 2Shanghai Key Laboratory of New Drug HIF-1 to promote glycolysis (12 14). Recent studies have Design, School of Pharmacy, East China University of Science and revealed that PDK1 is also regulated by oncogenic drivers at the 3 Technology, Shanghai, China. Drug Discovery and Design Center, posttranslational level. Diverse oncogenic tyrosine kinases, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. 4The Chem- including FGFR1, BCR-ABL, JAK2 V617F, and FLT3-ITD, which ical Proteomics Center, State Key Laboratory of Drug Research, are frequently aberrant in human cancers, activate PDK1 activity Shanghai Institute of Materia Medica, Chinese Academy of Sciences, by promoting its binding to ATP (15). Moreover, aberrant expres- Shanghai, China. sion of Lin28 facilitates aerobic glycolysis via targeting PDK1 by Note: Supplementary data for this article are available at Cancer Research microRNA let-7 in a HIF-1–independent manner (16). All this Online (http://cancerres.aacrjournals.org/). evidence suggests a model that oncogenic proteins rewire meta- W. Sun, Z. Xie, Y. Liu, and D. Zhao contributed equally to this article. bolic network via modulating PDK1 at multiple levels. PDK1 Corresponding Authors: Meiyu Geng, Shanghai Institute of Materia Medica, appears to be the Achilles heel in the reprogrammed glucose Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. metabolism in cancer cells (17). Indeed, the metabolic alterations Phone: 86-21-50806600-2426; Fax: 86-21-50807088; E-mail: caused by PDK1 inhibition in cancer cells provoke an increase in [email protected]; Min Huang, [email protected]; Jian Li, the intracellular reactive oxygen species (ROS; ref. 18), which [email protected]; and Cheng Luo, [email protected] further leads to mitochondria-dependent apoptosis (15, 19). doi: 10.1158/0008-5472.CAN-15-1023 Meanwhile, it has been also noticed that PDK1 inhibition causes 2015 American Association for Cancer Research. distinct responses in different cancer cells (20). It is thus crucial to

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know whether there exists PDK1 dependent cancer subset, which by analyzing the DNA profile of eight short tandem repeat (STR) will facilitate the translation of PDK1 inhibition to therapeutic loci plus amelogenin (Genesky Biotechnologies Inc.). Cells were benefits. maintained in appropriate culture medium as the suppliers A few PDK1 inhibitors have been reported, including the suggested. ATP competitive inhibitor radicicol, allosteric inhibitor dichloroacetate (DCA), and AZD7545 that disrupts PDK1 Glucose uptake measurement interaction with the PDC component (21–24). Among these After being treated with JX06 or vehicle control for approxi- inhibitors, DCA is the only one showing preliminary clinical mately 18 hours, cells were washed with PBS and cultured in efficacy in a small cohort of patients with glioblastoma (25). serum-free high-glucose medium for 6 more hours. Glucose levels The therapeutic promise of PDK1 inhibitors in cancer treat- in the medium were measured using a glucose assay kit (Beyo- ment needs to be further explored. Recently, covalent inhibi- time) and the readouts were normalized by the corresponding tors are believed to offer an alternative solution to kinase protein amounts of each sample. inhibition (26). The pharmacologic advantage of covalent inhibitors, such as high selectivity across kinase family and Intracellular lactate measurement sustained inhibitory effect, may provide new therapeutic Intracellular lactate levels were measured using a Lactate Col- opportunities for targeting PDK1. orimetric/Fluorometric Assay Kit (Biovision). After exposure to In an effort to discover new PDK1 inhibitors, we carried out an JX06 or vehicle control for 24 hours, cells were lysed and cen- enzymatic screen using a small-molecule library composed of trifuged at 12,000 g to collect the cell supernatant. The super- commercially available known drugs (Supplementary Table S1 natant of cell lysates was mixed with the assay solution. The and Supplementary Fig. S1). Thiram, a known pesticide with absorbance was measured at 570 nm and the readout was nor- anticancer activity, emerged prominently due to the remarkable malized by the protein amounts. activity against PDK1 (27, 28). Further chemical efforts based on thiram led to the discovery of a more potent new compound Intracellular ATP measurement designated as JX06. In this study, the unique molecular mecha- Intracellular ATP levels were assessed using an ATP assay kit nism of JX06 allowed us to probe the enzymatic regulation of (Beyotime). After exposure to JX06 or vehicle control for 24 PDK1. We discovered a highly conserved cysteine, which offered a hours, cells were lysed and centrifuged at 12,000 g to collect target for covalent modulation of PDK1. Moreover, our results the cell supernatant. An aliquot of ATP detection working uncovered a responsive cancer subset to PDK1 inhibition, which solution was added to a black 96-well culture plate and was fi might be translated to improve the clinical bene ts of PDK1 incubated for 5 minutes at room temperature. Then, the cell inhibitors. lysate was added to the wells, and the luminescence was measured immediately. The readout was normalized by the protein Materials and Methods amounts of each well. ELISA-based kinase activity assay The 6xHis-tagged full-length coding sequences of PDK1-4 Biotinylated JX06 pull-down assay and PDHA1 were expressed in E. coli and purified with Ni-NTA Cells were lysed with NP-40 lysis buffer (Beyotime). Biotiny- column following the manufacturer's instructions (Qiagen). lated pull-down assay was performed with biotinylated JX06 at m Enzymatic reaction was carried out with indicated enzyme indicated dose and 50 L streptavidin sepharose beads (Life (0.25 mg/well), substrate (0.5 mg/well) and ATP (10 mmol/L) Technologies) in NP-40 lysis buffer (Beyotime). Wash buffer contained extra 1% SDS in addition to the NP-40 buffer. in 100 mL buffer (50 mmol/L HEPES, 10 mmol/L MgCl2 and 1 mmol/L EGTA). After reaction, the plate was washed with Tween-PBS, followed by the incubation with primary antibody Oxygen consumption rate and extracellular acidification rate (p-PDHA1, S293; Abgent) and horseradish peroxidase–conju- analysis gated secondary antibody (Calbiochem). The plate was washed Cells were planted into XF96 cell culture plates (Seahorse and visualized with citrate buffer containing 0.1% H2O2 and Bioscience). Each XF96 assay well was equipped with a dispos- 2 mg/mL o-phenylenediamine and the absorbance was mea- able sensor cartridge and embedded with 96 pairs of fluorescent sured at 490 nm after termination. biosensors (oxygen and pH), coupled to fiber-optic waveguides. The measurement of oxygen consumption was expressed fi Cell culture in pmol/min and extracellular acidi cation rate was expressed EBC-1 cells were obtained from Japanese Research Resources in mpH/min. Bank (Tokyo, Japan). Kelly cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). ROS measurement 786-O, L02, and NCI-H460 cells were obtained from the cell Cells after JX06 treatment for 24 hours were incubated with bank of Chinese Academy of Sciences (Shanghai, China). SMMC- 3 mmol/L dihydroethidium (Invitrogen) in PBS for 20 minutes 7721 cells were gifted by the Second Military Medical University at 37 C. Intracellular ROS was measured by FACS analysis (Shanghai, China). HeLa cells were obtained from Shanghai (FACSCalibur flow cytometer; BD Biosciences). Cancer Institute (Shanghai, China). SKOV-3 cells were obtained from Fudan University Shanghai Cancer Center (Shanghai, Chi- Mitochondrial membrane potential na). GM00637 cell line was gifted by Dr. Yves Pommier (NCI, The mitochondrial membrane potential was determined by Bethesda, MD). Other cells used in this study were obtained from measuring TMRM retention (red fluorescence) using IN Cell American Type Culture Collection. Cell lines were authenticated Analyzer 2000 (GE Healthcare).

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Apoptosis assay reactivation of mitochondrial respiration resulted from PDK Apoptotic cells were measured by Annexin V and propidium inhibition. In the meanwhile, aerobic glycolysis determined by iodide (PI) dual-staining using the Annexin V-FITC Apoptosis the lactate production was significantly diminished by JX06 Detection Kit (Vazyme) followed by FACS analysis. treatment (Fig. 1H), which indicated a metabolic switch from aerobic glycolysis to oxidative phosphorylation (19, 30). Further, Cell viability assay JX06 dose-dependently suppressed the growth of A549 cells while Cells were seeded into a 96-well plate. After attachment, cells overexpression of PDK1 rescued cells from JX06-caused prolifer- were treated with JX06. CCK8 assay (Life Technologies) was ative inhibition (Fig. 1I). Consistently, depletion of PDK1 com- carried out after incubation for 72 hours. Untreated cells served promised cell sensitivity toward JX06 (Fig. 1J), suggesting that as the indicator of 100% cell viability. PDK1 inhibition largely accounted for the growth impendence of cancer cells caused by JX06. Molecular docking These results together demonstrated that JX06 was a selective Missing residues (residue 68–71, 168–169, 204–214, and PDK inhibitor. JX06 treatment led to metabolic alterations that 415–416) of PDK1 (PDB entry: 2Q8F1) were complemented redirected glucose flux from aerobic glycolysis to mitochondrial using "Build Homology Models" Module of Discovery Studio oxidation. PDK1 inhibition largely accounted for the cancer cell 2.5 (Accelrys Software Inc.). Then molecular docking was per- proliferative suppression caused by JX06. formed with Autodock 4.0 software. The binding site was defined as a sphere with a radius of 10 Å around the sulfur atom of Cys240. Cancer cells with high dependency on glycolysis are more AutoDock Tools (ADT) was used to prepare the grid parameter sensitive to PDK1 inhibition files (gpf) and the docking parameter files (dpf). The Lamarckian PDK1 inhibition is known to provoke an increase in intracel- genetic algorithm (LGA) was employed to explore the optimal lular ROS (18), which may result from decreased generation of chemical space in the binding site (29). Docking parameters were reducing substances, mainly NAPDH, for ROS detoxification kept at the default values. (31). Mechanistically, ROS generation and consequent mitochondrial membrane potential (MMP) reduction are believed to Statistical analysis account for PDK1 inhibition caused apoptosis of cancer cells (32). Statistical significance was analyzed using the Student t test or In an effort to examine the cellular impacts of JX06 in human one-way ANOVA, and P < 0.05 was considered significant. cancer cells, we unexpectedly discovered that cancer cell lines exhibited distinct outcomes in ROS generation caused by JX06, despite similarly inhibited PDK1 signaling (Fig. 2A and Supple- Results mentary Fig. S3). Moreover, the variation in the amount of ROS JX06 is a potent and selective inhibitor of PDK generation was closely associated with MMP reduction and apo- We first examined the activity of JX06 against all PDK isoforms ptotic occurrence (Fig. 2B and C). For example, A549 and EBC-1 using a biochemistry enzymatic assay. JX06 dose-dependently cells, which were more responsive than the other two cell lines in inhibited PDK1, PDK2, and PDK3, with a half-maximal inhibi- terms of ROS generation after exposure to JX06, showed a dra- tory concentration (IC50) value of 0.049 0.003 mmol/L, 0.101 matic decrease in MMP and an increase in the ratio of apoptotic 0.028 mmol/L, and 0.313 0.011 mmol/L, respectively (Fig. 1A). cells. In contrast, JX06 treatment–triggered responses of ROS The potency of JX06 was more than 100-fold higher than generation, MMP alteration, and cell apoptosis in H460 and DCA (Supplementary Table S2), a pan-PDK inhibitor known for HT29 cells were either marginal or undetectable (Fig. 2A–C). its benefits in glioblastoma patients (25). But JX06 barely Our results above suggested the existence of a responsive subset showed inhibitory activity against PDK4 at a concentration up to PDK1 inhibition among different cancer cell lines, which was to 10 mmol/L (Supplementary Table S2), suggesting JX06 as a pan- intriguing and barely explored yet. We first examined whether the inhibitor of PDK1, 2, and 3. Owing to the most significant role of variable PDK1 abundance could explain the diverse sensitivity of PDK1 among the PDK subtypes in cancer therapy, our efforts were cancer cells to JX06, however, the apparent association between thus particularly devoted to understand the impact of JX06 on PDK1 protein level and the JX06 sensitivity was not observed PDK1. Inhibition of PDK1 by JX06 led to the functional activation (Supplementary Fig. S4). We then speculated that basal glucose of PDC (Fig. 1B). To examine whether JX06 specifically targeted metabolic capacity of cancer cells might play a key role in PDKs, JX06 activity was profiled in a board panel of 323 kinases, determining the outcomes of PDK1 inhibition in cancer cells. To which covered most known kinases implicated in cancer malig- test this hypothesis, we used seahorse XF 96 Analyzer to measure nancy (Supplementary Table S3). Among the tested kinases, only the two major glucose metabolic pathways of 20 cell lines, PDKs and FAK were remarkably inhibited by JX06 at the concen- including various cancer and normal cells. The extracellular tration of 10 mmol/L (Fig. 1C). However, FAK inhibition could not acidification rate (ECAR) indicated the glycolytic capacity, where- be recapitulated at the cellular level (Supplementary Fig. S2). as the oxygen consumption rate (OCR) reflected the oxidative These data suggested that JX06 was a selective and potent inhibitor phosphorylation level of the tested cells (30, 33). The ratios of of PDK. ECAR to OCR (ECAR/OCR), which indicated the relative reliance The activity of JX06 toward PDK1 suggested its potential value of the cell lines on glycolysis or mitochondria respiration, were in anticancer therapy. We then proceeded to assess the impacts of compared among the cell lines. As expected, the three normal cell JX06 in A549 lung cancer cells. JX06 decreased PDHA1 phos- lines (L02, WI-38, and GM00637) all exhibited low ECAR/OCR phorylation at both serine 293 and serine 232 (S293 and S232) in ratios, which was in agreement with the current knowledge that a time- and dose-dependent manner (Fig. 1D and E). Glucose mitochondrial respiration plays a dominant role in glucose uptake and intracellular ATP level in A549 cells were significantly metabolism of normal cells. Meanwhile, we observed an apparent increased by JX06 treatment (Fig. 1F and G), suggesting the variation in ECAR/OCR ratios among these cancer cells. Cells like

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Figure 1. JX06 speciﬁcally inhibits PDK1 and causes metabolicswitch from aerobic glycolysis to oxidative phosphorylation in cancer cells. A, enzymatic activity againstPDK1,2,3.B,PDC reactivation. C, selectivity against a panel of kinases. D and E, time-, and dose-dependent inhibition of PDHA1 phosphorylation in A549 cells. Cells were treated with JX06 at 10 mmol/L for indicated durations (D) or indicated concentrations (E) for 24 hours. PDHA1 phosphorylation was detected using immunoblotting. F–H, metabolic alterations in glucose uptake (F), intracellular ATP amount (G), and extracellular lactate production (H). I, ectopic expression of PDK1 rescued JX06 inhibited cell growth. J, depletion of PDK1 compromised cell sensitivity toward JX06. All the results were normalized to the protein amounts. Mean SE (n ¼ 3); , P < 0.05; , P < 0.01..

A2058, BXPC-3, and A549 exhibited high ECAR/OCR ratios, suggesting they were more dependent on mitochondrial function suggesting a dominant reliance on glycolysis (Fig. 2D). In con- (Fig. 2D). We then subgrouped these cancer cells at a cutoff ECAR/ trast, HeLa and HT-29 cells harbored low ECAR/OCR ratios, OCR value of 0.5 and examined their sensitivity to JX06. Indeed,

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Figure 2. JX06 induces ROS generation and cell apoptosis in cancer cells with high ECAR/OCR. A, mitochondrial ROS generation. Cancer cells were treated with JX06 at 10 mmol/L for 24 hours. B, mitochondrial membrane potential. Cells were treated with JX06 at 10 mmol/L for 24 hours. C, cell apoptosis. Cells were treated with JX06 at 10 mmol/L for 48 hours. D, ratio of ECAR and OCR in a panel of cancer and normal cells. E, cell sensitivity toward JX06. Cell viability was measured using the CCK-8 assay. F, cell survival after depletion of PDK1. Mean SE (n ¼ 3); , P < 0.01. cells with higher ECAR/OCR ratio tended to be more responsive to JX06 (Fig. 2E). The variable dependency of these cells on PDK1 JX06 treatment. Normal cells and cancer cells with higher capacity activity was also conﬁrmed by PDK1 depletion using PDK siRNA in mitochondrial respiration exhibited much less sensitivity to (Fig. 2F and Supplementary Fig. S5).

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Our results above revealed an intriguing phenomenon that not observe the body weight loss in JX06-treated mice, suggesting cancer cells responded differently to PDK1 inhibition, and cells that JX06 was well tolerated at the administration dose (Fig. 3C). with higher dependency on aerobic glycolysis, which could be In agreement with the marked antitumor efficacy of JX06, the measured by ECAR/OCR ratio, might be the responsive cancer intratumoral PDK1 signaling was significantly inhibited, as subset to PDK1 inhibition. shown by the blockage of the PDHA1 phosphorylation as well as the decrease of plasma lactate level in JX06-treated mice (Fig. JX06 attenuates tumor growth in A549 xenograft models 3D and E). To further prove the therapeutic potential of JX06 via PDK1 Meanwhile, JX06 showed no significant antitumor activity in inhibition in the responsive cancer subset, we tested its antitumor HT-29 models despite of similar inhibition of PDK1 pathway, efficacy in A549 subcutaneous xenograft mice models. Tumor- further validating our above observations in cell lines (Fig. 3F and bearing mice were randomly divided into three groups and G). These results also largely excluded the involvement of other intraperitoneally received vehicle control or JX06 at 40 and potential targets in the mediating the anticancer efficacy of JX06. 80 mg/kg per day, respectively. A 21-day continuous treatment of 80 mg/kg JX06 considerably reduced 67.5% tumor volume Structure–activity relationship analysis identifies the compared with the vehicle control (Fig. 3A). Endpoint tumor pharmacophore of JX06 weights in the JX06 treated group were significantly less than those The remarkable selectivity and potency of JX06 against PDK1 treated with vehicle control (Fig. 3B). In the meanwhile, we did inspired us to further explore the molecular mechanisms of PDK1

Figure 3. JX06 inhibits tumor growth in vivo.A–E, A549 xenograft mouse models were treated with JX06 or vehicle control daily at indicated dosages for 21 consecutive days. A, tumor volume change. B, tumor weight after last dosing. C, body weight change. D, intratumoral PDHA1 phosphorylation. E, serum lactate level. F and G, HT-29 xenograft mouse models were treated with JX06 or vehicle control daily at indicated dosages for 21 consecutive days. F, tumor volume change. G, intratumoral PDHA1 phosphorylation. One-way ANOVA analysis, , P < 0.01.

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inhibition by JX06. To this end, we employed the structure– activity. In contrast, the replacement of the N-substituents with activity relationship (SAR) analysis to identify the key moieties other alkyls, such as -C2H5 (JX05), - (CH2)4- (JX07), -(CH2)5- of JX06 that might critically interact with PDK1. There are three (JX08), and -(CH2)2O(CH2)2- (JX06), showed only a marginal common moieties included in the chemical structures of both effect on the potency of the inhibitor, suggesting that the binding thiram and JX06, namely a central disulfide bond, two thioamides pocket of N-substituents was more accommodative. and symmetrical terminal N-methyl substituents. These moieties The SAR data aforementioned suggested a mechanism that were individually removed or modified to examine their impacts involved a disulfide bond and an adjacent thioamide in critically on the activity against PDK1 (Fig. 4A). Compounds JX02, 03, 04, interacting with PDK1. The susceptibility of this moiety to reduc- and 12, in which the disulfide bond was replaced, completely lost tion lightened us to propose a possibility of its covalent interac- the activity against PDK1, suggesting that the disulfide bond was tion with the thiol side chain of cysteine residuals in PDK1. Likely, essential for PDK1 inhibitory activity. Likewise, replacing sym- the thiol of cysteine attacks the chemically reactive thioamide and metrical thioamides with amide (JX09, JX10) or ketones (JX11) forms a new disulfide bond with JX compounds, resulting in the was not beneficial as well. However, it appeared that retaining one breakage of the disulfide bond of JX06 (Fig. 4B). To prove this thioamide was sufficient to sustain most of PDK1 inhibitory hypothesis, glutathione (GSH) that contains a reducing thiol

Figure 4. SAR analysis identifies pharmacophore of JX06. A, SAR analysis identified the key pharmacophore of JX06, i.e., a disulfide bonds (red) and two thioamides (magenta). B, proposed chemistry reaction between JX06 and sulfhydryl. C, inhibition curve of JX06 toward PDK1 under different concentrations of GSH.

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group was introduced to interrupt the disulfide bonds possibly protein and JX06 was submitted for mass spectrometry analysis. formed between PDK1 and JX06. As expected, the inhibitory The results showed that C240 residue in PDK1 was modified and activity of JX06 was dramatically reduced with the augment of the molecular weight shift matched with JX06 (Fig. 5H). Although GSH concentration in a dose-dependent manner, while in con- we could not rule out the possibility that similar modifications trast the efficacy of DCA was not affected (Fig. 4C). occurred on other cysteine, it largely appeared that covalent interaction with C240 fundamentally accounted for the enzymat- JX06 inhibits PDK1 by covalently binding to a conserved ic inhibition by JX06. cysteine residue Together, our data suggested that JX06 covalently bound to a To further understand the binding and inactivating steps of highly conserved cysteine residue, C240 in PDK1, which gave rise PDK1 inhibition by JX06, we examined the time-dependent to strong inhibition of the enzymatic activity. inhibition of PDK1 by JX06. The enzymatic assay of PDK1 was initiated in the presence of JX06 at 50 nmol/L following JX06 recognizes a hydrophobic pocket in PDK1 30-minute preincubation. The enzymatic activity was measured The specificity of covalent inhibitors is known to stem from the over time up to 60 minutes, and the noncovalent irreversible recognition of a binding pocket, which restricts its specific acces- inhibitor DCA was used as a control. The results suggested that sibility to the targeted residues to undergo a follow-up chemical distinct from DCA, preincubation of JX06 apparently influenced reaction. We next studied whether there existed a binding pocket the time-dependent inhibition curve of JX06, supporting the in the surroundings of the targeted cysteine that allowed the disulfide bond formation between JX06 and PDK1 (Fig. 5A). specifi c modification by JX06. For further confirmation, we generated a biotin-conjugated To address this question, we took the advantage of the previ- JX06 to test whether JX06 was able to covalently bind to endog- ously solved crystal structures of PDK1. Molecular docking enous PDK1 derived from cancer cells. Biotin-conjugated JX06, approach was used to analyze the binding mode of JX06 to PDK1. which retained the activity against PDK1 (Supplementary Fig. S6), JX06 was docked into PDK1 (PDB entry: 2Q8F1) with Autodock was preincubated with A549 cell lysates, and JX06-associated 4.0 (34) and a sphere with a radius of 10 Å around the sulfur atom proteins were enriched by affinity purification with streptavidin of C240 was defined as the binding site. JX06 suited quite well in beads. Afterwards, proteins possibly associated via noncovalent the binding pocket near C240 and formed hydrophobic interac- bonds were removed by sufficient wash using wash buffer con- tions with the surrounding residues (Fig. 6A and B). Remarkably, taining 1% SDS. The presence of PDK1 in the resultant complex the sulfur atom of JX06 was located within 3.8 Å distance of the was examined by immunoblotting. Indeed, PDK1 protein was sulfur atom of C240, offering a possibility to form a disulfide detected to be binding to biotin-conjugated JX06 in a dose- bond between them. To test the dependency of JX06 on this dependent manner (Fig. 5B), and this binding was largely reduced binding pocket, we designed a new JX06 derivative, named JX86, when biotin-conjugated JX06 was premixed with 30-fold of label- which contained the intact key moieties but was unable to fit into free JX06 during incubation with cell lysates (Fig. 5C). In contrast, this binding pocket. As expected, the inhibitory activity of JX86 the addition of label-free JX06 after a 12-hour incubation barely toward PDK1 was significantly reduced compared with JX06 (Fig. affected the binding between PDK1 and biotin-conjugated JX06 6C). Also, we introduced point mutations to the key surrounding (Fig. 5D), suggesting that regardless of the sufficient amount, residues that were identified to interact with JX06 in the binding label-free JX06 failed to interrupt the bond of biotin-labeled JX06 pocket, aiming at interfering JX06's docking by changing the space and PDK1 once the chemical reaction was completed. These data of the pocket or disrupting the electrostatic interactions. Accord- together strongly suggested the covalent association between JX06 ing to the sidechain conformations, M289F mutant was designed and PDK1. to reduce the space of the pocket, Y243A and Y289A mutants to We then proceeded to identify the cysteine residues involved in give rise to a bigger pocket, and E290K mutant to impact the the formation of the covalent bond with JX06. All the four cysteine electrostatic interactions. Indeed, all these point mutations more residues of PDK1 were individually mutated according to the or less affected the potency of JX06 toward PDK1 and dual prediction of Sorting Intolerant From Tolerant (SIFT) to retain mutations combining Y243A and M289A significantly reduced PDK1 activity maximally. Four PDK1 mutants, namely C71S, the potency of JX06 (Fig. 6D), although these mutations per se did C223T, C240L, and C421S, were generated. Enzyme amount not affect the catalytic activity of PDK1 (Fig. 6E). These data titration assay showed that C71S, C223T, and C421S maintained demonstrated that the inhibitory effect of JX06 on PDK1 required their enzymatic activity as the wild-type PDK1. The C240L mutant the specific recognition of the unique binding pocket. largely lost its catalytic capacity in an enzymatic assay (Fig. 5E), Importantly, the identification of this pocket provided answers and ectopic expression of C240L mutant failed to rescue the to a previous puzzle that JX06 was lacking the inhibitory capacity defects in PDHA1 phosphorylation or cell growth of PDK1 against PDK4 (Supplementary Table S2). PDK4 similarly pos- depleted cells (Supplementary Fig. S7). These data showed that sessed a conserved cysteine C215, which was essentially required C240 was crucial for the catalytic function of PDK1. We also for the enzymatic activity of PDK4 as well (Fig. 6F). Molecular noticed that C240 in PDK1 was highly conserved across evolu- modeling of JX06 to PDK4 indicated that the sulfur atoms of C215 tion, from Arabidopsis to human, supporting its critical role in and JX06 were in a distance of 8.8Å, which was beyond the range sustaining the catalytic function of PDK1 (Fig. 5F). We then possibly to form a chemical bond (Fig. 6G and H). These data determined the inhibitory activity of JX06 toward these four explained the selectivity of JX06 between PDK1 and PDK4. mutants. JX06 largely lost its activity toward C240L but not to other three mutants (Fig. 5G), whereas all these mutants Covalent modification by JX06 reduces ATP affinity of PDK1 responded similarly to DCA inhibition. These data suggested that Our data demonstrated the essential role of C240 in modulat- JX06 inhibited PDK1 activity via interacting with the C240 res- ing the enzymatic activity of PDK1. However, it remained unclear idue. For further validation, the mixture of recombinant PDK1 how this residue was critically involved in the catalytic function of

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Figure 5. JX06 inhibits PDK1 activity via covalently binding to a cysteine residue in an irreversible manner. A, covalent inhibition of PDK1 by JX06. B–D, PDK1 binding to biotin-conjugated JX06. PDK1 was copurified with biotin-conjugated JX06 in A549 cell lysate after incubation in the absence (B) or presence (C) of 30 folds of free JX06. D, coincubation with 30 folds of free JX06 was conducted after binding reaction. E, enzyme titration of indicated PDK1 mutants. F, alignment of PDK1 C240 across diverse species. G, IC50 of JX06 and DCA against mutated PDK1. H, mass spectrometry identified PDK1 C240 modification by JX06. Mean SE (n ¼ 3).

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Figure 6. The binding pocket of JX06 in PDK1. A, the binding pocket of JX06 (white sticks) in PDK1 (green surface). C240 is shown as green sticks. B, detailed interactions between JX06 (white sticks) and its surrounding residues in PDK1 (green sticks). PDK1 is shown as light green cartoon. C, superposed structures of JX86 (orange sticks) with JX06 (white sticks). D, JX06 activity against PDK1 mutants. E, enzymatic activity of PDK1 mutants. F, enzyme titration of PDK4 C215L. G, molecular model of the PDK4–JX06 complex. The binding pocket of JX06 (white sticks) in PDK4 (green surface). C215 is shown as green sticks. H, detailed interactions between JX06 (white sticks) and its surrounding residues in PDK4 (green sticks).

PDK1. PDK1 has been known to interchange between two con- compared the structures of open conformation of PDK1 (PDB formational states (24, 35–37). The closed conformation, where entry: 2Q8F) and closed conformation of PDK2 (PDB entry: the C-terminal tail of each PDK1 subunit loses its interaction with 1JM6; Fig. 7A; refs. 24, 38). It was observed that the sidechain the lipoyl-bearing domain (L2), has the active-site cleft closed orientations around C240 barely differed in these two conforma- inside. It exhibits a high binding affinity to ADP and hence stays in tions, suggesting that binding of JX06 with C240 unlikely inter- an inactive state (38). Upon the binding of L2 to PDK1, the C- fered with the transformation between the open and closed states. terminal tails form a crossover configuration and thus the active- We next attempted to seek for other possibilities. Notably, site cleft is widened, which leads to an open conformation. The N283/R286 in PDK1 differed dramatically in the sidechain ori- open conformation preferentially binds to ATP rather than ADP entation between the open and closed conformations (Fig. 7B and and activates its catalytic function. Comparing the closed and the C). Furthermore, we ran a 200-nanosecond molecular dynamic open conformations might provide important clues about how simulation of the open conformation of PDK1 and found out that C240 was involved in the regulation of PDK1 activity. We hence the sidechain of R286 swung flexibly (Fig. 7D and Supplementary

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Figure 7. JX06 binding reduces ATP afﬁnity of PDK1. A, superposed structures of residues around C240 in PDK1 open conformation and PDK2 closed conformation. Residues around C240 in PDK1 are shown as green sticks and labeled in green letters. Residues around C204 in PDK2 are shown as magenta sticks. The residues shown as sticks are R237/K201, R238/L202, I239/L203, C240/C204, D241/D205, L242/K206, Y243/Y207, Y244/Y208, I245/M209, N246/A210, S247/S211, P248/P212, M285/M249, and M289/V253 in PDK1/PDK2. PDK1 and PDK2 are shown as light green and magenta cartoons, respectively. B, the electrostatic surfaces of PDK1 (blue, positive charge; red, negative charge). JX06 and ATP are shown as sticks. Two residues (N283 and R286), which are likely involved in ATP binding are shown as green sticks. C, superposed structures of PDK1 open conformation and PDK2 closed conformation. C240/N283/R286 of PDK1, C204/N247/R250 of PDK2, JX06andATP are shown as sticks. PDK1 and PDK2 are shown as light green and magenta cartoons, respectively. D, structures extracted from the trajectory of molecular dynamic simulation. PDK1 at 0 nanoseconds is shown as green cartoon. C240, N283, and R286 at 0 nanoseconds are shown as green sticks and the ones extracted from molecular dynamic simulation are shown as magenta sticks. JX06 and ATP binding sites are shown as dotted ovals. E, PDK1 R286A and R286I enzymatic activity in different amounts of ATP. F, JX06 activity against PDK1 R286A and R286I mutants. G, a scheme showing proposed mechanism of JX06 in PDK1 inhibition.

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Movie S1; ref. 24). In addition, we noticed that R286 was located Theoretically, the cysteine should be essential for the enzymatic adjacent to both JX06-binding pocket and ATP-binding pocket activity of the targeted protein. In the meanwhile, surroundings (Fig. 7C). We hence speculated that conformational changes favorable for the inhibitors landing are required to allow the disturbed by JX06 might be transferred by R286 to the ATP- follow-up chemical reaction and confer the selectivity of the binding site, thus affecting the PDK1 activity. To test this possi- inhibitor (48). Lack of this knowledge has largely restricted the bility, we generated PDK1 R286A and R286I mutants and discovery of the covalent inhibitors for most kinases, which is observed that both mutants exhibited compromised enzymatic alsothecaseforPDK1.Inthisstudy,wediscoveredapreviously activity (Fig. 7E). In agreement with our speculation, the increase unappreciated cysteine, C240 in PDK1, which was essential for of ATP amount was able to rescue the enzymatic activity of these its enzymatic activity. Covalent modification on this residue two mutants, suggesting that R286 might play a critical role in induced conformational changes of arginine 286 through Van modulating affinity between PDK1 and ATP (Fig. 7E). More der Waals forces, which likely interfered the access of ATP to importantly, JX06 greatly lost its inhibitory activity toward PDK1 and in turn impaired the kinase activity of PDK1. To our R286 substitution mutants (R286A, R286I), whereas the allosteric knowledge, this study provided the first evidence suggesting the inhibitor DCA exhibited similar activity against the mutant and essential role of C240 in PDK1. We have also noticed that a wild-type PDK1 (Fig. 7F). These data together supported our previous study reported that oxidation of cysteine residues 45 hypothesis that R286 residue played an irreplaceable role in and 392 of PDK2 by hydrogen peroxide resulted in its catalytic mediating the inhibitory activity of JX06, and the conformational suppression, although the molecular mechanism remained changes of R286 caused by JX06 impaired PDK1 enzymatic unknown (49). The counterpart residues of these two cysteines activity via reducing the ATP affinity. in PDK1 are cysteine 71 and 421. Our data have shown that These data together provided important evidence to under- PDK1 C71S and C421S mutants retained their enzymatic stand the molecular basis of JX06. Covalent modification by JX06 activity, largely excluding their critical involvement in deter- on PDK1 C240 induced confirmation changes of R286 through mining the catalytic activity of PDK1 (Fig. 5E). Also, it would be Van der Waals forces, which was further transported to the ATP- interesting to investigate whether cysteine 240 in PDK1 can be binding site and thus affected the binding affinity between ATP posttranslationally modified to fine tune the enzymatic regu- and PDK1 (Fig. 7G). lation of PDK1. Moreover, we identified a unique hydrophobic pocket, which provided a venue for small molecules to access to C240 within a distance allowing chemical modification. This Discussion information could contribute to the rational design of covalent PDK1 is one of the most extensively investigated anticancer inhibitors of PDK1. Also, we proved that the first covalent targets in the aerobic glycolysis pathway, although most inhi- PDK1 inhibitor JX06 elicits potent inhibition to PDK1, which bitors are still at early stage preclinical research. The discovery could be translated to metabolic alterations and optimal anti- of these PDK1 inhibitors has benefited from the mechanistic tumor efficacy. insights into the enzymatic regulation of PDK1. Phosphoryla- A very recent study has shown that breast cancer T-47D and tion of PDHA1 by PDK1 occurs in the PDC, where PDK1 is MDA-MB-231 cells exhibited distinct metabolic responses to recruited to the complex through binding to E2 subunit (8, 39). DCA inhibition, suggesting the potential discrepancy in anti- Disrupting the interaction between PDK1 and E2 via binding to cancer efficacy of PDK1 inhibition among different cancer cells the lipoyl-binding pocket in the PDK1 N-terminal has given rise (20). Our study extended this observation by proving the to the discovery of AZD7545 (23, 40). Despite its remarkable variations in the antitumor efficacy of PDK1 inhibition in a glucose-lowering effect, the anticancer efficacy of AZD7545 broad panel of cancer cells, and identifying a subset cancer cells seems obscure (23). Like other kinases, binding to ATP-binding more responsive to PDK1 inhibition. These results intrigued us pocket of PDK1, also known as GHKL (gyrase, Hsp90, histidine to investigate the molecular indicators that might possibly kinase, and MutL) domain, to block ATP entry also results in allow us to identify the responsive subset. Indeed, the ratios PDK1 inhibition (41). However, the fact that GHKL domain of ECAR to OCR were closely related to the sensitivities to PDK1 widely exists in several other kinases compromised the speci- inhibition. According to our results, equivalent PDK inhibition ficity of ATP-completive PDK1 inhibitors such as radicicol caused variable responses in ROS generation and MMP level (38, 42). DCA is the most advanced PDK1 inhibitor that has change in cancer cells. In the subset cells with less reliance on shown preliminary benefits in glioblastoma patients (25). glycolysis, such as H460 cells, the MMP level posttreatment Mechanistically, DCA is a pyruvate analog that promotes con- intended to remain at a hyperpolarizing state (Supplementary formational changes at the active-site cleft of PDK1 and hinders Fig. S8), which explained the resistance to the apoptosis induc- thedissociationofADPfromtheactivesite(24,43).However, tion in these cells (19, 25). Encouragingly, technical campaign its potency in PDK1 inhibition is relatively low. The IC50 value aiming at directly measuring the ECAR/OCR ratio of fresh of DCA for enzymatic inhibition is at millimole level (19, 22). tumor tissue is undergoing at the moment. And its practice Thus far, explorations into all these known mechanisms appear can be expected in the very near future. This technical break- not very successful. through will make it possible to test our observations in clinical Lately, covalent inhibitors have gained increasing attention settings. for their pharmacologic advantages (44). Covalent inhibitors In summary, our fi ndings might contribute to the field in three have been demonstrated to hold the promise in overcoming the aspects: first, we gained new insights into the internal enzymatic challenges of potency, selectivity, efficacy, and resistance faced regulation of PDK1, which involved a previously unappreciated by noncovalent inhibitors (45–47). Currently, structure-based cysteine; second, we identified the first PDK1 covalent inhibitor approaches are employed to design small molecules that target and proved the possibility of the rationale design of PDK1 kinases through covalent attachment to a specificcysteine. covalent inhibitors; third, we showed that the intrinsic metabolic

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status of cancer cells varied among the cells and determined their Administrative, technical, or material support (i.e., reporting or organizing responsiveness to PDK1 inhibition. ECAR/OCR ratio reflected data, constructing databases): D. Zhao, D. Zhang, S. Tang addiction to aerobic glycolysis and might be used to select cancer Study supervision: J. Ding, J. Li, M. Huang, M. Geng patients receiving metabolic-modulating drugs. Grant Support fl This work was supported by the National Program on Key Basic Research Disclosure of Potential Con icts of Interest Project of China (No.2012CB910704 to M. Geng), grants from National fl No potential con icts of interest were disclosed. Natural Science Foundation of China (No. 81222049 to M. Huang, No. 21222211 to J. Li, and No. 81202549 to Z. Xie), and the Natural Science Authors' Contributions Foundation of China for Innovation Research Group (No. 81321092 to J. Ding). fi Conception and design: Z. Xie, D. Zhao, J. Li, M. Huang, M. Geng The PDK enzymatic assay was established with the nancial support by Actelion Development of methodology: W. Sun, Z. Xie, Y. Liu, D. Zhao, H. Lv Pharmaceuticals Ltd. Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the provided facilities, etc.): W. Sun, Z. Xie, D. Zhao, Z. Wu, N. Jin, M. Tan, J. Li payment of page charges. This article must therefore be hereby marked Analysis and interpretation of data (e.g., statistical analysis, biostatistics, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate computational analysis): W. Sun, Z. Xie, Y. Liu, D. Zhao, Z. Wu, H. Jiang, this fact. M. Tan, C. Luo, J. Li Writing, review, and/or revision of the manuscript: W. Sun, Z. Xie, D. Zhao, Received April 16, 2015; revised August 2, 2015; accepted August 4, 2015; Z. Wu, C. Luo, J. Li, M. Huang, M. Geng published OnlineFirst October 19, 2015.

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OF14 Cancer Res; 75(22) November 15, 2015 Cancer Research

JX06 Selectively Inhibits Pyruvate Dehydrogenase Kinase PDK1 by a Covalent Cysteine Modification

Wenyi Sun, Zuoquan Xie, Yifu Liu, et al.

Cancer Res Published OnlineFirst October 19, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1023

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