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A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals

Open AccessPublished:August 14, 2019DOI:https://doi.org/10.1074/jbc.RA119.008777
      The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKCδI is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKCδI activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKCδI isozymes and a caspase-3 binding site on the PKCδI hinge domain with Schrödinger software and molecular dynamics simulations, we identified NP627, an organic small-molecule inhibitor of PKCδI. Characterization of NP627 by surface plasmon resonance (SPR) revealed that PKCδI and NP627 interact with each other with high affinity and specificity, SPR kinetics revealed that NP627 disrupts caspase-3 binding to PKCδI, and in vitro kinase assays demonstrated that NP627 specifically inhibits PKCδI activity. The SPR results also indicated that NP627 affects macromolecular interactions between protein surfaces. Of note, release of the PKCδI catalytic fragment was sufficient to induce apoptosis and inflammation in adipocytes. NP627 treatment of adipocytes from obese individuals significantly inhibited PKCδI catalytic fragment release, decreased inflammation and apoptosis, and significantly improved mitochondrial metabolism. These results indicate that PKCδI is a robust candidate for targeted interventions to manage obesity-associated chronic inflammatory diseases. We propose that NP627 may also be used in other biological systems to better understand the impact of caspase-3–mediated activation of kinase activity.

      Introduction

      The obesity epidemic continues to rise in populations worldwide. Obesity per se is not fatal; however, the metabolic consequences and sequalae of obesity promote life-threatening morbidities such as cardiovascular diseases, metabolic syndrome, type 2 diabetes, gout, and osteoarthritis. Adipose tissue is an important endocrine regulator of energy homeostasis and glucose metabolism. Fat accumulation in omental and subcutaneous abdominal depots is central to obesity and its associated diseases. Adipose tissue maintains a balance between lipogenesis (energy preservation during the postprandial period) and lipolysis (energy expenditure) via hormones and signaling pathways. Obesity results in altered profiles of adipokines (hormone and cytokines) secreted by adipose tissue. Adipose-specific adipokines include leptin and adiponectin, whereas adipocytokines such as TNFα
      The abbreviations used are: TNF
      tumor necrosis factor
      ASC
      adipose stem cell
      ASO
      antisense oligomer
      BMI
      body mass index
      SVF
      stromal vascular fraction
      qPCR
      quantitative PCR
      PI
      Propidium iodide
      MD
      molecular dynamics
      PARP
      poly ADP ribose polymerase
      RMSD
      root mean square deviation
      SPR
      surface plasmon resonance
      RU
      response unit
      MBP
      myelin basic protein
      TMB
      tetramethylbenzidine substrate
      OCR
      oxygen consumption rate
      IRB
      institutional review board
      DAPI
      4′,6-diamidino-2-phenylindole.
      and interleukins are secreted by adipose tissue. Adipose tissue responds to excess energy or nutrition deficiency by increasing or decreasing cell size and mass. This remodeling by adipose tissue is a dynamic process and restricts inflammation and promotes insulin sensitivity. Under conditions of excess energy leading to hypertrophy of adipose tissue, the associated macrophages acquire a pro-inflammatory phenotype. Obesity is accompanied by a chronic low level of inflammation. Apoptosis is also markedly increased in adipose tissue from obese humans and in high-fat diet–induced obesity in rodent models (
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      • Carter G.
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      ). We have shown that PKCδII (mouse splice variant) and PKCδVIII (human splice variant) function as prosurvival proteins (
      • Patel N.A.
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      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ); the functions of other PKCδ splice variants are have not yet been established. PKCδI participates in the regulation of early stages of apoptosis by phosphorylating key apoptotic proteins or in later events by acting downstream of caspases.
      PKCδI has been shown to mediate inflammation in several cell types, including adipocytes, macrophages, vascular smooth muscle cells, and hepatocytes (
      • Tang Y.
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      ). PKCδI is proteolytically cleaved by caspase-3 at its hinge region, separating the regulatory domain from the catalytic domain (
      • Emoto Y.
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      ). The release of the catalytically active fragment induces nuclear fragmentation and apoptosis (
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      • Kanthasamy A.G.
      Caspase-3-dependent proteolytic cleavage of protein kinase Cδ is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl.
      ). Cleavage and activation of PKCδI are critical for its function in mediating signaling cascades in cells (
      • Denning M.F.
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      The protein kinase Cδ catalytic fragment targets Mcl-1 for degradation to trigger apoptosis.
      ).
      For in vivo therapeutic applications, Rottlerin, the previously used inhibitor, has been shown not to be specific for PKCδ (
      • Soltoff S.P.
      Rottlerin: an inappropriate and ineffective inhibitor of PKCδ.
      ). We previously published antisense oligomers (ASOs, Ionis Pharmaceuticals) directed toward the splice sites for PKCδ that could selectively inhibit the specific splice variant (
      • Jiang K.
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      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ). We showed that ASO71 decreased PKCδI, which resulted in a decrease in PARP cleavage (apoptosis marker). The antisense oligomer ASO71 (which decreases PKCδI) and the commercially available PRKCD siRNA (which decreases both PKCδI and PKCδVIII) are not suitable, as these approaches decrease the total PKCδI levels and cannot delineate the role of PKCδI kinase activity specifically in obesity. Moreover, PKCδI levels regulate the cell cycle and adipogenesis (
      • Fukumoto S.
      • Nishizawa Y.
      • Hosoi M.
      • Koyama H.
      • Yamakawa K.
      • Ohno S.
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      Protein kinase Cδ inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression.
      ,
      • Patel R.S.
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      • Apostolatos H.
      • Patel N.A.
      Transformer 2β homolog (Drosophila) (TRA2B) regulates protein kinase CδI (PKCδI) splice variant expression during 3T3L1 preadipocyte cell cycle.
      ); hence, is not advisable to deplete cells of total PKCδI.
      Our strategy was to inhibit the kinase activity of PKCδI, as it has been shown that PKCδI activity is increased in adipocytes of obese mice (
      • Talior I.
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      PKC-δ-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase.
      ,
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      PKCδ regulates hepatic triglyceride accumulation and insulin signaling in Lepr(db/db) mice.
      ). As described above, PKCδI is a serine/threonine kinase that is activated by cleavage at its hinge region, releasing its C-terminal catalytic fragment. This fragment, freed from inhibition by the regulatory domain, is sufficient for its function (
      • Brodie C.
      • Blumberg P.M.
      Regulation of cell apoptosis by protein kinase Cδ.
      ). The cleavage and activation of PKCδI set up a positive feedback loop that impinges upon upstream components of the death effector pathway, thereby amplifying the caspase cascade and helping cells commit to apoptosis (
      • Denning M.F.
      • Wang Y.
      • Tibudan S.
      • Alkan S.
      • Nickoloff B.J.
      • Qin J.Z.
      Caspase activation and disruption of mitochondrial membrane potential during UV radiation-induced apoptosis of human keratinocytes requires activation of protein kinase C.
      ,
      • Sitailo L.A.
      • Tibudan S.S.
      • Denning M.F.
      Bax activation and induction of apoptosis in human keratinocytes by protein kinase Cδ catalytic domain.
      • Sitailo L.A.
      • Tibudan S.S.
      • Denning M.F.
      The protein kinase Cδ catalytic fragment targets Mcl-1 for degradation to trigger apoptosis.
      ). Hence, to specifically inhibit PKCδI-mediated pathways, we chose to inhibit the kinase activity rather than decreasing its levels.
      Here we identified a small molecule that would inhibit cleavage of PKCδI in adipocytes and thereby decrease its kinase activity. We investigated the binding affinity of this small molecule to PKCδI and its ability to inhibit binding of caspase-3 to PKCδI. Using this small molecule to inhibit PKCδI kinase activity, we investigated whether inflammation, apoptosis, and metabolic dysfunction associated with obesity could be reduced in human adipocytes.

      Results

      PKCδI is increased in obese human ASCs and adipocytes

      We determined the expression of PKCδI in human adipose tissues obtained from obese individuals. Omental and subcutaneous adipose tissue from six lean and six obese patients (IRB 20295; obese BMIs between 43 and 45 kg/m2; lean BMIs between 22 and 23 kg/m2; nondiabetic, nonsmokers, other criteria matched; designated as subcutaneous lean, subcutaneous obese, omental lean, and omental obese according to depot and lean/obese status) were evaluated for PKCδI levels, and our results showed increased PKCδI levels in both subcutaneous and omental depots of obese subjects (Fig. 1a). Adipose tissue was digested with collagenase and purified to obtain adipocytes (free from other cells and macrophages) in supernatant, whereas the pellet contained the SVF fraction from which ASCs were isolated. The omental adipocytes were further analyzed by Western blotting. Our results showed increased PKCδI expression and release of its C-terminal catalytic fragment in obese adipocytes. Obese adipocytes had increased cleavage of caspase-3 (increased apoptosis) and higher expression of the adipokine TNFα, indicating increased inflammation (Fig. 1b). Next, the omental ASCs were differentiated into mature adipocytes in vitro as described previously by our laboratory (
      • Carter G.
      • Apostolatos A.
      • Patel R.
      • Mathur A.
      • Cooper D.
      • Murr M.
      • Patel N.A.
      Dysregulated alternative splicing pattern of PKCδ during differentiation of human preadipocytes represents distinct differences between lean and obese adipocytes.
      ). We measured PKCδI levels using SYBR Green qPCR, and our results showed that PKCδI was increased in obese human ASCs and adipocytes (Fig. 1c).
      Figure thumbnail gr1
      Figure 1PKCδI is increased in adipose tissue and adipocytes in obesity. a, adipose tissue was obtained from lean (BMI 22–23 kg/m2) and obese (BMI 43–45 kg/m2) donors (n = 6 each group), digested with collagenase, and purified to obtain adipocytes and ASCs. Total RNA was isolated from different depots: subcutaneous lean, subcutaneous obese, omental lean, and omental obese. Real-time qPCR was performed in triplicate to measure absolute quantification of PKCδI expression using β-actin as an internal control. b, Western blotting was performed on omental adipocytes that were immunoblotted using antibodies against PKCδI, TNFα, caspase-3, and β-actin. Experiments were repeated five times with similar results. The graph represents normalized densitometric units of PKCδ_F (full-length) and PKCδI_C (cleaved) normalized to β-actin obtained in the immunoblots. c, omental ASCs were differentiated in vitro to mature adipocytes. RNA was isolated from ASCs, and adipocytes and PKCδI expression was measured using SYBR Green qPCR. The experiment was repeated five times with similar results. Statistical analysis was performed by two-way analysis of variance. ***, p < 0.001.

      Homology modeling and screening of small molecules binding to PKCδI

      Schrödinger PRIME software was used to perform homology modeling of PKCδI. 3D structures of PKCδ isoforms (
      • Steinberg S.F.
      Structural basis of protein kinase C isoform function.
      ) and crystal structures available on PDB, including 1YRK and 1PTQ, were utilized to create computational chimeras to establish a suitable protein scaffold for docking. These structures were then minimized using the Schrödinger Protein Preparation Wizard and the OPLS 2005 forcefields. De novo protein predictions were also prepared and minimized using Protein Preparation Wizard I-TASSER (
      • Yang J.
      • Yan R.
      • Roy A.
      • Xu D.
      • Poisson J.
      • Zhang Y.
      The I-TASSER suite: protein structure and function prediction.
      ) and RAPTOR X (
      • Gao Y.
      • Wang S.
      • Deng M.
      • Xu J.
      RaptorX-Angle: real-value prediction of protein backbone dihedral angles through a hybrid method of clustering and deep learning.
      ).
      Compounds contained in Chembridge Microformat were prepared for docking to PKCδI by Schrödinger LigPrep. Schrödinger Glide XP was used to screen successful docking events based on the criteria for ligand binding to key PKCδI amino acids and affinity to DXXD(P4-P1)/X (the caspase-3 recognition sequence on PKCδI (
      • Emoto Y.
      • Manome Y.
      • Meinhardt G.
      • Kisaki H.
      • Kharbanda S.
      • Robertson M.
      • Ghayur T.
      • Wong W.W.
      • Kamen R.
      • Weichselbaum R.
      Proteolytic activation of protein kinase Cδ by an ICE-like protease in apoptotic cells.
      )). Results were tabulated using a spreadsheet, and compounds with low ΔG across the models created were selected for in vitro analysis. The leading hits were tested for their ability to inhibit release of the catalytic fragment of PKCδI by treating obese adipocytes with 10 nm compounds. The top candidate, N′∼1∼,N′∼5∼-bis[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanimidoyl] pentanedihydrazide (hereafter referred to as NP627), was selected (Fig. 2, a and b) as a small molecule inhibiting the release of the PKCδI C-terminal fragment. Compound 594 (Cpd594) and compound 118 (Cpd118) were from the same screening process. Cpd594 was used as a control in subsequent experiments, as it is essentially half of the symmetrical NP627 and did not block release of the C-terminal PKCδI fragment in adipocytes (as shown in Fig. 2, a and b).
      Figure thumbnail gr2
      Figure 2NP627 decreased cleavage of the PKCδI C-terminal fragment. a, structure of the symmetrical PKCδI inhibitor NP627 and the control compound Cpd594, which is a monomer of NP627. b, Cpd118 and Cpd594 are from the same screening process as NP627. Western blots were performed on omental obese adipocytes treated with 10 nm NP627, cpd118, or cpd594 and immunoblotted using antibodies against PKCδI and β-actin. The graph shows normalized densitometric units of experiments repeated five times with similar results. Statistical analysis was performed by two-way analysis of variance. **, p < 0.01.

      Molecular dynamics (MD) simulation for PKCδI and NP627

      The structure of PKCδI obtained from homology modeling was optimized using the QwikMD plugin for Visual Molecular Dynamics (VMD) (
      • Ribeiro J.V.
      • Bernardi R.C.
      • Rudack T.
      • Stone J.E.
      • Phillips J.C.
      • Freddolino P.L.
      • Schulten K.
      QwikMD: integrative molecular dynamics toolkit for novices and experts.
      ). The PKCδ1 model was equilibrated in solvent containing 150 mm NaCl for 25 ns with the root mean square deviation (RMSD) over time, indicating a deviation from the originally produced model (Fig. 3a). This is due to compaction of the protein over the course of the MD simulation, as indicated by a lowering of dihedral angle energy (Fig. 3b), as shown in Fig. 3c at 5 ns of simulation compared with Fig. 3d, which has some secondary structure additions from the original structure and is in a more compact or well-folded state.
      Figure thumbnail gr3
      Figure 3MD refined model for NP627 binding to PKCδI. a, RMSD trajectory over the course of the 25-ns MD simulation run on PKCδI using an I-TASSER–developed homology model of PKCδI equilibrated with QwikMD. b, dihedral energy of PKCδI over the course of the 25-ns MD simulation of PKCδI. c, DMQD sequence of PKCδI at 5 ns, with DMQD shown in balloon form and colored red and the C2 domain on the left. d, DMQD sequence of PKCδI at 25 ns, with DMQD shown in balloon form and colored red and the C2 domain on the left. e, averaged docking scores of NP627 to a 30 × 30 × 30 Å grid centered at residues DMQD from the PKCδI MD equilibrated homology model. f, ligand interaction diagram of NP627 to PKCδI depicting the pose with the highest-affinity g score from Schrödinger XP of −9.2 kcal/mol. g, ligand interaction diagram of NP627 to PKCδI depicting the pose with the second-highest affinity g score from Schrödinger XP of −9 kcal/mol.
      To probe the binding motifs of PKCδI with NP627, we docked static .pdb files taken from the MD simulation at 5-ns intervals (Fig. 3e). These docking results were based on a box centered at residues DMQD of PKCδI. The predicted ΔG for binding to this region had the highest affinity at 15 ns, with the ΔG of the highest affinity in two poses indicating a predicted KD of 627 to PKCδI of ∼1 nm. Both poses are shown with ligand interaction diagrams (Fig. 3, f and g) to show the important residues for NP627 binding to the model hinge region of PKCδI.

      Binding affinity of small molecules designed for PKCδI

      To characterize the interaction between the lead compound NP627 (or control Cpd594) and PKCδI, surface plasmon resonance (SPR) was used. SPR has emerged as a leading powerful technique to determine the interaction and affinity between molecules, as the ligand or the enzyme need not be labeled, thus eliminating possible changes to its molecular properties. Pure recombinant PKCδI was immobilized on a chip containing a gold film layered over glass, and the compounds were introduced in a liquid phase. The changes induced in surface plasmon resonance with binding are monitored and measured as real-time detection with high sensitivity, providing information regarding the affinity, specificity, and kinetics of biomolecular interactions. The binding affinity of pure recombinant PKCδI protein to NP627 was measured using SPR, where affinity was established via a steady state at 4 s before injection stop for NP627 or Cpd594. Binding was measured as relative response units (RU) versus time and represented in a sensorgram (Fig. 4a). This real-time measurement of association and dissociation of the binding allows calculation of association and dissociation rate constants. The kinetics for NP627 were analyzed and calculated by KD/KA. The KD for NP627 was at 1.2 nm for the steady state calculation and 610 pm using the kinetics data fitting a 1:1 Langmuir binding model, with Rmax set to local. Bivalent analyte fits additionally yielded a KD of about 1.2 nm, similar to steady-state calculations, which indicates symmetry of NP627 to the PKCδI binding site. Binding of the control compound Cpd594 to PKCδI showed lower-affinity binding, with steady-state affinity calculated to be 2 ± 1.8 μm. These results (Fig. 4b) demonstrate a high binding affinity of NP627 to PKCδI. Further, they indicate the importance of symmetry and avidity for robust interaction of NP627 with the binding site of PKCδI, including the DXXD(P4-P1)/X region modeled, as indicated by cpd594 binding with less affinity to PKCδI.
      Figure thumbnail gr4
      Figure 4Surface plasmon resonance–determined steady-state binding affinity of NP627 and Cpd594 to PKCδI. a, sensorgrams performed in triplicates for NP627 (0.12207, 0.488281, 0.976563, 1.953125, 15.625, 31.25, and 125 nm) binding to ∼12,000 RU PKCδI cross-linked on a CM5 chip at a flow rate of 30 μl/min with 120-s association. Fitting at 4 s before injection stop from sensorgrams was exported from BIAevaluate into GraphPad and fit using one site-specific binding model, yielding a KD of 1.2 ± 0.76 nm. b, sensorgrams in triplicates for Cpd594 (0.78125, 1.5625, 3.125, 6.25, and 12.5 μm) at the same flow rate as in a, yielding a KD of 2 ± 1.8 μm. Experiments were repeated four times with similar results. c, using SPR, caspase-3 was flowed alone or in the presence of 1 nm NP627 (red) on a CM5 chip with 800 RU of PKCδI cross-linked at a flow rate of 60 μl/min in triplicates. The steady state was taken 4 s before injection stop and exported from BiaEvaluate into GraphPad for caspase-3 (Cas3) alone (blue), caspase-3 with 1 nm NP627 (red), and NP627 alone and fit using one site-specific binding model, yielding a KD of 815 ± 699 pm for caspase-3 binding to PKCδI in the absence of NP627, with no saturation curve for caspase-3 in the presence of NP627. A graph of exported steady-state data from caspase-3 binding in the presence and absence of 1 nm NP627 and 5 nm caspase-3 is shown. Experiments were repeated three times with similar results. d, lean human adipocyte stem cells were grown on an eight-chamber slide and in the presence of 10 nm NP627 and/or 5 nm caspase-3 (casp3). Cells were fixed and probed using an antibody against the hinge region of pPKCδI. Cells were visualized at ×20 and ×4 (scale bars = 200 μm) using a Nikon A1R confocal microscope with DAPI staining for nuclei. Experiments were repeated three times with similar results.

      NP627 disrupts binding of caspase-3 to PKCδI

      The small molecule NP627 was designed to inhibit binding of caspase-3 to the hinge region of PKCδI, which results in release of the catalytic domain. Hence, the binding of PKCδI to caspase-3 was determined using SPR (as described above). The results indicate a high-affinity interaction between PKCδI and caspase-3 of 815 ± 69 pm (Fig. 4c). Results incorporating NP627 in the same run demonstrate nearly complete inhibition of the interaction between caspase-3 and PKCδI by addition of NP627. To visualize the reduction of PKCδI cleavage in the hinge region with NP627, immunochemistry was performed using an antibody specific to the target site of caspase-3 on the PKCδI hinge region (amino acids 309–318). Lean ASCs were either pretreated with 10 nm NP627 or left untreated for 24 h, followed by addition of 5 nm caspase-3 for 30 min. Cells were washed and fixed with 4% paraformaldehyde, and images were obtained using a Nikon A1R confocal microscope. Results show decreased staining of PKCδI in caspase-3–treated cells, indicating cleavage at the PKCδI hinge region. Treatment with NP627 prevented caspase-3–mediated cleavage of PKCδI, as seen in Fig. 4d.

      NP627 inhibits the kinase activity of PKCδI

      Because we established that NP627 has robust affinity for PKCδI using SPR, we sought to evaluate its effect on PKCδI activity in cells. Obese adipocytes were treated with increasing doses (1–100 nm) of NP627 for 24 h. The expression levels of PKCδI were not affected, but NP627 inhibited cleavage and release of the PKCδI C-terminal fragment. Additionally, its splice variant PKCδVIII (lacking the caspase-3 cleavage sequence), which is present in human adipocytes, was not affected by NP627 treatment (Fig. 5a). 10 nm NP627 was sufficient to inhibit release of the catalytic fragment, and this dose was used in all experiments.
      Figure thumbnail gr5
      Figure 5NP627 specifically inhibits PKCδI kinase activity and is nontoxic. a, Western blotting was performed using obese adipocytes which were untreated (C = control) or treated with increasing doses of NP627 (1, 10, and 100 nm) and immunoblotted using antibodies against PKCδI, PKCδVIII, and β-actin, as indicated in the figure. The experiment was repeated five times with similar results. Shown is a graphical representation of densitometric units normalized to β-actin, representative of five experiments performed independently. b, in vitro kinase assay performed using pure recombinant PKCδI and MBP treated with or without 10 nm NP627. Western blot analysis was performed three times with similar results using antibodies against phospho-MBP or MBP. c, relative PKC kinase activity was assayed using pure recombinant PKC proteins and NP627. TMB substrate was added for 30 min, and absorbance was read at 450 nm. The graph represents experiments repeated three times with similar results. d, adipocytes were treated with 10 nm NP627 and then analyzed using a WST1 assay for cell viability. The graph represents experiments repeated five times with similar results. Statistical analysis was performed by two-way analysis of variance. *, p < 0.05; ***, p < 0.001.
      To determine the effect of NP627 on the kinase activity of PKCδI, in vitro kinase assays were performed with the recombinant proteins PKCδI and myelin basic protein (MBP, a known PKCδI substrate) in a kinase buffer containing phosphatidyl serine and ATP with and without 10 nm NP627 (30-min incubation prior to the assay). The results show that NP627 treatment decreased phosphorylation of myelin basic protein (Fig. 5b).

      NP627 is specific for PKCδI

      Next we sought to evaluate whether 10 nm NP627 could affect other PKC isozymes. Using a PKC activity kit, a colorimetric assay (ENZO, run in triplicate) was performed; this incorporates recombinant PKC isoforms and tetramethylbenzidine substrate (TMB), and a color develops in proportion to PKC phosphotransferase activity. Relative kinase activity is calculated as follows: (average absorbance of PKC isozyme − average absorbance of blank)/quantity of pure kinase used per assay. Our data show specificity for PKCδI; other PKC isozyme activities were not inhibited by NP627 (Fig. 5c).

      NP627 is not cytotoxic

      To determine the effect of NP627 on cellular viability and cytotoxicity in ASCs and adipocytes, we performed the WST-1 assay (Roche) according to the manufacturer's instructions. The amount of formazan dye formed directly correlated with the number of viable cells as measured by absorbance. The WST-1 assay indicated that NP627 did not cause cellular toxicity (Fig. 5d).

      Inhibiting PKCδI with NP627 decreases inflammation in obese adipocytes

      PKCδI is an important mediator of increased levels of inflammation in obese adipocytes (
      • Wallerstedt E.
      • Smith U.
      • Andersson C.X.
      Protein kinase C-δ is involved in the inflammatory effect of IL-6 in mouse adipose cells.
      ). To evaluate the effect of inhibition of release of the PKCδI catalytic fragment by NP627 on inflammation, obese adipocytes were treated with 10 nm NP627 for 24 h and used in the human inflammation array (Abcam, ab134003). Among the cytokines, TNFα, MCP-1, and IL6 were dramatically reduced upon treatment with NP627. We verified the results individually using SYBR Green real-time qPCR. The macrophage chemotactic protein 1 (MCP-1) promotes macrophage infiltration in adipocytes (
      • Bruun J.M.
      • Lihn A.S.
      • Pedersen S.B.
      • Richelsen B.
      Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT.
      ) to sustain chronic inflammation. Our results (Fig. 6a) demonstrated that the levels of MCP-1 and the pro-inflammatory adipokines TNFα and IL6 declined in NP627-treated obese adipocytes.
      Figure thumbnail gr6
      Figure 6NP627 reduces inflammation and increases mitochondrial respiratory fitness. a, obese adipocytes were treated with 10 nm NP627 for 24 h. RNA was isolated, and expression levels of TNFα, MCP1, and IL6 were measured using SYBR Green qPCR using β-actin as an internal control. The experiment was performed in triplicate and repeated five times. b, obese and lean human adipocyte stem cells were grown on an eight-chamber slide and in the presence of 10 nm NP627 and/or 5 nm caspase-3. Cells were fixed and probed for cleaved PARP. Cells were visualized at ×20x and ×4 (scale bar = 200 μm) using a Nikon A1R confocal microscope with DAPI staining for nuclei. Experiments were repeated three times with similar results. c, human ASCs from omental lean, omental obese, and omental obese treated with 10 nm NP627 were seeded at 6000 cells/well and differentiated into mature adipocytes in a Seahorse XFp cell miniplate in triplicates, and the experiment was repeated three times. Treatment with 10 nm NP627 was maintained throughout differentiation. A mitochondrial stress test was performed according to the manufacturer's instructions, and the OCR was measured. The measurements were normalized to cell count, and analysis was performed using Seahorse Wave software and GraphPad by two-way analysis of variance. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

      NP627 decreases apoptosis in obese adipocytes

      Pre-adipocytes in obesity undergo ongoing apoptosis mediated by caspase-3 (
      • Nishimura T.
      • Hashimoto H.
      • Nakanishi I.
      • Furukawa M.
      Microvascular angiogenesis and apoptosis in the survival of free fat grafts.
      ) and have been shown to be a key link between obesity and insulin resistance (
      • Alkhouri N.
      • Gornicka A.
      • Berk M.P.
      • Thapaliya S.
      • Dixon L.J.
      • Kashyap S.
      • Schauer P.R.
      • Feldstein A.E.
      Adipocyte apoptosis, a link between obesity, insulin resistance, and hepatic steatosis.
      ). To determine whether NP627 inhibited PKCδI-mediated apoptosis, lean ASCs were treated with 10 nm NP627 for 24 h, followed by caspase-3 for 30 min. Cells were washed and fixed with 4% paraformaldehyde, and immunochemistry was performed for PARP. Images were obtained using a Nikon A1R confocal microscope. Treatment with NP627 reduced PARP levels (Fig. 6b), indicating decreased apoptosis levels. These results indicate that release of the PKCδI catalytic fragment induced apoptosis and that this could be inhibited by NP627.

      NP627 improves cellular respiration

      Obesity is associated with decreased cellular respiration. PKCδI has been shown to improve cellular respiration in obesity (
      • Talior I.
      • Tennenbaum T.
      • Kuroki T.
      • Eldar-Finkelman H.
      PKC-δ-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase.
      ). To determine the effect of NP627 on cellular respiration in obese adipocytes, we determined the oxygen consumption rate (OCR) using a Seahorse Bioscience XF Extracellular Flux Analyzer. ASCs from obese donors were treated with 10 nm NP627, and treatment was maintained through differentiation into adipocytes. Simultaneously, lean and obese ASCs without treatments were differentiated into adipocytes. Mature adipocytes were then analyzed for mitochondrial stress. Our results show a decreased OCR in obese adipocytes compared with lean adipocytes. Treatment of obese adipocytes with NP627 increased the OCR. Additionally, treatment with NP627 improved ATP production, maximum respiration, and percent spare respiratory capacity of obese adipocytes (Fig. 6c). The results show that decreasing PKCδI activity by treatment with NP627 improves cellular respiration and improves mitochondrial function in obese adipocytes.

      NP627 decreases PKCδI activity in obese adipose tissue

      Last, adipocytes were freshly isolated from human omental adipose tissue of obese donors (IRB 20295 as described above) and were treated with 10 nm NP627 for 48 h or remained untreated. Our results showed that NP627 treatment decreased PKCδI cleavage and decreased TNFα levels (Fig. 7a).
      Figure thumbnail gr7
      Figure 7NP627 decreases PKCδI activity in human adipose tissue. a, omental adipose tissue was obtained from human obese subjects, and adipocytes were isolated and treated with or without 10 nm NP627 for 48 h. Western blot analysis was performed and immunoblotted against PKCδI, TNFα, and β-actin. Shown is a graphical representation of densitometric units in the Western blots normalized to β-actin in five experiments performed independently. b, adipocytes isolated from human obese subjects treated with 10 nm NP627 for 24 h. Cells were gated for Annexin and PI to measure apoptosis. The experiment was performed in triplicates and repeated five times. The graph is representative of experiments repeated five times with similar results. Statistical analysis was performed by two-way analysis of variance. **, p < 0.01; ***, p < 0.001.
      We simultaneously used annexin/PI flow cytometry to determine the effect on ongoing apoptosis in the above adipocytes isolated from obese donors with or without NP627 treatment. Our results showed decreased apoptosis with NP627 treatment in obese patient adipocytes (Fig. 7b).

      Discussion

      Adipose tissue plays an important role in developing a systemic inflammatory state that significantly contributes to obesity-related morbidities such as cardiovascular risk and metabolic syndrome. PKCδI is implicated in insulin resistance, diabetes, and vascular function (
      • Bowles D.K.
      • Maddali K.K.
      • Dhulipala V.C.
      • Korzick D.H.
      PKCδ mediates anti-proliferative, pro-apoptotic effects of testosterone on coronary smooth muscle.
      ,
      • Greene M.W.
      • Burrington C.M.
      • Lynch D.T.
      • Davenport S.K.
      • Johnson A.K.
      • Horsman M.J.
      • Chowdhry S.
      • Zhang J.
      • Sparks J.D.
      • Tirrell P.C.
      Lipid metabolism, oxidative stress and cell death are regulated by PKCδ in a dietary model of nonalcoholic steatohepatitis.
      ,
      • Peluso J.J.
      • Pappalardo A.
      • Fernandez G.
      Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKCδ-dependent pathway.
      ) related to obesity and presents itself as an important kinase in the manifestation of obesity-related morbidities. PKCδI can be activated by several mechanisms. Diacylglycerol and phorbol 12-myristate 13-acetate promote its translocation to the membrane and thereby enable substrate interaction. PKCδI is activated by cleavage at its hinge region, releasing its C-terminal catalytic fragment. This fragment, freed from inhibition by the regulatory domain, is sufficient for its catalytic function (
      • Brodie C.
      • Blumberg P.M.
      Regulation of cell apoptosis by protein kinase Cδ.
      ). PKCδI activity is also modulated by tyrosine phosphorylation via Src-related kinases (
      • Shanmugam M.
      • Krett N.L.
      • Peters C.A.
      • Maizels E.T.
      • Murad F.M.
      • Kawakatsu H.
      • Rosen S.T.
      • Hunzicker-Dunn M.
      Association of PKCδ and active Src in PMA-treated MCF-7 human breast cancer cells.
      ,
      • Sumandea M.P.
      • Rybin V.O.
      • Hinken A.C.
      • Wang C.
      • Kobayashi T.
      • Harleton E.
      • Sievert G.
      • Balke C.W.
      • Feinmark S.J.
      • Solaro R.J.
      • Steinberg S.F.
      Tyrosine phosphorylation modifies protein kinase Cδ-dependent phosphorylation of cardiac troponin I.
      ). In this study, our results showed that obese adipocytes have increased cleavage of the PKCδI catalytic domain and that release of the catalytic fragment of PKCδI promoted pro-apoptosis and pro-inflammatory states in obese adipocytes. Hence, we evaluated the effect of inhibiting release of the PKCδI kinase domain in obese adipocytes. Of interest was cleavage mediated by caspase-3 on the DXXD(P4-P1)/X site on the hinge region of PKCδI because we had shown previously that the PKCδVIII alternatively spliced variant in humans, in which the caspase-3 cleavage site is disrupted, is a prosurvival protein (
      • Jiang K.
      • Apostolatos A.H.
      • Ghansah T.
      • Watson J.E.
      • Vickers T.
      • Cooper D.R.
      • Epling-Burnette P.K.
      • Patel N.A.
      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ,
      • Carter G.
      • Patel R.
      • Apostolatos A.
      • Murr M.
      • Cooper D.R.
      • Patel N.A.
      Protein kinase Cδ (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells.
      ).
      Many different strategies have been employed to increase the specificity of small molecules to particular kinases, and here we employed a drug discovery approach taking advantage of the unique caspase-3 activation mechanism of PKCδI. We screened and identified a small molecule with dual specificity for PKCδI and the DXXD sequence (caspase-3 recognition site) so that it distinguishes its binding specifically to PKCδI and not to other PKC isoforms or the alternatively spliced variant PKCδVIII in human adipocytes. NP627 binds PKCδI with high affinity (KD 1.3 nm), and it also shows high specificity for PKCδI over other PKCs, indicating that it is a good lead compound for therapies dependent on specific capase-3 cleavage–mediated kinase activity of PKCδI. The SPR data indicated that the PKCδI DXXD(P4-P1)/X region is symmetrical, having an aspartate separated by two amino acids in alpha helices. MD simulations showed the interaction of NP627 with PKCδI and the DMQD region. NP627 has a dramatically higher affinity for PKCδI over control the compound Cpd594 (KD2 μm), according to SPR results. NP627 has essentially two arms of the control molecule Cpd594 attached together. The affinity loss of Cpd594 resulting from lack of an opposite symmetrical side of the molecule is over 3-fold, which could indicate importance of the linked portion of the molecule. The importance of symmetry and avidity for robust interaction of NP627 to the binding site of PKCδI and experiments demonstrating inhibition of PKCδI kinase activity further underscore the structure–activity relationship. The importance of symmetry for NP627 could be probed in the future by replacing the propyl group with different linkers between the two amide bonds of NP627.
      Our results using SPR demonstrate nearly complete inhibition of the interaction between caspase-3 and PKCδI by NP627, indicative of protein–protein inhibition, which makes NP627 one of a select few small organic molecules able to affect macromolecular interactions, such as those between protein surfaces (
      • Arkin M.R.
      • Tang Y.
      • Wells J.A.
      Small-molecule inhibitors of protein-protein interactions: progressing toward the reality.
      ). SPR data indicated binding of NP627 to PKCδI at 1 nm; however, our cellular assays showed efficacy at 10 nm. It may be postulated that, because of the relatively high hydrophobicity of NP627, a higher dose was required under experimental conditions. A literature search did not reveal any studies utilizing the compound (referred to as NP627 here) in cell culture or animal models of disease. Future experiments are planned to test the effectiveness, pharmacokinetics/pharmacodynamics (PK/PD) and absorption, distribution, metabolism and excretion studies of NP627 in rodent models of obesity. Our results shown here demonstrate that NP627 is not toxic to cells.
      The results presented here demonstrate that NP627 disrupts caspase-3–mediated activation of PKCδI. NP627 is highly specific for PKCδI over other human PKCδ splice variants and PKC isoforms. Obese adipocytes treated with NP627 show decreased apoptosis and inflammation. Moreover, metabolic stress is reduced, demonstrating a metabolically healthier state of obese adipocytes. Using a Seahorse XFp Analyzer, we determined that the OCRs of obese adipocytes were drastically reduced compared with lean adipocytes, reflecting the compromised metabolic state in obesity. Treatment with NP627 induced a significant increase in mitochondrial OCR, especially ATP production, maximum respiration, and percent spare capacity. The results from treatment with NP627 are consistent with an improvement in ATP production because activated PKCδI has been shown to move to the mitochondrial membrane and inhibit ATP synthase (
      • Nguyen T.T.
      • Ogbi M.
      • Yu Q.
      • Fishman J.B.
      • Thomas W.
      • Harvey B.J.
      • Fulton D.
      • Johnson J.A.
      Modulation of the protein kinase Cδ interaction with the “d” subunit of F1F0-ATP synthase in neonatal cardiac myocytes: development of cell-permeable, mitochondrially targeted inhibitor and facilitator peptides.
      ). Improved maximum respiration and percent spare capacity reflect the cell's enhanced flexibility to compensate for metabolic challenges. Our results from the Seahorse mitochondrial stress test are supported by previous studies (
      • Yang Y.C.
      • Tsai C.Y.
      • Chen C.L.
      • Kuo C.H.
      • Hou C.W.
      • Cheng S.Y.
      • Aneja R.
      • Huang C.Y.
      • Kuo W.W.
      PKCδ activation is involved in ROS-mediated mitochondrial dysfunction and apoptosis in cardiomyocytes exposed to advanced glycation end products (Ages).
      ) showing that reduced PKCδI activity improves the OCR and overall mitochondrial metabolism in cardiomyocytes. Other studies have shown that metabolically healthy obese subjects have lower inflammation markers (
      • Karelis A.D.
      • Faraj M.
      • Bastard J.P.
      • St-Pierre D.H.
      • Brochu M.
      • Prud'homme D.
      • Rabasa-Lhoret R.
      The metabolically healthy but obese individual presents a favorable inflammation profile.
      ). Future studies will characterize whether OCR improvement with inhibition of PKCδI kinase activity by NP627 is due to increased mitochondrial functionality, mitochondrial number, or other changes within the adipocyte itself. Our results presented here demonstrate that PKCδI is a robust candidate for targeted intervention for management of chronic inflammatory diseases associated with obesity, such as cardiovascular diseases and diabetes. As a broader effect, the strategy of dual binding for targeting a protein kinase may result in highly specific therapy where the role of protein kinases is pivotal in disease manifestation.

      Experimental procedures

      Adipose tissue samples

      White adipose tissue was obtained as discarded tissue from surgeries performed at Tampa General Hospital by Dr. Murr. Donors consented to their waste tissue being used in research. The subcutaneous and omental depots were collected from the same subject. Adipose tissue was obtained from lean (BMI 22–23 kg/m2) and obese (BMI 43–45 kg/m2) donors (n = 6 each group); both groups were nonsmokers, had no cancers, and other criteria matched. The deidentified samples were obtained under an institutional review board–approved protocol (University of South Florida IRB 20295) with a “not human research activities” determination and were transported to the laboratory and processed within 24 h of collection. Additional tissue with the same criteria was also obtained from ZenBio.

      ASCs

      ASCs were isolated as described previously by our laboratory (
      • Watson J.E.
      • Patel N.A.
      • Carter G.
      • Moor A.
      • Patel R.
      • Ghansah T.
      • Mathur A.
      • Murr M.M.
      • Bickford P.
      • Gould L.J.
      • Cooper D.R.
      Comparison of markers and functional attributes of human adipose-derived stem cells and dedifferentiated adipocyte cells from subcutaneous fat of an obese diabetic donor.
      ). Briefly, adipose tissue was cut into small pieces and digested with 0.075% collagenase type 1 (Worthington) in modified PBS for 2 h at 37 °C. Digestion was stopped by adding α-minimum Eagle's medium and 20% heat-inactivated FBS. The suspension was filtered and centrifuged at 400 × g at room temperature. The pellet contained the stromal vascular fraction (SVF). The pellet was resuspended in 1 ml of erythrocyte lysis buffer (Stem Cell Technologies) for 10 min and washed in 20 ml of PBS with 2% P/S/A before centrifugation (300–500 × g, 5 min). The supernatant was aspirated, and the cell pellet was resuspended in 3 ml of stromal medium (α-minimum Eagle's medium, Mediatech) with 20% FBS, 1% l-glutamine (Mediatech), and 1% penicillin/streptomycin/amphotericin B. Following three rinses in stromal medium, SVF cells were plated for initial cell culture at 37 °C with 5% CO2 in ASC medium from ZenBio (catalog no. PM-1). Subconfluent cells were passaged by trypsinization. Experiments were conducted within passages 2–3.

      In vitro differentiation of ASCs to adipocytes

      Adipose stem cells derived from normal or obese patients were purchased from Zenbio. These were tested in culture to differentiate into mature adipocytes, show accumulation of lipid, and secrete adiponectin and leptin. At the start of all experiments, cells were grown to confluency so that all cells were synchronized and then differentiated. The cells were cultured as follows. ASCs were passaged with preadipocyte medium (PM-1; DMEM/Ham's F-12 medium, HEPES, FBS, penicillin, streptomycin, and amphotericin B; ZenBio) and then plated at 50,000 cells/cm2 with PM-1. Cells were fed every other day with PM-1 until confluent. To induce differentiation, PM-1 was replaced with differentiation medium (DM2, ZenBio), which included biotin, pantothenate, human insulin, dexamethasone, isobutylmethylxanthine, and a PPARγ agonist (days 0–7). After 7 days, DM-2 was replaced with adipocyte medium (AM1, ZenBio, days 7–14), which included PM-1, biotin, pantothenate, human insulin, and dexamethasone. By day 14, cells contained large lipid droplets and were considered mature adipocytes. Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.

      Quantitative real-time qPCR

      Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions. 1 μg of RNA was used to synthesize first-strand cDNA using oligo(dT) primers and the OmniscriptTM kit (Qiagen). 1 μl of cDNA was amplified by real-time quantitative PCR using Maxima SYBR Green/Rox qPCR Master Mix (Thermo Scientific) in an ABI ViiA7 sequence detection system (PE Applied Biosystems) to quantify the relative levels of transcripts in the samples. The primers were as follows: PKCδI, 5′-ACATCCTAGGTACAACAACGGGAC-3′ (sense) and 5′-ACCACGTCCTTCTTCAGACAC-3′ (antisense); MCP1, 5′-CTCATAGCAGCCACCTTCATTCC-3′ (sense) and 5′-TCAAGTCTTCGGAGTTTGGGTTT-3′ (antisense); IL6, 5′-AGACAGCCACTCACCTCTTCAG-3′ (sense) and 5′-TTCTGCCAGTGCCTCTTTGCTG-3′ (antisense); TNFα, 5′-CTCTTCTGCCTGCTGCACTTTG-3′ (sense) and 5′-ATGGGCTACAGGCTTGTCACTC-3′ (antisense); β-actin, 5′-CTCTTCCAGCCTTCCTTCCT-3′ (sense) and 5′-AGCACTGTGTTGGCGTACAG-3′ (antisense); GAPDH, 5′-TGACGTGCCGCCTGGAGAAAC-3′ (sense) and 5′-CCGGCATCGAAGGTGGAAGAG-3′ (antisense). Amplification was performed on a ViiaA 7 (Applied Biosystems). Real-time PCR was then performed in triplicate on samples and standards. The plate setup included a standard series, no template control, no RNA control, no reverse transcriptase control, and no amplification control. After primer concentrations were optimized to give the desired standard curve and a single melt curve, the relative quotient was determined using the ΔΔCT method with β-actin or GAPDH as the endogenous control and ASC control samples as calibrator samples. For absolute quantification, a standard curve was generated for PKCδI in every assay. To do so, the PKCδI-pTracer plasmid was used to obtain a standard curve correlating the amounts with the threshold cycle number (CT values). A linear relationship (r2 > 0.96) was obtained for PKCδI. Real-time qPCR was then performed on samples and standards in triplicates. The plate setup also included a standard series, no template control, no RNA control, no reverse transcriptase control, and no amplification control. The dissociation curve was analyzed for each sample. Absolute quantification of PKCδI expression levels was calculated by normalizing the values to GAPDH.

      Western blot analysis

      Cell lysates (40 μg) were separated on a 10% SDS-PAGE gel. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with Tris-buffered saline/0.1% Tween 20 containing 5% bovine serum albumin, washed, and incubated in primary antibody at 4 °C overnight. Membranes were then washed and incubated in secondary antibody (1:5000) for 1 h at room temperature, and enhanced chemiluminescence (Pierce) was used for detection. The FluorChem MTM (Protein Simple) imaging system was used to capture digital chemiluminescence images and process Western blots. Data were analyzed using AlphaView® software. Primary antibodies included PARP (Cell Signaling Technology, 9542), PKCδI (Cell Signaling Technology, 2058S), MBP and pMBP (Upstate, 05675 and 05429, respectively), caspase-3 (Santa Cruz Biotechnology, 56053), TNFα (Novus, 19532), actin (Sigma, A3884), GAPDH (Santa Cruz Biotechnology, 25778), and PKCδVIII raised in the Patel laboratory (
      • Jiang K.
      • Apostolatos A.H.
      • Ghansah T.
      • Watson J.E.
      • Vickers T.
      • Cooper D.R.
      • Epling-Burnette P.K.
      • Patel N.A.
      Identification of a novel antiapoptotic human protein kinase C δ isoform, PKCδVIII in NT2 cells.
      ).

      Drug discovery

      Schrödinger's Maestro program (version 9.3.5) was used as the graphical user interface, and compounds contained in the ChemBridge (ChemBridge Chemicals) Microformat library were prepared for virtual screening with Schrödinger's LigPrep program as in Ref.
      • Sparks D.K.
      • Johnson B.R.
      Two-dimensional quantum propagation using wavelets in space and time.
      . Virtual screening was performed using Schrödinger's GLIDE software (
      • Friesner R.A.
      • Murphy R.B.
      • Repasky M.P.
      • Frye L.L.
      • Greenwood J.R.
      • Halgren T.A.
      • Sanschagrin P.C.
      • Mainz D.T.
      Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes.
      ) on an α-helical construct of the amino acid sequence DXXD(P4-P1)/X, generated using Schrödinger Prime (
      • Jacobson M.P.
      • Pincus D.L.
      • Rapp C.S.
      • Day T.J.
      • Honig B.
      • Shaw D.E.
      • Friesner R.A.
      A hierarchical approach to all-atom protein loop prediction.
      ). Homology modeling was performed using PDB and Schrödinger Prime to form a PKCδI proteinaceous region for potential DXXD(P4-P1)/X docking, and grids were generated using GLIDE both with and without homology-modeled PKCδI. Schrödinger SiteMap was used on both grids to determine areas of the model suitable for binding, and docking was performed on the DXXD(P4-P1)/X region from three potential trajectories suitable for the proteinaceous DXXD(P4-P1)/X–PKCδI homology model. Results were then compared, and g scores were analyzed prior to ordering potential leads from ChemBridge Chemicals based on affinity for either DXXD(P4-P1)/X–PKCδI homology model. I-TASSER results for the PKCδ1 sequence taken from Uniport were equilibrated for 25 ns with constant temperature with NAMD 2.12 (
      • Phillips J.C.
      • Braun R.
      • Wang W.
      • Gumbart J.
      • Tajkhorshid E.
      • Villa E.
      • Chipot C.
      • Skeel R.D.
      • Kalé L.
      • Schulten K.
      Scalable molecular dynamics with NAMD.
      ) using the CHARMM36m force field (
      • Huang J.
      • Rauscher S.
      • Nawrocki G.
      • Ran T.
      • Feig M.
      • de Groot B.L.
      • Grubmüller H.
      • MacKerell Jr., A.D.
      CHARMM36m: an improved force field for folded and intrinsically disordered proteins.
      ) and as described in Ref.
      • Starr M.L.
      • Sparks R.P.
      • Arango A.S.
      • Hurst L.R.
      • Zhao Z.
      • Lihan M.
      • Jenkins J.L.
      • Tajkhorshid E.
      • Fratti R.A.
      Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.
      . The long-range electrostatic forces were evaluated using the particle mesh Ewald method (
      • Cerutti D.S.
      • Duke R.E.
      • Darden T.A.
      • Lybrand T.P.
      Staggered mesh Ewald: an extension of the smooth particle-mesh Ewald method adding great versatility.
      ). VMD 1.9.3 was used for MD RMSD trajectory visualization and analysis.

      Surface plasmon resonance

      SPR measurements were performed on a Biacore T200 instrument equipped with CM-5 chips. Small-molecule disassociation constants were obtained by cross-linking ∼12,000 RU of PKCδI to the CM5 chip using 1-ethyl-3-(3-dimethylamino) propyl carbodiimide and N-Hydroxysuccinimide (EDC/NHS) chemistry. Small molecules were dissolved in running buffer of HEPES (pH 7.4) and 150 mm NaCl (HBS buffer) according to the relative logP supplied by Chembridge. Steady-state injections were performed in HBS buffer at a flow rate of 30 μl/min with an association time of 120 s and dissociation time of 120 s, and binding was measured in relative response units as described in Ref.
      • Starr M.L.
      • Sparks R.P.
      • Arango A.S.
      • Hurst L.R.
      • Zhao Z.
      • Lihan M.
      • Jenkins J.L.
      • Tajkhorshid E.
      • Fratti R.A.
      Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.
      . Regeneration with 10 mm NaOH in HBS buffer was performed at flow rate of 30 μl/s for 30 s after each small-molecule injection. The steady state was obtained using GE BIAcore T200 evaluation software version 3.0 (BIAevaluate), and steady-state data were fitted and exported using BiaEvaluate software into GraphPad Prism 7.00 for Windows (GraphPad Software, La Jolla, CA). Kinetic injections were performed in HBS buffer at 60 μl/min with an association time of 300 s and disassociation time of 120 s, followed by a 30-s injection of 10 mm NaOH in HBS buffer. Kinetics were fit using a BIAevaluate 1:1 binding model with Rmax set to local, and the resulting sensorgrams and fits were exported into GraphPad. Caspase-3 binding to PKCδI was performed by cross-linking ∼800 RU of PKCδI to a CM-5 chip using EDC/NHS chemistry. Binding of caspase-3 to PKCδI and inhibition of caspase-3 binding by NP627 was performed in the presence and absence of small molecules injected at 60 μl/min with an association time of 90 s and dissociation time of 150 s as described above.

      Immunochemistry

      Adipocytes were plated in a 96 well-plate and treated with 10 nm NP627 for 24 h. Caspase-3 (15 units) was added for 30 min, and cells were fixed by removing medium, washing three times with PBS, and adding 4% paraformaldehyde for 30 min. Cells were blocked with 10% goat serum for 1 h and incubated overnight with primary antibodies (1:500 PARP, Upstate, 04-576; 1:500 for the pPKCδ hinge region, Santa Cruz Biotechnology, sc-377560). Cells were washed with PBS three times and incubated for 1 h with Alexa Fluor 488 secondary antibody (1:2000, Invitrogen) at room temperature. Cells were washed once more with PBS and stained with DAPI. Images were captured using a Nikon A1R confocal microscope and analyzed using NIS software.

      PKC kinase activity assay using a kit from ENZO and performed according to the manufacturer's instructions

      Briefly, the assay is based on a solid-phase ELISA that utilizes a specific synthetic peptide as a substrate for PKC and a polyclonal antibody that recognizes the phosphorylated form of the substrate. The assay is designed for analysis of PKC isozyme activity in the solution phase. In the assay, the substrate, which is readily phosphorylated by PKCs, is precoated on the wells of the provided PKC substrate microtiter plate. The samples to be assayed are added to the appropriate wells in triplicate, followed by addition of ATP to initiate the reaction. The kinase reaction is terminated, and a phosphospecific substrate antibody is added to the wells that binds specifically to the phosphorylated peptide substrate. The phosphospecific antibody is subsequently bound by a peroxidase-conjugated secondary antibody. The assay is developed with TMB, and a color develops in proportion to PKC phosphotransferase activity. Color development is stopped with acid stop solution, and the intensity of the color is measured in a microplate reader at 450 nm.

      Annexin V/PI apoptosis assay

      Adipocytes from obese were maintained in (AM) adipocyte maintenance medium (Zenbio) and treated with 10 nm NP627 for 48 h. Medium was collected, and cells were washed once with Hank's balanced salt soultion (HBSS) and then trypsinized for 5.0 min. 5 ml of complete medium was added to neutralize the trypsin. Medium and washes were pooled and centrifuged at 1200 rpm for 5 min. Cells were washed once with PBS and once with binding buffer and then incubated for 15 min with 5.0 μl of Annexin V–FITC and 5.0 μl of PI in 100 μl of binding buffer (BD Pharmingen) at room temperature in the dark. 400 μl of binding buffer was added, and cells were analyzed by flow cytometry within 1 h. Annexin V–FITC and PI fluorescence were measured using an Accuri C6 flow cytometer.

      Oxygen consumption rate

      ASCs from lean and obese donors were plated into a Seahorse XFp cell culture miniplate (Agilent Technologies) at a density of 6000 cells/well as determined by optimization cycles. The following day, NP627 was added to pre-adipocyte medium at a concentration of 10 nm and maintained with medium changes and differentiation into mature adipocytes. Adipocytes were incubated in Seahorse XF medium (supplemented with 100 mm pyruvate, 200 mm glutamine, and 2.5 m glucose) in a non-CO2 incubator at 37° for 1 h. Seahorse sensor cartridges were prepared and loaded into ports as described for the mitochondrial stress test (100 μm oligomycin, 100 μm carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and 50 μm antimycin A/rotenone). Cells were run in the Seahorse XFp Analyzer. After the Seahorse run, adipocytes were counted using a Cellometer Vision CBA Image cytometer (Nexcelom). The measurements were normalized to cell counts, and data were analyzed using Agilent Wave software.

      Statistical analysis

      All experiments were repeated three to five times to ensure reproducibility of results. Analyses were performed using PRISMTM software and analyzed using two-tailed Student's t test or two-way analysis of variance as indicated in the figure legends; *, p < 0.05 (significant); **, p < 0.001 (highly significant); ***, p < 0.001 (extremely significant). Analysis was performed either within groups or between groups as determined by the experiment.

      Author contributions

      R. P. S., A. L., R. A. F., and N. A. P. formal analysis; R. P. S., R. A. F., and N. A. P. validation; R. P. S., A. L., D. B., and R. P. investigation; R. P. S., A. L., D. B., R. P., W. G., and N. A. P. methodology; M. M., W. G., and N. A. P. conceptualization; M. M., R. A. F., and N. A. P. resources; W. G., R. A. F., and N. A. P. supervision; R. A. F. and N. A. P. data curation; R. A. F. software; R. A. F. and N. A. P. funding acquisition; N. A. P. writing-original draft; N. A. P. project administration; N. A. P. writing-review and editing.

      Acknowledgments

      The equilibrium MD simulations were developed with the help of Andres Arango and Emad Tajkhorshid.

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