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

Abstract

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.

Keywords: NP627; PKC; adipocyte; adipose tissue; adipose tissue metabolism; apoptosis; inflammation; metabolic disease; metabolic disorder; obesity.

Figure 1.

PKCδI 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.

Figure 2.

NP627 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.

Figure 3.

MD 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.

Figure 4.

Surface 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 K_D 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 K_D 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 K_D 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.

Figure 5.

NP627 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.

Figure 6.

NP627 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.

Figure 7.

NP627 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.