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. 2024 Jan 11;73(4):585–591. doi: 10.2337/db23-0506

KD025 Is a Casein Kinase 2 Inhibitor That Protects Against Glucolipotoxicity in β-Cells

Ranjan Devkota 1, Jonnell C Small 1, Kaycee Carbone 1, Michael A Glass 1, Amedeo Vetere 1, Bridget K Wagner 1,✉
PMCID: PMC10958584  PMID: 38211571

Abstract

Glucolipotoxicity (GLT), in which elevated levels of glucose and fatty acids have deleterious effects on β-cell biology, is thought to be one of the major contributors in progression of type 2 diabetes. In search of novel small molecules that protect β-cells against GLT, we previously discovered KD025, an inhibitor of Rho-associated coiled-coil–containing kinase isoform 2 (ROCK2), as a GLT-protective compound in INS-1E cells and dissociated human islets. To further understand the mechanism of action of KD025, we found that pharmacological and genetic inhibition of ROCK2 was not responsible for the protective effects of KD025 against GLT. Instead, kinase profiling revealed that KD025 potently inhibits catalytic subunits of casein kinase 2 (CK2), a constitutively active serine/threonine kinase. We experimentally verified that the inhibition of one of the catalytic subunits of casein kinase 2, CK2A1, but not CK2A2, improved cell viability when challenged with GLT. We conclude that KD025 inhibits CK2 to protect β-cells from GLT.

Article Highlights

  • We previously identified the small molecule KD025 in a high-throughput phenotypic screen and found that it protects both rodent cell lines and human islets from the deleterious effects of glucolipotoxicity.

  • Here, we focus on the mechanism of action of KD025.

  • KD025 inhibits casein kinase 2 to protect β-cells against glucolipotoxicity.

  • Due to limited therapeutic options to protect β-cell loss in type 2 diabetes, selective inhibition of casein kinase 2 maybe a potential therapeutic strategy to preserve β-cell dysfunction in type 2 diabetes.

Introduction

The global epidemics of obesity and type 2 diabetes (T2D) continue to grow (1). In 2045, it is estimated 783 million adults will live with T2D (2). While there are many approved therapeutic strategies to treat T2D (3), they do not address the underlying issue of β-cell dysfunction, failure, and ultimate death in patients with diabetes (1). The onset of obesity is associated with elevated free fatty acids (FFAs) due to reduced FFA clearance; these elevated FFAs are thought to contribute to T2D progression by promoting insulin resistance and pancreatic β-cell dysfunction (4). Elevated plasma glucose in conjunction with elevated FFAs lead to β-cell failure, known as glucolipotoxicity (GLT) (5). GLT decreases insulin gene expression, impairs glucose-stimulated insulin secretion (GSIS), and activates β-cell apoptotic machinery (6). We previously performed a high-throughput phenotypic screen in INS-1E, a rodent pancreatic β-cell line, to discover novel small molecules that suppress GLT-induced β-cell apoptosis. We identified KD025, also known as SLx-2119 or belumosudil, as one of the most potent GLT-protective compounds (7).

KD025 is an Rho-associated coiled-coil–containing kinase isoform 2 (ROCK2) inhibitor, with a specificity toward ROCK2 that is rare among the ROCK inhibitor class (8). When bound to GTP, RhoA activates ROCK1 and ROCK2, which then phosphorylate several substrates (9). The isoforms ROCK1 and ROCK2 share ∼90% of sequence homology in their kinase domain, making it quite challenging to develop isoform-specific inhibitors. ROCK inhibition as a therapeutic strategy gained popularity with the discovery of ρ/ROCK pathway involvement in several diseases, including vascular disease, cancer, asthma, glaucoma, kidney failure, osteoporosis, and neuronal degenerative disease (9). The activation of ROCK signaling has also been implicated in the pathogenesis of diabetes (10), but the specific role of ROCK2 in β-cell biology has not been fully characterized. The pan-ROCK inhibit or fasudil was the first to be clinically approved in Japan in 1995 for the treatment of cerebral vasospasm. More recently, KD025 has been approved by the U.S. Food and Drug Administration for the treatment of chronic graft-versus-host disease (11). The ROCK2 specificity of this small molecule sets it apart from several family members; therefore, the discovery of KD025 as a GLT-protective compound initially led us to reason that inhibition of ROCK2 may be beneficial against GLT.

In this study, we tested several pan-ROCK inhibitors in INS-1E cells and human islets, and found that KD025 uniquely rescues GLT-mediated decrease in cell viability and insulin secretion. Further, kinase profiling studies indicated that KD025 is also an inhibitor of casein kinase 2 (CK2). Using several different strategies to modulate CK2 activity, we uncovered a role of CK2 in mediating β-cell viability when challenged with GLT conditions. Our results suggest that CK2 inhibition may be a feasible therapeutic strategy for mitigating β-cell death in T2D.

Research Design and Methods

Refer to Supplementary Methods for detailed cell culture conditions and experimental information.

High-Content Fluorescent Microscopy Assay

INS-1E cells were stained with Hoechst 33342, Caspase-3/7 dye, and DNA dye DRAQ7, all at 1:5,000 dilution for 1.5 h. Cells were imaged using an Opera Phenix High-Content Imaging Instrument (PerkinElmer). Live cell number was calculated by selecting the population of cells negative for the DRAQ7 and caspase 3/7 dyes using Harmony software (PerkinElmer), and percent viability was quantified normalizing the live cell number to the total number of cells.

Immunofluorescence and Human Islet Staining

INS-1E cells and dissociated human islets were fixed using paraformaldehyde, permeabilized with Triton X-100, and blocked with BSA. Cells were incubated with primary antibody overnight at 4°C, washed, and incubated with secondary antibody. Hoechst 33342 was added for 20 min, and cells were washed and stored at 4°C. Imaging was done using an Opera Phenix High-Content Imaging Instrument. Refer to Supplementary Methods for additional details.

Kinase Profiling

Kinase profiling of KD025, SR3677, and H-1152 was performed by Eurofins DiscoverX KINOMEscan scanMAX against 468 targets. KD025, SR3677, and H-1152 were all screened at 1 μmol/L. Refer to Supplementary Methods for additional details.

siRNA Knockdown

Lipofectamine RNAiMAX was used for siRNA transfection. INS-1E cells were plated 5,000 cells per well in 96-well plate format and transfected with 10 pmol siRNA. Transfected cells were incubated for 72–96 h. For GLT treatment, basal media was removed, GLT media was added, and cells were incubated for another 48 h. siRNA was purchased from Sigma-Aldrich: Rock2 (SASI_Rn01_00030374), Csnk2a1 (SASI_Rn01_00051446), and Csnk2a2 (SASI_Rn02_00228536).

Overexpression

Lipofectamine RNAiMAX was used for transfection of INS-1E cells with Csnk2a1-Flag clone EX-Rn14990-M14 (Genecopoeia). Cells were plated 5,000 cells per well in 96-well plate format and transfected with 300 ng of EX-Rn14990-M14 vector. Transfected cells were incubated for 72 h. For GLT treatment, basal media was removed, GLT media was added, and cells were incubated for another 48 h.

GSIS

INS-1E-PIG cells or dissociated human islets were subjected to GSIS as previously described (12). For INS-1E-PIG cells, secreted luciferase was measured using native coelenterazine. For human islets, secreted insulin was determined by colorimetric or chemiluminescence insulin ELISA (Alpco) according to the manufacturer’s instructions.

Data and Resource Availability

All data generated or analyzed during this study are included in the published article (and its Supplementary Material). No applicable resources were generated or analyzed during the current study.

Results

KD025 Protects Against GLT Independently of ROCK2

KD025 (Fig. 1A) is a GLT-protective small molecule in both INS-1E and human islets (7). Here, we also found that KD025 is protective against GLT-mediated apoptosis (Supplementary Fig. 1A) and partially rescues GLT-induced GSIS defect in both INS-1E cells (Fig. 1B and Supplementary Fig. 1B) and dissociated human islets (Fig. 1C). Interestingly, we found that KD025 had no effect on cell viability in basal conditions (Supplementary Fig. 1C) and GLT-induced reduction in insulin content (Supplementary Fig. 1D). Since KD025 is annotated as a ROCK2 inhibitor, we investigated whether KD025-mediated cellular protection is a result of ROCK2 inhibition. We tested and compared KD025 with eight well-known pan-ROCK inhibitors including SR3677, H-1152, Y-27632, fasudil, Rho Kinase Inhibitor V, AS-1892802, GSK429286A, and RKI-1447 (9) in INS-1E and human islets treated with GLT-inducing media. We found that KD025 was the only compound capable of rescuing β-cell number and viability when challenged with GLT conditions (Fig. 1D and E). These data suggest that KD025-induced protection against GLT is likely to be mediated through another target, and not through ROCK2. Consistently, we found that siRNA-mediated knockdown of Rock2 in INS-1E cells had no effect on β-cell number or viability under GLT conditions (Fig. 1F–H).

Figure 1.

Figure 1

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KD025 protects against GLT in INS-1E cells and human islets. (A) Chemical structure of KD025. (B) INS-1E cells were starved at 2.8 mmol/L glucose for 1 h and stimulated at 16.7 mmol/L glucose for 2 h. Ratio of luciferase activity between high and low glucose is shown. See Supplementary Fig. 1B for luciferase activity at high (16.7 mmol/L) and low (2.8 mmol/L) glucose; 10 μmol/L KD025 rescues GLT-mediated insulin secretion defect in INS-1E cells (n = 5). (C) Dissociated human islets were starved at 2.8 mmol/L glucose for 1 h and stimulated at 16.7 mmol/L glucose for 2 h. Ratio of insulin secretion between high and low glucose is shown; 10 μmol/L KD025 rescues GLT-mediated insulin secretion defect in dissociated human islets (n = 5). (D and E) KD025 improves the viability of INS-1E cells and dissociated human islets treated with GLT media for 48 h (n = 3). Black dotted line (n = 24) represents percent viability of cells incubated in basal culture media. Gray dotted line (n = 24) represents percent viability of INS-1E cells or human islets treated with GLT media and DMSO. (F) siRNA against Rock2 reduces ROCK2 protein expression in INS-1E. (G and H) Rock2 knockdown is not sufficient to rescue GLT-induced reduction in INS-1E cell number, number of cells negative for DRAQ7 and caspase 3/7 dyes, and viability, percentage of cells normalized to total number of cells (n = 8). Statistical analysis was performed using unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001).

KD025 Is a Casein Kinase 2 Inhibitor

Based on these results, we reasoned that KD025 activity may be due to its effect on other targets. To test this hypothesis, we performed head-to-head kinase profiling to compare the activity of KD025 with two pan-ROCK inhibitors, SR3677 and H-1152. A total of 468 human kinase targets were tested, and kinases with <35% remaining activity compared with control were identified as hits. As expected, we found that all three compounds are potent ROCK2 inhibitors (Fig. 2 and Supplementary Table 1). However, KD025 is unique among these three compounds in specificity and selectivity, with potent activity toward three other kinases, the catalytic subunits of CK2 (CK2A1 and CK2A2) and MRCKB (Fig. 2A and Supplementary Table 1). Since the other reported inhibitors of MRCKB, Y-27632 and fasudil (13), did not confer any protection against GLT (Fig. 1D and E), we focused on understanding the involvement of CK2. CK2 is a serine/threonine kinase involved in important cellular processes (14). We then tested CX4945, a known CK2 inhibitor (15), in INS-1E cells and found that CX4945 was as effective as KD025 at restoring viability and GSIS, when challenged with GLT (Fig. 3A–C).

Figure 2.

Figure 2

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KD025 is a CK2 inhibitor. TREEspot visualization of kinase inhibitory activity of 1 mmol/L KD025 (A), SR3677 (B), or H-1152 (C), tested against 468 human kinases. Inhibited kinases (percent activity <35%) are shown as yellow (ROCK2), blue (CK2), or red (kinases other than ROCK2/CK2) circles; unaffected kinases (percent activity >35%) are depicted as green dots. All three compounds are potent ROCK2 inhibitors, shown as yellow circles whereas KD025 uniquely inhibits catalytic subunits of casein kinase 2 (CK2A1 and CK2A2), shown as blue circles. Image generated using TREEspot software tool and reprinted with permission from KINOMEScan, a division of DiscoverX Corporation, © DiscoverX Corporation 2010.

Figure 3.

Figure 3

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Csnk2a1 silencing improves GLT-treated INS-1E viability. (A and B) Ten micromoles per liter CX4945 rescues cell number and viability in INS-1E treated with GLT media (n ≥ 6). (C) INS-1E cells were starved at 2.8 mmol/L glucose for 1 h and stimulated at 16.7 mmol/L glucose for 2 h. Ratio of secretion between high and low glucose is shown; 10 μmol/L CX4945 rescues GLT-mediated insulin secretion defect (n = 5). (D) Quantitative PCR results showing the degree of Csnk2a1 silencing. (E) Csnk2a1 silencing leads to reduced CK2A1 protein expression. (F) Live cell number for INS-1E cells treated with basal or GLT media, with or without Csnk2a1 knockdown, and with or without 10 μmol/L KD025 treatment (n = 6). (G) Percent viability for INS-1E cells treated with basal or GLT media, with or without Csnk2a1 knockdown, and with or without 10 μmol/L KD025 treatment (n = 6). (H) Quantitative PCR results showing the degree of Csnk2a2 silencing. (I) Csnk2a2 silencing leads to reduced CK2A2 protein expression. (J) Live cell number for INS-1E cells treated with basal or GLT media, with or without Csnk2a2 knockdown, and with or without 10 μmol/L KD025 treatment (n = 6). (K) Percent viability for INS-1E cells treated with basal or GLT media, with or without Csnk2a2 knockdown, and with or without 10 μmol/L KD025 treatment (n = 6). Live cell number represents the number of cells negative for DRAQ7 and caspase 3/7 dyes, and percent viability denotes the number of live cells normalized to total number of cells. Statistical analysis was performed using unpaired t test (ns, not significant; **P < 0.01; ***P < 0.001).

Knockdown of Csnk2a1, but Not Csnk2a2, Partially Protects β-Cells Against GLT

CK2 is ubiquitously expressed in eukaryotes and exists as a tetrameric complex consisting of two catalytic α subunits (α and α′) and two regulatory β subunits (16). Based on kinase profiling, we used siRNA to silence each of the catalytic α subunits (Csnk2a1 and Csnk2a2) in INS-1E cells (Fig. 3D, E, H, and I). We found that silencing of Csnk2a1, but not Csnk2a2, improved INS-1E viability when challenged with GLT. Although Csnk2a1 silencing was not able to increase GLT-treated INS-1E cell number to similar levels as KD025, there was a significant improvement in INS-1E viability, to ∼65%, compared with GLT control (∼55%) (Fig. 3F and G). Knockdown of Csnk2a2 had no effect on GLT-treated INS-1E viability (Fig. 3J and K). Although siRNA against Csnk2a2 slightly increased INS-1E cell number compared with the scrambled control, the percent viability of these cells was similar to controls. These data suggest differential functions for the two catalytic subunits of CK2 in β-cells, consistent with our observation that knockdown of Csnk2a1, but not Csnk2a2, partially improved GLT-induced defect in insulin secretion (Supplementary Fig. 1E). Interestingly, knockdown of either of these kinases had no effect on GLT-mediated reduction in insulin content (Supplementary Fig. 1F). We then evaluated whether combining KD025 treatment and Csnk2a1 knockdown would have an additive protective effect against GLT, but we did not observe a difference when compared with cells with KD025 alone (Supplementary Fig. 1G and H). These results further suggest that KD025-mediated protection against GLT is partially achieved through inhibition of Csnk2a1.

Casein Kinase 2 Overexpression Eliminates KD025-Mediated Protection

To confirm the role of CK2 in KD025 activity, we overexpressed CK2A1::FLAG fusion protein in INS-1E (Fig. 4A and B) and observed a significant decrease in cell viability, both in the basal state and when challenged with GLT. Consistent with these results, CK2A1::FLAG overexpression completely eliminated the protective effects of KD025 against GLT (Fig. 4C and D).

Figure 4.

Figure 4

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CK2A1 overexpression eliminates KD025-mediated protection against GLT. (A) Western blot confirming transient expression of C-terminal CK2A1::FLAG fusion protein in INS-1E cells. (B) Representative images of immunofluorescence confirm CK2A1::FLAG expression, shown with white arrowheads. (C) CK2A1 overexpression decreases the live cell number of basal and GLT-treated INS-1E. CK2A1 overexpression eliminated KD025-mediated protection against GLT (n = 4). (D) CK2A1 overexpression decreases percent viability of basal and GLT-treated INS-1E. CK2A1 overexpression eliminated KD025-mediated protection against GLT (n = 4). Statistical analysis was performed using unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001; ###P < 0.001, GLT vs. GLT+KD025).

Discussion

Recently, a number of high-throughput screens have been reported to identify small molecules that can suppress GLT-induced loss of β-cell function and mass (17,18). Using the same approach, we previously identified KD025 as a GLT-protective compound in INS-1E and dissociated human islets (7). Although KD025 is annotated as a ROCK2 inhibitor, we found that 1) KD025-mediated protection against GLT is not ROCK2-dependent, 2) KD025 is a casein kinase 2 inhibitor as well as a ROCK2 inhibitor, and 3) inhibition of a catalytic subunit of casein kinase 2 plays a role in protecting β-cells from the effects of GLT.

ROCK2 influences a variety of cellular processes, including actin cytoskeleton organization, cell adhesion, motility, proliferation, and apoptosis (19). Therefore, it is not a surprise that ROCK2 is implicated in different pathological conditions, including cardiovascular disease, Alzheimer’s disease, and cancer (9). However, the role of ROCK2 in pancreatic β-cell viability has not been well described. With the discovery of KD025 as a GLT-protective compound, we tested several pan-ROCK inhibitors to identify GLT-protective ROCK2 inhibitors. To our surprise, we found that other ROCK inhibitors did not confer cellular protection from GLT. This observation was consistent with the results from siRNA-based knockdown of Rock2, where silencing Rock2 had no effect on cell viability upon GLT treatment.

Large-scale kinase profiling has emerged as an effective approach to interrogate compounds against a large number of targets (20). Hence, we performed kinase profiling of KD025 along with two other pan-ROCK inhibitors, SR3677 and H-1152. We noticed that all three compounds are highly potent ROCK2 inhibitors; more importantly, KD025 has unique targets: CSNK2A1 and CSNK2A2, the α and α′ catalytic subunits of CK2, respectively. Our results are consistent with a previous study demonstrating CK2 as a novel target of KD025 for adipocyte differentiation (21). The selectivity and specificity of our kinase profiling results (Fig. 2) directly reflects the concentration of the compounds used (1 μmol/L in this study, compared with 10 μmol/L in the previous study [21]).

CK2 is a constitutively active serine/threonine kinase, known to have hundreds of substrates involved in diverse cellular processes such as transcription, translation, protein stability and degradation, cell cycle progression, and cell survival (14). CK2 is implicated in a number of pathologies, including cancer, neurodegenerative diseases, cardiovascular diseases, inflammatory diseases, and diabetes (14). In T2D mice, the liver has increased Csnk2a1 gene expression and Ck2α protein levels compared with controls (22). This observation is consistent with increased Csnk2a1 gene expression in β-cells isolated from human T2D donors compared with nondiabetic individuals (23). These results are also consistent with our observation that silencing Csnk2a1, but not Csnk2a2, improves β-cell viability when challenged with GLT. Further, overexpression of a C-terminal FLAG-tag Csnk2a1 in GLT-treated INS-1E completely ablated KD025-mediated GLT protectivity, confirming CK2α as the GLT-relevant target for KD025 in β-cells. Since our results show that treatment with KD025 provides significantly greater protection against GLT than siRNA-based inhibition of Csnk2a1, it is possible that Csnk2a1 might not be the sole contributor to the effects of KD025. Future research using the CK2 knockout cells would be essential for a thorough and comprehensive understanding. Our observation regarding KD025-mediated recovery of GSIS under GLT conditions is consistent with the role of CK2 in insulin secretion (24) as well as the observation that CX-4945 treatment in rat and human islets exposed to elevated glucose and fatty acids improves insulin secretion (25).

Overall, our findings show that KD025-mediated protection against GLT is not ROCK2 dependent and instead is partially due to CK2α inhibition. Our results further reinforce the role of CK2 in β-cell biology and present CK2 as a potential therapeutic target. Future research on the identification of T2D-relevant CK2 phosphorylation substrates will be essential for complete understanding of β-cell dysfunction in T2D.

This article contains supplementary material online at https://doi.org/10.2337/figshare.24968748.

Article Information

Acknowledgments. The authors thank Maria Kost-Alimova (Broad Institute) for assistance and technical support for high-content fluorescent microscopy.

Funding. This work was supported by the National Institutes of Health Human Islet Research Network (U01DK123717 [B.K.W.]). R.D. was supported by Knut och Alice Wallenberg Stiftelse, Sweden (KAW 2019.0580). The authors gratefully acknowledge the use of the Opera Phenix High-Content/High-Throughput imaging system at the Broad Institute, funded by grant S10-OD-026839-01 (B.K.W.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. R.D., J.C.S., K.C., M.A.G., and A.V. performed experiments and analyzed data. R.D., J.C.S., and B.K.W. contributed to discussions and data interpretation. R.D., J.C.S., and B.K.W. wrote the manuscript. All authors reviewed and edited the manuscript. B.K.W. is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Funding Statement

This work was supported by the National Institutes of Health Human Islet Research Network (U01DK123717 [B.K.W.]). R.D. was supported by Knut och Alice Wallenberg Stiftelse, Sweden (KAW 2019.0580). The authors gratefully acknowledge the use of the Opera Phenix High-Content/High-Throughput imaging system at the Broad Institute, funded by grant S10-OD-026839-01 (B.K.W.).

Footnotes

R.D. and J.C.S. contributed equally.

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