Targeting Casein Kinase 1 (CK1) in Hematological Cancers

The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.     

Casein Kinase 1 Epsilon Expression Predicts Poorer Prognosis in Low T-Stage Oral Cancer Patients

Casein kinase 1 is a group of ubiquitous serine/threonine kinases that are involved in normal cellular functions and several pathological conditions, such as DNA repair, cell cycle progression, cytokinesis, differentiation, and apoptosis. Recent studies have indicated that casein kinase 1-epsilon (CK1ɛ) and casein kinase 1-delta (CK1δ) expression has a role in human cancers. We investigated the associations between CK1ɛ and CK1δ expression and the clinical parameters of oral cancer using immunohistochemical study methods on oral squamous cell carcinoma specimens. The results of our immunohistochemical analysis showed that the loss of CK1ɛ expression was greatly associated with a poor four-year survival rate in oral cancer patients (p = 0.002). A Kaplan-Meier analysis showed that patients who had a loss of CK1ɛ expression had a considerably poorer overall survival rate than patients who had positive CK1ɛ expressions (p = 0.022). A univariate analysis revealed that patients who had a loss of CK1ɛ expression had considerably poorer overall survival (OS) than patients who had positive expression (p = 0.024, hazard ratio (HR) = 1.7). In conclusion, our data indicated that the loss of cytoplasmic CK1ɛ expression is greatly associated with poor survival and might be an adverse survival factor.     

A Computational Workflow for the Identification of Novel Fragments Acting as Inhibitors of the Activity of Protein Kinase CK1δ

Fragment-Based Drug Discovery (FBDD) has become, in recent years, a consolidated approach in the drug discovery process, leading to several drug candidates under investigation in clinical trials and some approved drugs. Among these successful applications of the FBDD approach, kinases represent a class of targets where this strategy has demonstrated its real potential with the approved kinase inhibitor Vemurafenib. In the Kinase family, protein kinase CK1 isoform δ (CK1δ) has become a promising target in the treatment of different neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. In the present work, we set up and applied a computational workflow for the identification of putative fragment binders in large virtual databases. To validate the method, the selected compounds were tested in vitro to assess the CK1δ inhibition.     

Long Non-Coding RNA KCNQ1OT1 Regulates Protein Kinase CK2 Via miR-760 in Senescence and Calorie Restriction

Long non-coding RNAs (lncRNAs) play important biological roles. Here, the roles of the lncRNA KCNQ1OT1 in cellular senescence and calorie restriction were determined. KCNQ1OT1 knockdown mediated various senescence markers (increased senescence-associated β-galactosidase staining, the p53-p21^Cip1/WAF1 pathway, H3K9 trimethylation, and expression of the senescence-associated secretory phenotype) and reactive oxygen species generation via CK2α downregulation in human cancer HCT116 and MCF-7 cells. Additionally, KCNQ1OT1 was downregulated during replicative senescence, and its silencing induced senescence in human lung fibroblast IMR-90 cells. Additionally, an miR-760 mimic suppressed KCNQ1OT1-mediated CK2α upregulation, indicating that KCNQ1OT1 upregulated CK2α by sponging miR-760. Finally, the KCNQ1OT1–miR-760 axis was involved in both lipopolysaccharide-mediated CK2α reduction and calorie restriction (CR)-mediated CK2α induction in these cells. Therefore, for the first time, this study demonstrates that the KCNQ1OT1–miR-760–CK2α pathway plays essential roles in senescence and CR, thereby suggesting that KCNQ1OT1 is a novel therapeutic target for an alternative treatment that mimics the effects of anti-aging and CR.     

CK1δ-Derived Peptides as Novel Tools Inhibiting the Interactions between CK1δ and APP695 to Modulate the Pathogenic Metabolism of APP

Alzheimer’s disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-β (Aβ), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aβ levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.     

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca²⁺-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca²⁺ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca²⁺ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca²⁺ entry were investigated in Fura-2 Ca²⁺-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca²⁺ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S₁₁₇₂A mutant displayed enhanced Ca²⁺ entry. The TRPM3-mediated Ca²⁺ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.     

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.     

Diabetic Kinome Inhibitors—A New Opportunity for β-Cells Restoration

Diabetes, and several diseases related to diabetes, including cancer, cardiovascular diseases and neurological disorders, represent one of the major ongoing threats to human life, becoming a true pandemic of the 21st century. Current treatment strategies for diabetes mainly involve promoting β-cell differentiation, and one of the most widely studied targets for β-cell regeneration is DYRK1A kinase, a member of the DYRK family. DYRK1A has been characterized as a key regulator of cell growth, differentiation, and signal transduction in various organisms, while further roles and substrates are the subjects of extensive investigation. The targets of interest in this review are implicated in the regulation of β-cells through DYRK1A inhibition—through driving their transition from highly inefficient and death-prone populations into efficient and sufficient precursors of islet regeneration. Increasing evidence for the role of DYRK1A in diabetes progression and β-cell proliferation expands the potential for pharmaceutical applications of DYRK1A inhibitors. The variety of new compounds and binding modes, determined by crystal structure and in vitro studies, may lead to new strategies for diabetes treatment. This review provides recent insights into the initial self-activation of DYRK1A by tyrosine autophosphorylation. Moreover, the importance of developing novel DYRK1A inhibitors and their implications for the treatment of diabetes are thoroughly discussed. The evolving understanding of DYRK kinase structure and function and emerging high-throughput screening technologies have been described. As a final point of this work, we intend to promote the term “diabetic kinome” as part of scientific terminology to emphasize the role of the synergistic action of multiple kinases in governing the molecular processes that underlie this particular group of diseases.     

Synthesis of Novel Acyl Derivatives of 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)propan-1-ols—Intracellular TBBi-Based CK2 Inhibitors with Proapoptotic Properties

Protein kinase CK2 has been considered as an attractive drug target for anti-cancer therapy. The synthesis of N-hydroxypropyl TBBi and 2MeTBBi derivatives as well as their respective esters was carried out by using chemoenzymatic methods. Concomitantly with kinetic studies toward recombinant CK2, the influence of the obtained compounds on the viability of two human breast carcinoma cell lines (MCF-7 and MDA-MB-231) was evaluated using MTT assay. Additionally, an intracellular inhibition of CK2 as well as an induction of apoptosis in the examined cells after the treatment with the most active compounds were studied by Western blot analysis, phase-contrast microscopy and flow cytometry method. The results of the MTT test revealed potent cytotoxic activities for most of the newly synthesized compounds (EC₅₀ 4.90 to 32.77 µM), corresponding to their solubility in biological media. We concluded that derivatives with the methyl group decrease the viability of both cell lines more efficiently than their non-methylated analogs. Furthermore, inhibition of CK2 in breast cancer cells treated with the tested compounds at the concentrations equal to their EC₅₀ values correlates well with their lipophilicity since derivatives with higher values of logP are more potent intracellular inhibitors of CK2 with better proapoptotic properties than their parental hydroxyl compounds.     

Anti-Growth,Anti-Angiogenic,and Pro-Apoptotic Effects by CX-4945, an Inhibitor of Casein Kinase 2, on HuCCT-1 Human Cholangiocarcinoma Cells via Control of Caspase-9/3, DR-4, STAT-3/STAT-5, Mcl-1, eIF-2α, and HIF-1α

Overexpression of casein kinase 2 (CK2) has an oncogenic and pro-survival role in many cancers. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-angiogenic effects. Up to date, the anti-cancer effect and mechanism of CX-4945 on human cholangiocarcinoma (CCA) remain unclear. This study investigated whether CX-4945 inhibits growth and induces apoptosis of HuCCT-1 cells, a human CCA cell line. Of note, treatment with CX-4945 at 20 μM markedly reduced survival and induced apoptosis of HuCCT-1 cells, as evidenced by nuclear DNA fragmentation, PARP cleavage, activation of caspase-9/3, and up-regulation of DR-4. Although CX-4945 did not affect the phosphorylation and expression of CK2, it vastly inhibited the phosphorylation of CK2 substrates, supporting the drug’s efficacy in inhibiting CK2 and its downstream pathway. Importantly, knockdown of CK2 that partially suppressed the phosphorylation of CK2 substrates resulted in a significant reduction of HuCCT-1 cell survival. In addition, CX-4945 reduced the phosphorylation and expression of STAT-3 and STAT-5 in HuCCT-1 cells, and pharmacological inhibition or respective knockdown of these proteins resulted in significant growth suppression of HuCCT-1 cells. CX-4945 also had abilities to decrease Mcl-1 expression while increasing eIF-2α phosphorylation in HuCCT-1 cells. Furthermore, there was a time-differential negative regulation of HIF-1α expression by CX-4945 in HuCCT-1 cells, and knockdown of HIF-1α caused a significant reduction of the cell survival. In summary, these results demonstrated that CX-4945 has anti-growth, anti-angiogenic, and pro-apoptotic effects on HuCCT-1 cells, which are mediated through control of CK2, caspase-9/3, DR-4, STAT-3/5, Mcl-1, eIF-2α, and HIF-1α.     

Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic β-Cells

Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells.     

Anti-adipogenic and Pro-lipolytic Effects on 3T3-L1 Preadipocytes by CX-4945, an Inhibitor of Casein Kinase 2

Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-adipogenic activities. However, the anti-adipogenic and pro-lipolytic effects and the mode of action of CX-4945 in (pre)adipocytes remain elusive. Here, we explored the effects of CX-4945 on adipogenesis and lipolysis in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte cell line. CX-4945 at 15 μM strongly reduced lipid droplet (LD) accumulation and triglyceride (TG) content in differentiating 3T3-L1 cells, indicating the drug’s anti-adipogenic effect. Mechanistically, CX-4945 reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and perilipin A in differentiating 3T3-L1 cells. Strikingly, CX-4945 further increased the phosphorylation levels of cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) while decreasing the intracellular ATP content in differentiating 3T3-L1 cells. In differentiated 3T3-L1 cells, CX-4945 had abilities to stimulate glycerol release and elevate the phosphorylation levels of hormone-sensitive lipase (HSL), pointing to the drug’s pro-lipolytic effect. In addition, CX-4945 induced the activation of extracellular signal-regulated kinase-1/2 (ERK-1/2), and PD98059, an inhibitor of ERK-1/2, attenuated the CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 cells, indicating the drug’s ERK-1/2-dependent lipolysis. In summary, this investigation shows that CX-4945 has strong anti-adipogenic and pro-lipolytic effects on differentiating and differentiated 3T3-L1 cells, mediated by control of the expression and phosphorylation levels of CK2, C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, AMPK, LKB-1, ERK-1/2, and HSL.     

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson’s disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2^G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) “cell-autonomous”. Here, we explored whether ΔLRRK2^G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2^G2019S or its “dead” kinase form, ΔLRRK2^DK, and human α-syn with the A53T mutation (AAV-α-syn^A53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-syn^A53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-syn^A53T with AAV-ΔLRRK2^G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-syn^A53T alone or with AAV-ΔLRRK2^DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2^G2019 alone^, through cell-autonomous mechanisms.     

Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy

Eukaryotic elongation factor 2 kinase (eEF2K or Ca²⁺/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure–activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.     

Early Gβγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gβγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gβγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gβγ-GRK2 inhibition at the time of DMM by administering the Gβγ inhibitor “gallein” and the GRK2 inhibitor “paroxetine” daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gβγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gβγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gβγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gβγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.     

Glycogen Synthase Kinase 3β Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

Alternative strategies beyond current chemotherapy and radiation therapy regimens are needed in the treatment of advanced stage and recurrent endometrial cancers. There is considerable promise for biologic agents targeting the extracellular signal-regulated kinase (ERK) pathway for treatment of these cancers. Many downstream substrates of the ERK signaling pathway, such as glycogen synthase kinase 3β (GSK3β), and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3β inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3β by either lithium chloride (LiCl) or specific GSK3β inhibitor VIII showed cytostatic and cytotoxic effects on multiple endometrial cancer cell lines, with little effect on the immortalized normal endometrial cell line. Flow cytometry and immunofluorescence revealed a G2/M cell cycle arrest in both type I (AN3CA, KLE, and RL952) and type II (ARK1) endometrial cancer cell lines. In addition, LiCl pre-treatment sensitized AN3CA cells to the chemotherapy agent paclitaxel. Administration of LiCl to AN3CA tumor-bearing mice resulted in partial or complete regression of some tumors. Thus, GSK3β activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.     

Design of CK2β-Mimicking Peptides as Tools To Study the CK2α/CK2β Interaction in Cancer Cells

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2α/CK2β interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2β-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2α using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2α ligand (K_i=0.622 μm ). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC₅₀=37 μm ) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement.     

Quantitative Analysis of the Potency of Equimolar Two-Drug Combinations and Combi-Molecules Involving Kinase Inhibitors In Vitro: The Concept of Balanced Targeting

The median-effect principle proposed by Chou and Talalay is the most effective approach to parameterize interactions between several agents in combination. However, this method cannot be used to evaluate the effectiveness of equimolar drug combinations, which are comparative references for dual-targeting molecular design. Here, using data acquired through the development of “combi-molecules” blocking two kinases (e.g., EGFR-c-Src and EGFR-c-Met), we established potency indices for equimolar and dual-targeted inhibitors. If the fold difference (κ) between the IC50 of the two individual kinase inhibitors was >6, the IC50 of their equimolar combination resembled that of the more potent inhibitor. Hence, the “combi-targeting” of the two kinases was considered “imbalanced” and the combination ineffective. However, if κ ≤ 6, the IC50 of the combination fell below that of each individual drug and the combi-targeting was considered “balanced” and the combination effective. We also showed that combi-molecules should be compared with equimolar combinations only under balanced conditions and propose a new parameter Ω for validating their effectiveness. A multi-targeted drug is effective if Ω < 1, where Ω is defined as the IC50 of the drug divided by that of the corresponding equimolar combination. Our study provides a methodology to determine the in vitro potency of equimolar two-drug combinations as well as combi-/hybrid molecules inhibiting two different kinase targets.