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


Abstract

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.

Keywords: balanced targeting; combi-molecules; equimolar combinations; hybrid molecules; kinase inhibitors; multi-targeting.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) IC50 values of tumor cell growth inhibition on a panel of human cancer cell lines using gefitinib (EGFR kinase inhibitor), dasatinib (c-Src kinase inhibitor), and their equimolar combination. The fold difference between the IC50 values of the two individual drugs is denoted by κ. (b–g) Relative IC50 values (IC50 of individual drug minus the average IC50 of the drug on the entire panel of cell lines) of gefitinib, dasatinib, and their equimolar combination for (b–d) κ > 6 and (e–g) κ ≤ 6. 1 Rao S. et al., 2015 [19].
Figure 2
Figure 2
(a) IC50 values of tumor cell growth inhibition on a panel of human cancer cell lines using gefitinib, crizotinib (c-Met kinase inhibitor), and their equimolar combination. The fold difference between the IC50 values of the two individual drugs is denoted by κ. (b–g) Relative IC50 values (IC50 of individual drug minus the average IC50 of the drug on the entire panel of cell lines) of gefitinib, crizotinib, and their equimolar combination for (b–d) κ > 6, and (e–g) κ ≤ 6. 1 Rao et al. 2015 [19].
Figure 3
Figure 3
(a) IC50 values of growth inhibition assay targeting c-Met and c-Src using crizotinib, dasatinib, and their equimolar combination. (b) IC50 values of growth inhibition targeting EGFR and DNA using gefitinib, temozolomide (DNA alkylating agent), and their equimolar combination. (c) IC50 values of growth inhibition assay targeting c-Met and DNA using crizotinib, temozolomide, and their equimolar combination. The fold difference between the IC50 values of the two individual drugs is denoted by κ. 1 Rao et al. 2015 [19].
Figure 4
Figure 4
Comparing the distribution of IC50 values of kinase inhibitor-1 (γ1), kinase inhibitor-2 (γ2), and their equimolar combination (γ3) across all cell lines exhibiting a fold difference in IC50 of (a) > 6 and (b) ≤ 6. Note that γ1 > γ2 and κ represents the fold difference, which is calculated as γ1/γ2. Statistical analysis was carried out using unpaired two-tailed Student t-test.
Figure 5
Figure 5
(a) Structures of EGFR-c-Src (AL660-AL776) and EGFR-c-Met (LP121) combi-molecules. E = EGFR targeting head, S = c-Src targeting head, and M = c-Met targeting head (structures shown in Figure S1 and Schemes S1–S4 in the supplementary section). (b) Immunoblot analysis of inhibition of EGFR and c-Src phosphorylation by 1 μM dose of EGFR-c-Src-targeting combi-molecules (i.e., AL660, AL690, AL692, AL739) and the clinical inhibitors gefitinib (G) and dasatinib (D) in NIH3T3-Her14 (EGFR transfected) cells stimulated with 50 ng/mL of EGF. (c) IC50 values of growth inhibition and the combi-targeting effect, Ω (γ4/γ3) for imbalanced targeting combi-molecule on the NIH3T3-wild type and Her14 (EGFR transfected) cells. (d) EGFR and c-Met kinase inhibitory potency of LP121 using an in vitro kinase assay. (e) Target modulation by LP121, C—crizotinib (5 μM), G—gefitinib (5 μM), and C + G—an equimolar combination of crizotinib + gefitinib (5 μM each) in 4T1 cells using western blot analysis under conditions of EGF + HGF (50 ng/mL each) stimulation. (f,g) IC50 values of growth inhibition of the balance-targeted combi-molecule AL776 and the equivalent equimolar drug combination. Ω (γ4/γ3) was calculated for cell lines with κ ≤ 6. (h,i) IC50 values of growth inhibition of the balanced-targeted combi-molecule LP121 and the equivalent equimolar drug combination. Ω (γ4/γ3) was calculated for cell lines with κ ≤ 6. 1 Rao et al. 2015 [19].
Figure 6
Figure 6
Flow chart summarizing the sequence of events leading to the design and synthesis of unimolecular analogs (i.e., hybrid drugs, chimeric molecules, and combi-molecules) as predicted on the basis of the fold difference between individual drugs (κ). γ1 = IC50 of drug 1, γ2 = IC50 of drug 2, γ3 = IC50 of equimolar combination of drug 1 + drug 2, γ4 = IC50 of unimolecular analog (e.g., combi-molecule), κ = γ1/γ2 where γ1 > γ2, and Ω = combi-targeting effect of unimolecular analog calculated using the equation Ω = γ4/γ3.
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