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Target modulation by a kinase inhibitor engineered to induce a tandem blockade of the epidermal growth factor receptor (EGFR) and c-Src: the concept of type III combi-targeting.

Rao S, Larroque-Lombard AL, Peyrard L, Thauvin C, Rachid Z, Williams C, Jean-Claude BJ - PLoS ONE (2015)

Bottom Line: Cancer cells are characterized by a complex network of interrelated and compensatory signaling driven by multiple kinases that reduce their sensitivity to targeted therapy.Variation of K1-K2 linkers led to AL776, our first optimized EGFR-c-Src targeting prototype.We now term this new targeting model consisting of designing a kinase inhibitor K1-K2 to target Kin-1 and Kin-2, and to further release two inhibitors K1 and K2 of the latter kinases, "type III combi-targeting".

View Article: PubMed Central - PubMed

Affiliation: Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal Victoria Hospital, 687 Pine Avenue West Rm M7.19, Montreal, Quebec, H3A 1A1 Canada.

ABSTRACT
Cancer cells are characterized by a complex network of interrelated and compensatory signaling driven by multiple kinases that reduce their sensitivity to targeted therapy. Therefore, strategies directed at inhibiting two or more kinases are required to robustly block the growth of refractory tumour cells. Here we report on a novel strategy to promote sustained inhibition of two oncogenic kinases (Kin-1 and Kin-2) by designing a molecule K1-K2, termed "combi-molecule", to induce a tandem blockade of Kin-1 and Kin-2, as an intact structure and to be further hydrolyzed to two inhibitors K1 and K2 directed at Kin-1 and Kin-2, respectively. We chose to target EGFR (Kin-1) and c-Src (Kin-2), two tyrosine kinases known to synergize to promote tumour growth and progression. Variation of K1-K2 linkers led to AL776, our first optimized EGFR-c-Src targeting prototype. Here we showed that: (a) AL776 blocked EGFR and c-Src as an intact structure using an in vitro kinase assay (IC50 EGFR = 0.12 μM and IC50 c-Src = 3 nM), (b) it could release K1 (AL621, a nanomolar EGFR inhibitor) and K2 (dasatinib, a clinically approved Abl/c-Src inhibitor) by hydrolytic cleavage both in vitro and in vivo, (c) it could robustly inhibit phosphorylation of EGFR and c-Src (0.25-1 μM) in cells, (d) it induced 2-4 fold stronger growth inhibition than gefitinib or dasatinib and apoptosis at concentrations as low as 1 μM, and, (e) blocked motility and invasion at sub-micromolar doses in the highly invasive 4T1 and MDA-MB-231 cells. Despite its size (MW = 1032), AL776 blocked phosphorylation of EGFR and c-Src in 4T1 tumours in vivo. We now term this new targeting model consisting of designing a kinase inhibitor K1-K2 to target Kin-1 and Kin-2, and to further release two inhibitors K1 and K2 of the latter kinases, "type III combi-targeting".

No MeSH data available.


Related in: MedlinePlus

Growth inhibitory and apoptotic properties of AL776.(A) Growth inhibition was carried out in NIH3T3 wild typeand Her14 (EGFR transfected), 4T1 and MDA-MB-231 cell lines using thesulforhodamine B (SRB) assay. Cells were treated with varying doses ofAL776, gefitinib or dasatinib for a period of 5 days following whichcells were fixed, stained and quantified. Each experiment was repeatedat least four times and carried out in triplicates. (B)Comparison of the growth inhibition curves and correspondingIC50 values of AL776 in the isogenic NIH3T3 wild type andEGFR transfected cell lines. Each point represents the average ±SEM of five independent experiments carried out in triplicates. Thedifference between their average IC50 values wasstatistically significant with p < 0.05. (C,D) Annexin V and propidium iodide staining (PI) ofcells was used to determine the percentage of apoptosis (early + late)induced by AL776, gefitinib and dasatinib. Cells were treated withdifferent doses of AL776 for 48h, collected, stained and analyzed usingflow cytometry. The histogram represents the average ± SEM ofthree independent experiments with p < 0.05 in the NIH3T3-EGFRtransfected cell line.
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pone.0117215.g009: Growth inhibitory and apoptotic properties of AL776.(A) Growth inhibition was carried out in NIH3T3 wild typeand Her14 (EGFR transfected), 4T1 and MDA-MB-231 cell lines using thesulforhodamine B (SRB) assay. Cells were treated with varying doses ofAL776, gefitinib or dasatinib for a period of 5 days following whichcells were fixed, stained and quantified. Each experiment was repeatedat least four times and carried out in triplicates. (B)Comparison of the growth inhibition curves and correspondingIC50 values of AL776 in the isogenic NIH3T3 wild type andEGFR transfected cell lines. Each point represents the average ±SEM of five independent experiments carried out in triplicates. Thedifference between their average IC50 values wasstatistically significant with p < 0.05. (C,D) Annexin V and propidium iodide staining (PI) ofcells was used to determine the percentage of apoptosis (early + late)induced by AL776, gefitinib and dasatinib. Cells were treated withdifferent doses of AL776 for 48h, collected, stained and analyzed usingflow cytometry. The histogram represents the average ± SEM ofthree independent experiments with p < 0.05 in the NIH3T3-EGFRtransfected cell line.

Mentions: Its growth inhibitory property was tested by treating the NIH3T3 wild type, Her14(EGFR transfected), MDA-MB-231 and 4T1 cell lines with a dose range of AL776 orgefitinib or dasatinib for a period of 5 days and the IC50 values forgrowth inhibition were determined. The results showed that AL776 induced stronggrowth inhibition in NIH3T3-Her14 cells with an IC50 of 0.18μM and showed 2–4 fold higher potency compared with clinical drugsgefitinib or dasatinib. In MDA-MB-231 and 4T1 cell lines, it induced stronggrowth inhibition, like dasatinib, with IC50 values in thesub-micromolar range (Fig.9A). More importantly, AL776 was selectively more potent in NIH3T3cells transfected to overexpress EGFR when compared with their wild typecounterpart (Fig. 9B).


Target modulation by a kinase inhibitor engineered to induce a tandem blockade of the epidermal growth factor receptor (EGFR) and c-Src: the concept of type III combi-targeting.

Rao S, Larroque-Lombard AL, Peyrard L, Thauvin C, Rachid Z, Williams C, Jean-Claude BJ - PLoS ONE (2015)

Growth inhibitory and apoptotic properties of AL776.(A) Growth inhibition was carried out in NIH3T3 wild typeand Her14 (EGFR transfected), 4T1 and MDA-MB-231 cell lines using thesulforhodamine B (SRB) assay. Cells were treated with varying doses ofAL776, gefitinib or dasatinib for a period of 5 days following whichcells were fixed, stained and quantified. Each experiment was repeatedat least four times and carried out in triplicates. (B)Comparison of the growth inhibition curves and correspondingIC50 values of AL776 in the isogenic NIH3T3 wild type andEGFR transfected cell lines. Each point represents the average ±SEM of five independent experiments carried out in triplicates. Thedifference between their average IC50 values wasstatistically significant with p < 0.05. (C,D) Annexin V and propidium iodide staining (PI) ofcells was used to determine the percentage of apoptosis (early + late)induced by AL776, gefitinib and dasatinib. Cells were treated withdifferent doses of AL776 for 48h, collected, stained and analyzed usingflow cytometry. The histogram represents the average ± SEM ofthree independent experiments with p < 0.05 in the NIH3T3-EGFRtransfected cell line.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414309&req=5

pone.0117215.g009: Growth inhibitory and apoptotic properties of AL776.(A) Growth inhibition was carried out in NIH3T3 wild typeand Her14 (EGFR transfected), 4T1 and MDA-MB-231 cell lines using thesulforhodamine B (SRB) assay. Cells were treated with varying doses ofAL776, gefitinib or dasatinib for a period of 5 days following whichcells were fixed, stained and quantified. Each experiment was repeatedat least four times and carried out in triplicates. (B)Comparison of the growth inhibition curves and correspondingIC50 values of AL776 in the isogenic NIH3T3 wild type andEGFR transfected cell lines. Each point represents the average ±SEM of five independent experiments carried out in triplicates. Thedifference between their average IC50 values wasstatistically significant with p < 0.05. (C,D) Annexin V and propidium iodide staining (PI) ofcells was used to determine the percentage of apoptosis (early + late)induced by AL776, gefitinib and dasatinib. Cells were treated withdifferent doses of AL776 for 48h, collected, stained and analyzed usingflow cytometry. The histogram represents the average ± SEM ofthree independent experiments with p < 0.05 in the NIH3T3-EGFRtransfected cell line.
Mentions: Its growth inhibitory property was tested by treating the NIH3T3 wild type, Her14(EGFR transfected), MDA-MB-231 and 4T1 cell lines with a dose range of AL776 orgefitinib or dasatinib for a period of 5 days and the IC50 values forgrowth inhibition were determined. The results showed that AL776 induced stronggrowth inhibition in NIH3T3-Her14 cells with an IC50 of 0.18μM and showed 2–4 fold higher potency compared with clinical drugsgefitinib or dasatinib. In MDA-MB-231 and 4T1 cell lines, it induced stronggrowth inhibition, like dasatinib, with IC50 values in thesub-micromolar range (Fig.9A). More importantly, AL776 was selectively more potent in NIH3T3cells transfected to overexpress EGFR when compared with their wild typecounterpart (Fig. 9B).

Bottom Line: Cancer cells are characterized by a complex network of interrelated and compensatory signaling driven by multiple kinases that reduce their sensitivity to targeted therapy.Variation of K1-K2 linkers led to AL776, our first optimized EGFR-c-Src targeting prototype.We now term this new targeting model consisting of designing a kinase inhibitor K1-K2 to target Kin-1 and Kin-2, and to further release two inhibitors K1 and K2 of the latter kinases, "type III combi-targeting".

View Article: PubMed Central - PubMed

Affiliation: Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal Victoria Hospital, 687 Pine Avenue West Rm M7.19, Montreal, Quebec, H3A 1A1 Canada.

ABSTRACT
Cancer cells are characterized by a complex network of interrelated and compensatory signaling driven by multiple kinases that reduce their sensitivity to targeted therapy. Therefore, strategies directed at inhibiting two or more kinases are required to robustly block the growth of refractory tumour cells. Here we report on a novel strategy to promote sustained inhibition of two oncogenic kinases (Kin-1 and Kin-2) by designing a molecule K1-K2, termed "combi-molecule", to induce a tandem blockade of Kin-1 and Kin-2, as an intact structure and to be further hydrolyzed to two inhibitors K1 and K2 directed at Kin-1 and Kin-2, respectively. We chose to target EGFR (Kin-1) and c-Src (Kin-2), two tyrosine kinases known to synergize to promote tumour growth and progression. Variation of K1-K2 linkers led to AL776, our first optimized EGFR-c-Src targeting prototype. Here we showed that: (a) AL776 blocked EGFR and c-Src as an intact structure using an in vitro kinase assay (IC50 EGFR = 0.12 μM and IC50 c-Src = 3 nM), (b) it could release K1 (AL621, a nanomolar EGFR inhibitor) and K2 (dasatinib, a clinically approved Abl/c-Src inhibitor) by hydrolytic cleavage both in vitro and in vivo, (c) it could robustly inhibit phosphorylation of EGFR and c-Src (0.25-1 μM) in cells, (d) it induced 2-4 fold stronger growth inhibition than gefitinib or dasatinib and apoptosis at concentrations as low as 1 μM, and, (e) blocked motility and invasion at sub-micromolar doses in the highly invasive 4T1 and MDA-MB-231 cells. Despite its size (MW = 1032), AL776 blocked phosphorylation of EGFR and c-Src in 4T1 tumours in vivo. We now term this new targeting model consisting of designing a kinase inhibitor K1-K2 to target Kin-1 and Kin-2, and to further release two inhibitors K1 and K2 of the latter kinases, "type III combi-targeting".

No MeSH data available.


Related in: MedlinePlus