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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

Series of EGFR-c-Src targeting type III molecules and their kinaseinhibitory potency in vitro.(A) EGFR-c-Src targeting type III molecules were designedand synthesized in our laboratory using quinazoline moieties (red) asthe EGFR targeting head and dasatinib as the c-Src inhibitory arm(green), connected through different hydrolysable linkers.(B) In vitro kinase assay was used todetermine the potency of each molecule in the series to competitivelybind and inhibit the ATP binding pocket of the tyrosine kinase domainsof EGFR and c-Src. Gefitinib and dasatinib were used as control drugsfor comparison, and the IC50 values of kinase inhibition weredetermined using the GraphPad Prism 6.0 software. Each value representsthe average IC50 from three independent experiments, carriedout in duplicate.
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pone.0117215.g002: Series of EGFR-c-Src targeting type III molecules and their kinaseinhibitory potency in vitro.(A) EGFR-c-Src targeting type III molecules were designedand synthesized in our laboratory using quinazoline moieties (red) asthe EGFR targeting head and dasatinib as the c-Src inhibitory arm(green), connected through different hydrolysable linkers.(B) In vitro kinase assay was used todetermine the potency of each molecule in the series to competitivelybind and inhibit the ATP binding pocket of the tyrosine kinase domainsof EGFR and c-Src. Gefitinib and dasatinib were used as control drugsfor comparison, and the IC50 values of kinase inhibition weredetermined using the GraphPad Prism 6.0 software. Each value representsthe average IC50 from three independent experiments, carriedout in duplicate.

Mentions: A series of K1-K2 molecules designed and synthesized in our laboratory ispresented in Fig. 2A. Thequinazoline backbone being highly tolerant of bulky substituents at the6-position [23], waschosen as the EGFR targeting scaffold. This scaffold is common to many clinicalinhibitors of EGFR including gefitinib, erlotinib, lapatinib and afatinib and isknown to anchor into the ATP binding site of the receptor [24]. In the past, we foundthe pyrazolo pyrimidine class of inhibitors of c-Src to be extremely sensitiveto substituent changes [11]. Therefore, we selected the thiazolylaminopyrimidine backbone ofdasatinib, a clinically approved and potent inhibitor of Abl and c-Src, as thec-Src targeting scaffold [25]. To facilitate intracellular hydrolysis, we selected ester- orcarbonate-based linkers between K1 and K2. This led to the structures shown inFig. 2A, withIC50 values for EGFR and c-Src inhibition measured by anin vitro kinase assay. Of all the linkers studied, thesuccinic acid one led to the most potent dual EGFR-c-Src targeting molecule. Thelatter, AL776 showed an IC50 of 0.12 μM for EGFR kinaseinhibition and 3 nM for c-Src kinase inhibition (Fig. 2B). Therefore, AL776 was selected as our K1-K2prototype in the study.


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)

Series of EGFR-c-Src targeting type III molecules and their kinaseinhibitory potency in vitro.(A) EGFR-c-Src targeting type III molecules were designedand synthesized in our laboratory using quinazoline moieties (red) asthe EGFR targeting head and dasatinib as the c-Src inhibitory arm(green), connected through different hydrolysable linkers.(B) In vitro kinase assay was used todetermine the potency of each molecule in the series to competitivelybind and inhibit the ATP binding pocket of the tyrosine kinase domainsof EGFR and c-Src. Gefitinib and dasatinib were used as control drugsfor comparison, and the IC50 values of kinase inhibition weredetermined using the GraphPad Prism 6.0 software. Each value representsthe average IC50 from three independent experiments, carriedout in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117215.g002: Series of EGFR-c-Src targeting type III molecules and their kinaseinhibitory potency in vitro.(A) EGFR-c-Src targeting type III molecules were designedand synthesized in our laboratory using quinazoline moieties (red) asthe EGFR targeting head and dasatinib as the c-Src inhibitory arm(green), connected through different hydrolysable linkers.(B) In vitro kinase assay was used todetermine the potency of each molecule in the series to competitivelybind and inhibit the ATP binding pocket of the tyrosine kinase domainsof EGFR and c-Src. Gefitinib and dasatinib were used as control drugsfor comparison, and the IC50 values of kinase inhibition weredetermined using the GraphPad Prism 6.0 software. Each value representsthe average IC50 from three independent experiments, carriedout in duplicate.
Mentions: A series of K1-K2 molecules designed and synthesized in our laboratory ispresented in Fig. 2A. Thequinazoline backbone being highly tolerant of bulky substituents at the6-position [23], waschosen as the EGFR targeting scaffold. This scaffold is common to many clinicalinhibitors of EGFR including gefitinib, erlotinib, lapatinib and afatinib and isknown to anchor into the ATP binding site of the receptor [24]. In the past, we foundthe pyrazolo pyrimidine class of inhibitors of c-Src to be extremely sensitiveto substituent changes [11]. Therefore, we selected the thiazolylaminopyrimidine backbone ofdasatinib, a clinically approved and potent inhibitor of Abl and c-Src, as thec-Src targeting scaffold [25]. To facilitate intracellular hydrolysis, we selected ester- orcarbonate-based linkers between K1 and K2. This led to the structures shown inFig. 2A, withIC50 values for EGFR and c-Src inhibition measured by anin vitro kinase assay. Of all the linkers studied, thesuccinic acid one led to the most potent dual EGFR-c-Src targeting molecule. Thelatter, AL776 showed an IC50 of 0.12 μM for EGFR kinaseinhibition and 3 nM for c-Src kinase inhibition (Fig. 2B). Therefore, AL776 was selected as our K1-K2prototype in the study.

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