Limits...
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

In vitro and in vivo hydrolysis ofAL776 using high performance liquid chromatography (HPLC) and massspectrometry (MS) analyses.(A) The kinetics of entry into the cells and degradation ofAL776 inside the cells were monitored using HPLC analysis. NIH3T3-Her14(EGFR transfected) cells were treated with 25 μM of AL776 for 1h,2h, 6h, 24h and 48h, after which the cells and the correspondingextracellular media were collected and processed according to theprocedure described in the Materials and Method section. The area underthe curve (AUC) for the AL776 peak was determined and its percentagecompared with all the other peaks was calculated and plotted.(B) A representative spectrum obtained from liquidchromatography (LC)-mass spectrometry (MS) analysis in cells treatedwith AL776 for 48h is shown with m/z = 462 (AL621), m/z = 488(dasatinib) and m/2z = 517 (AL776). (C) The kinetics ofAL776 hydrolysis in the plasma of CD-1 mice injected with 80 mg/kg ofthe drug was monitored 5, 15 and 30 min post-administration. LC-MSchromatograms at different time points with m/z values for intact AL776and its metabolites are shown: m/z = 462 for AL621, m/z = 562 forAL621-L (succinic acid linked-AL621), m/z = 488 for dasatinib, m/z = 588for dasatinib-L (succinic acid linked-dasatinib), m/2z = 516 forAL776.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414309&req=5

pone.0117215.g004: In vitro and in vivo hydrolysis ofAL776 using high performance liquid chromatography (HPLC) and massspectrometry (MS) analyses.(A) The kinetics of entry into the cells and degradation ofAL776 inside the cells were monitored using HPLC analysis. NIH3T3-Her14(EGFR transfected) cells were treated with 25 μM of AL776 for 1h,2h, 6h, 24h and 48h, after which the cells and the correspondingextracellular media were collected and processed according to theprocedure described in the Materials and Method section. The area underthe curve (AUC) for the AL776 peak was determined and its percentagecompared with all the other peaks was calculated and plotted.(B) A representative spectrum obtained from liquidchromatography (LC)-mass spectrometry (MS) analysis in cells treatedwith AL776 for 48h is shown with m/z = 462 (AL621), m/z = 488(dasatinib) and m/2z = 517 (AL776). (C) The kinetics ofAL776 hydrolysis in the plasma of CD-1 mice injected with 80 mg/kg ofthe drug was monitored 5, 15 and 30 min post-administration. LC-MSchromatograms at different time points with m/z values for intact AL776and its metabolites are shown: m/z = 462 for AL621, m/z = 562 forAL621-L (succinic acid linked-AL621), m/z = 488 for dasatinib, m/z = 588for dasatinib-L (succinic acid linked-dasatinib), m/2z = 516 forAL776.

Mentions: The kinetics of hydrolysis of AL776 was studied both in vitrousing NIH3T3-Her14 (EGFR transfected) cells and in vivo in CD-1mice following i.p. and i.v. injection. In vitro, highperformance liquid chromatography (HPLC) analysis of the extracellular mediumand isolated whole cells revealed that AL776 was stable enough to slowly diffuseinto the cells with minimal extracellular decomposition. As shown in Fig. 4A, 24–48h later,AL776 was detectable inside the cells but not in the extracellular medium,indicating that the absorption equilibrium was shifted towards intracellularretention of the molecule. AL776 slowly degraded inside the cells and liquidchromatography-mass spectrometry (LC-MS) analysis confirmed that the tworeleased metabolites were AL621 and dasatinib (Fig. 4B). The observation of detectable levels ofAL776 as long as 48h after treatment indicates that, as predicted, in additionto the individual metabolites K1 and K2 released inside the cells, intact K1-K2may also contribute to their response to drug treatment. A representativespectrum is shown in Fig. 4Bwith m/z corresponding to the major metabolites along with intact AL776.


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)

In vitro and in vivo hydrolysis ofAL776 using high performance liquid chromatography (HPLC) and massspectrometry (MS) analyses.(A) The kinetics of entry into the cells and degradation ofAL776 inside the cells were monitored using HPLC analysis. NIH3T3-Her14(EGFR transfected) cells were treated with 25 μM of AL776 for 1h,2h, 6h, 24h and 48h, after which the cells and the correspondingextracellular media were collected and processed according to theprocedure described in the Materials and Method section. The area underthe curve (AUC) for the AL776 peak was determined and its percentagecompared with all the other peaks was calculated and plotted.(B) A representative spectrum obtained from liquidchromatography (LC)-mass spectrometry (MS) analysis in cells treatedwith AL776 for 48h is shown with m/z = 462 (AL621), m/z = 488(dasatinib) and m/2z = 517 (AL776). (C) The kinetics ofAL776 hydrolysis in the plasma of CD-1 mice injected with 80 mg/kg ofthe drug was monitored 5, 15 and 30 min post-administration. LC-MSchromatograms at different time points with m/z values for intact AL776and its metabolites are shown: m/z = 462 for AL621, m/z = 562 forAL621-L (succinic acid linked-AL621), m/z = 488 for dasatinib, m/z = 588for dasatinib-L (succinic acid linked-dasatinib), m/2z = 516 forAL776.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117215.g004: In vitro and in vivo hydrolysis ofAL776 using high performance liquid chromatography (HPLC) and massspectrometry (MS) analyses.(A) The kinetics of entry into the cells and degradation ofAL776 inside the cells were monitored using HPLC analysis. NIH3T3-Her14(EGFR transfected) cells were treated with 25 μM of AL776 for 1h,2h, 6h, 24h and 48h, after which the cells and the correspondingextracellular media were collected and processed according to theprocedure described in the Materials and Method section. The area underthe curve (AUC) for the AL776 peak was determined and its percentagecompared with all the other peaks was calculated and plotted.(B) A representative spectrum obtained from liquidchromatography (LC)-mass spectrometry (MS) analysis in cells treatedwith AL776 for 48h is shown with m/z = 462 (AL621), m/z = 488(dasatinib) and m/2z = 517 (AL776). (C) The kinetics ofAL776 hydrolysis in the plasma of CD-1 mice injected with 80 mg/kg ofthe drug was monitored 5, 15 and 30 min post-administration. LC-MSchromatograms at different time points with m/z values for intact AL776and its metabolites are shown: m/z = 462 for AL621, m/z = 562 forAL621-L (succinic acid linked-AL621), m/z = 488 for dasatinib, m/z = 588for dasatinib-L (succinic acid linked-dasatinib), m/2z = 516 forAL776.
Mentions: The kinetics of hydrolysis of AL776 was studied both in vitrousing NIH3T3-Her14 (EGFR transfected) cells and in vivo in CD-1mice following i.p. and i.v. injection. In vitro, highperformance liquid chromatography (HPLC) analysis of the extracellular mediumand isolated whole cells revealed that AL776 was stable enough to slowly diffuseinto the cells with minimal extracellular decomposition. As shown in Fig. 4A, 24–48h later,AL776 was detectable inside the cells but not in the extracellular medium,indicating that the absorption equilibrium was shifted towards intracellularretention of the molecule. AL776 slowly degraded inside the cells and liquidchromatography-mass spectrometry (LC-MS) analysis confirmed that the tworeleased metabolites were AL621 and dasatinib (Fig. 4B). The observation of detectable levels ofAL776 as long as 48h after treatment indicates that, as predicted, in additionto the individual metabolites K1 and K2 released inside the cells, intact K1-K2may also contribute to their response to drug treatment. A representativespectrum is shown in Fig. 4Bwith m/z corresponding to the major metabolites along with intact AL776.

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