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

Anti-motility and anti-invasive properties of AL776 in 4T1 mousemammary tumour and MDA-MB-231 triple negative breast cancer celllines.(A) 4T1 and (B) MDA-MB-231 cells were treatedwith 100 nM of AL776 or control drugs gefitinib or dasatinib for aperiod of 24h and the wound-closure (scratch) was monitored at both 0hand 24h time points. (C) 4T1 or (D) MDA-MB-231cells were treated with varying doses of AL776 (in comparison withdasatinib or gefitinib) for a period of 24h in a Boyden Chamber invasionassay. Cells were plated in serum-free media (top chamber) and allowedto invade across the layer of matrigel towards media containing 10% FBS(bottom chamber) through chemotaxis. Drugs were added to both the topand bottom chambers to maintain a uniform distribution. Cells werefixed, stained and quantified to generate the percentage of invadingcells across the matrigel in comparison with untreated control cells.The histograms represent the average ± SEM of three independentexperiments. Statistical analysis was performed using the unpairedtwo-tailed t-test and p values were obtained (p < 0.05 issignificant): 4T1 ** (p = 0.001), MDA-MB-231 * (p< 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117215.g008: Anti-motility and anti-invasive properties of AL776 in 4T1 mousemammary tumour and MDA-MB-231 triple negative breast cancer celllines.(A) 4T1 and (B) MDA-MB-231 cells were treatedwith 100 nM of AL776 or control drugs gefitinib or dasatinib for aperiod of 24h and the wound-closure (scratch) was monitored at both 0hand 24h time points. (C) 4T1 or (D) MDA-MB-231cells were treated with varying doses of AL776 (in comparison withdasatinib or gefitinib) for a period of 24h in a Boyden Chamber invasionassay. Cells were plated in serum-free media (top chamber) and allowedto invade across the layer of matrigel towards media containing 10% FBS(bottom chamber) through chemotaxis. Drugs were added to both the topand bottom chambers to maintain a uniform distribution. Cells werefixed, stained and quantified to generate the percentage of invadingcells across the matrigel in comparison with untreated control cells.The histograms represent the average ± SEM of three independentexperiments. Statistical analysis was performed using the unpairedtwo-tailed t-test and p values were obtained (p < 0.05 issignificant): 4T1 ** (p = 0.001), MDA-MB-231 * (p< 0.05).

Mentions: c-Src being a key tyrosine kinase in the signaling pathways associated withmotility and invasion, we thought it of interest to evaluate the effects ofAL776 on motility and invasion using the wound-healing and the Boyden chamberassay respectively. These experiments were performed in the highly invasive 4T1and MDA-MB-231 breast cancer cell lines. Both cell lines were used in theseassays due to their high levels of c-Src expression, which is a key oncogene indriving tumour invasion and metastasis [28,29]. The assay was carried out by exposing the cells to the drug for24h, a time point at which 50% of intact AL776 was found in the cells.Wound-healing assay results showed that AL776 at 0.1 μM blockedwound-closure after a 24h drug exposure in both cell lines (Fig. 8A and B). Boyden chamberinvasion assay results showed that AL776, like dasatinib, strongly inhibitedinvasion at a concentration as low as 0.1 μM. However, gefitinib wasmostly unable to block invasion at such low doses (Fig. 8C and D), indicating that c-Src and not EGFR isprimarily responsible for the invasive properties of these cells.


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)

Anti-motility and anti-invasive properties of AL776 in 4T1 mousemammary tumour and MDA-MB-231 triple negative breast cancer celllines.(A) 4T1 and (B) MDA-MB-231 cells were treatedwith 100 nM of AL776 or control drugs gefitinib or dasatinib for aperiod of 24h and the wound-closure (scratch) was monitored at both 0hand 24h time points. (C) 4T1 or (D) MDA-MB-231cells were treated with varying doses of AL776 (in comparison withdasatinib or gefitinib) for a period of 24h in a Boyden Chamber invasionassay. Cells were plated in serum-free media (top chamber) and allowedto invade across the layer of matrigel towards media containing 10% FBS(bottom chamber) through chemotaxis. Drugs were added to both the topand bottom chambers to maintain a uniform distribution. Cells werefixed, stained and quantified to generate the percentage of invadingcells across the matrigel in comparison with untreated control cells.The histograms represent the average ± SEM of three independentexperiments. Statistical analysis was performed using the unpairedtwo-tailed t-test and p values were obtained (p < 0.05 issignificant): 4T1 ** (p = 0.001), MDA-MB-231 * (p< 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117215.g008: Anti-motility and anti-invasive properties of AL776 in 4T1 mousemammary tumour and MDA-MB-231 triple negative breast cancer celllines.(A) 4T1 and (B) MDA-MB-231 cells were treatedwith 100 nM of AL776 or control drugs gefitinib or dasatinib for aperiod of 24h and the wound-closure (scratch) was monitored at both 0hand 24h time points. (C) 4T1 or (D) MDA-MB-231cells were treated with varying doses of AL776 (in comparison withdasatinib or gefitinib) for a period of 24h in a Boyden Chamber invasionassay. Cells were plated in serum-free media (top chamber) and allowedto invade across the layer of matrigel towards media containing 10% FBS(bottom chamber) through chemotaxis. Drugs were added to both the topand bottom chambers to maintain a uniform distribution. Cells werefixed, stained and quantified to generate the percentage of invadingcells across the matrigel in comparison with untreated control cells.The histograms represent the average ± SEM of three independentexperiments. Statistical analysis was performed using the unpairedtwo-tailed t-test and p values were obtained (p < 0.05 issignificant): 4T1 ** (p = 0.001), MDA-MB-231 * (p< 0.05).
Mentions: c-Src being a key tyrosine kinase in the signaling pathways associated withmotility and invasion, we thought it of interest to evaluate the effects ofAL776 on motility and invasion using the wound-healing and the Boyden chamberassay respectively. These experiments were performed in the highly invasive 4T1and MDA-MB-231 breast cancer cell lines. Both cell lines were used in theseassays due to their high levels of c-Src expression, which is a key oncogene indriving tumour invasion and metastasis [28,29]. The assay was carried out by exposing the cells to the drug for24h, a time point at which 50% of intact AL776 was found in the cells.Wound-healing assay results showed that AL776 at 0.1 μM blockedwound-closure after a 24h drug exposure in both cell lines (Fig. 8A and B). Boyden chamberinvasion assay results showed that AL776, like dasatinib, strongly inhibitedinvasion at a concentration as low as 0.1 μM. However, gefitinib wasmostly unable to block invasion at such low doses (Fig. 8C and D), indicating that c-Src and not EGFR isprimarily responsible for the invasive properties of these cells.

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