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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

Prototypes of type-I, type-II and the novel type-III targetingmolecules.(A) The type-I molecule (I-Tz) was designed to contain an EGFRtyrosine kinase inhibitor (I) and a DNA alkylating triazene (Tz) moietybridged by a hydrolysable linker. The type-I molecule can inhibit EGFR as anintact structure (step 2), or upon undergoing hydrolysis to release the twomoieties (I + Tz), but the molecule is only capable of targeting DNA throughthe release of its Tz moiety (step 1). (B) The type-II moleculewas designed to contain the EGFR tyrosine kinase inhibitor (I) and the DNAalkylating triazene moiety (Tz) connected via a non-hydrolysable linker.This type-II molecule is capable of targeting both EGFR and DNA as an intactstructure through each of its targeting arm (steps 1 and 2).(C) The novel type-III molecule (K1-K2) is designed to containtwo tyrosine kinase inhibitors connected via a hydrolysable linker where K1is targeted to Kin-1 (EGFR) and K2 to Kin-2 (c-Src). This molecule is“programmed” to exhibit both type-I and type-II likeproperties by inhibiting its two targets both as an intact structure (steps2, 3) as well as upon undergoing hydrolysis to release inhibitors of EGFRand c-Src (step 1).
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pone.0117215.g001: Prototypes of type-I, type-II and the novel type-III targetingmolecules.(A) The type-I molecule (I-Tz) was designed to contain an EGFRtyrosine kinase inhibitor (I) and a DNA alkylating triazene (Tz) moietybridged by a hydrolysable linker. The type-I molecule can inhibit EGFR as anintact structure (step 2), or upon undergoing hydrolysis to release the twomoieties (I + Tz), but the molecule is only capable of targeting DNA throughthe release of its Tz moiety (step 1). (B) The type-II moleculewas designed to contain the EGFR tyrosine kinase inhibitor (I) and the DNAalkylating triazene moiety (Tz) connected via a non-hydrolysable linker.This type-II molecule is capable of targeting both EGFR and DNA as an intactstructure through each of its targeting arm (steps 1 and 2).(C) The novel type-III molecule (K1-K2) is designed to containtwo tyrosine kinase inhibitors connected via a hydrolysable linker where K1is targeted to Kin-1 (EGFR) and K2 to Kin-2 (c-Src). This molecule is“programmed” to exhibit both type-I and type-II likeproperties by inhibiting its two targets both as an intact structure (steps2, 3) as well as upon undergoing hydrolysis to release inhibitors of EGFRand c-Src (step 1).

Mentions: Current trend in cancer drug discovery is towards the design of multi-targeted agents[1]. This trend is drivenby the observation that the attrition rates in the development of multi-targetedagents are significantly lower than that of single-targeted agents [2]. Indeed analysis of 974anticancer agents from 1995 to 2007, in developmental phases (phase I toregistration) led to an overall attrition rate of 82%. However, this rate fell toonly 52% when the analysis was restricted to a subset of multi-targeted kinaseinhibitors [2]. The clinicalefficacy of multi-targeted agents is partly imputed to their ability to induce atandem blockade of multiple targets that drive tumour progression and resistance toapoptosis in refractory tumours. However, despite the acknowledged potency ofmulti-targeted drugs, their rational design to inhibit specific oncogenic targetsremains a tremendous challenge [3]. In the past, in the context of a novel multi-targeted approachtermed “combi-targeting”, we designed inhibitors termed“combi-molecules” that can block targets as divergent as tyrosinekinase receptors and genomic DNA. We demonstrated their ability to kill tumour cellsby blocking receptor phosphorylation, damaging DNA and down-regulating DNA repairproteins [4,5]. We classified suchmolecules as type I (i.e., those that require hydrolysis to fully exhibit their dualpotency) and type II (i.e., those that could induce DNA damage and a tandem blockadeof receptor mediated signaling without requirement for hydrolysis). As depicted inFig. 1A, the type I moleculeI-Tz was designed to release an EGFR tyrosine kinase inhibitor (I) and a DNAdamaging species Tz (step 1). I-Tz was also designed to interact with EGFR as anintact structure (step 2) [6–8].Conversely, I-Tz in its type II form is designed to inhibit EGFR tyrosine kinase anddamage DNA without requirement for hydrolysis (Fig. 1B, steps 1 and 2) [9,10]. While this classification includes several types of agents directedat the epidermal growth factor receptor (EGFR) and DNA, the demonstration of theapproach with two different tyrosine kinase targets remained a challenge [11–13]. Here, we designed arational approach to give rise to a novel type of chimeric kinase inhibitor, thatreconciles the type I and II targeting models. As shown in Fig. 1C (step 1), to target a cellexpressing kinase 1 (Kin-1) and kinase 2 (Kin-2), we wish to design the moleculeK1-K2 to behave like a type I targeted molecule, by conferring it a hydrolysablelinker, which upon hydrolysis will release free K1 and K2 (inhibitors of kinases 1and 2 respectively). In addition, the molecule is designed to possess an intrinsicdual K1/K2 targeting property as an intact molecule, thereby behaving as a type IImolecule (Fig. 1C, steps 2 and3). The expected advantage of the latter property lies in the fact that in the eventthat the hydrolysis of K1-K2 is slow inside the tumour cell, the intact structurecan still induce a tandem blockade of the oncogenic targets Kin-1 and Kin-2.Overall, this novel targeting approach, which is now designated as type IIIcombi-targeting, is designed to induce multispecies dynamic inside the cells withthe dual tyrosine kinase Kin-1 and Kin-2 inhibition as a constant. Here we challengethis concept using an optimized molecule AL776, which was designed to block areceptor tyrosine kinase, EGFR, as Kin-1, and a non-receptor tyrosine kinase, c-Src,as Kin-2.


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)

Prototypes of type-I, type-II and the novel type-III targetingmolecules.(A) The type-I molecule (I-Tz) was designed to contain an EGFRtyrosine kinase inhibitor (I) and a DNA alkylating triazene (Tz) moietybridged by a hydrolysable linker. The type-I molecule can inhibit EGFR as anintact structure (step 2), or upon undergoing hydrolysis to release the twomoieties (I + Tz), but the molecule is only capable of targeting DNA throughthe release of its Tz moiety (step 1). (B) The type-II moleculewas designed to contain the EGFR tyrosine kinase inhibitor (I) and the DNAalkylating triazene moiety (Tz) connected via a non-hydrolysable linker.This type-II molecule is capable of targeting both EGFR and DNA as an intactstructure through each of its targeting arm (steps 1 and 2).(C) The novel type-III molecule (K1-K2) is designed to containtwo tyrosine kinase inhibitors connected via a hydrolysable linker where K1is targeted to Kin-1 (EGFR) and K2 to Kin-2 (c-Src). This molecule is“programmed” to exhibit both type-I and type-II likeproperties by inhibiting its two targets both as an intact structure (steps2, 3) as well as upon undergoing hydrolysis to release inhibitors of EGFRand c-Src (step 1).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0117215.g001: Prototypes of type-I, type-II and the novel type-III targetingmolecules.(A) The type-I molecule (I-Tz) was designed to contain an EGFRtyrosine kinase inhibitor (I) and a DNA alkylating triazene (Tz) moietybridged by a hydrolysable linker. The type-I molecule can inhibit EGFR as anintact structure (step 2), or upon undergoing hydrolysis to release the twomoieties (I + Tz), but the molecule is only capable of targeting DNA throughthe release of its Tz moiety (step 1). (B) The type-II moleculewas designed to contain the EGFR tyrosine kinase inhibitor (I) and the DNAalkylating triazene moiety (Tz) connected via a non-hydrolysable linker.This type-II molecule is capable of targeting both EGFR and DNA as an intactstructure through each of its targeting arm (steps 1 and 2).(C) The novel type-III molecule (K1-K2) is designed to containtwo tyrosine kinase inhibitors connected via a hydrolysable linker where K1is targeted to Kin-1 (EGFR) and K2 to Kin-2 (c-Src). This molecule is“programmed” to exhibit both type-I and type-II likeproperties by inhibiting its two targets both as an intact structure (steps2, 3) as well as upon undergoing hydrolysis to release inhibitors of EGFRand c-Src (step 1).
Mentions: Current trend in cancer drug discovery is towards the design of multi-targeted agents[1]. This trend is drivenby the observation that the attrition rates in the development of multi-targetedagents are significantly lower than that of single-targeted agents [2]. Indeed analysis of 974anticancer agents from 1995 to 2007, in developmental phases (phase I toregistration) led to an overall attrition rate of 82%. However, this rate fell toonly 52% when the analysis was restricted to a subset of multi-targeted kinaseinhibitors [2]. The clinicalefficacy of multi-targeted agents is partly imputed to their ability to induce atandem blockade of multiple targets that drive tumour progression and resistance toapoptosis in refractory tumours. However, despite the acknowledged potency ofmulti-targeted drugs, their rational design to inhibit specific oncogenic targetsremains a tremendous challenge [3]. In the past, in the context of a novel multi-targeted approachtermed “combi-targeting”, we designed inhibitors termed“combi-molecules” that can block targets as divergent as tyrosinekinase receptors and genomic DNA. We demonstrated their ability to kill tumour cellsby blocking receptor phosphorylation, damaging DNA and down-regulating DNA repairproteins [4,5]. We classified suchmolecules as type I (i.e., those that require hydrolysis to fully exhibit their dualpotency) and type II (i.e., those that could induce DNA damage and a tandem blockadeof receptor mediated signaling without requirement for hydrolysis). As depicted inFig. 1A, the type I moleculeI-Tz was designed to release an EGFR tyrosine kinase inhibitor (I) and a DNAdamaging species Tz (step 1). I-Tz was also designed to interact with EGFR as anintact structure (step 2) [6–8].Conversely, I-Tz in its type II form is designed to inhibit EGFR tyrosine kinase anddamage DNA without requirement for hydrolysis (Fig. 1B, steps 1 and 2) [9,10]. While this classification includes several types of agents directedat the epidermal growth factor receptor (EGFR) and DNA, the demonstration of theapproach with two different tyrosine kinase targets remained a challenge [11–13]. Here, we designed arational approach to give rise to a novel type of chimeric kinase inhibitor, thatreconciles the type I and II targeting models. As shown in Fig. 1C (step 1), to target a cellexpressing kinase 1 (Kin-1) and kinase 2 (Kin-2), we wish to design the moleculeK1-K2 to behave like a type I targeted molecule, by conferring it a hydrolysablelinker, which upon hydrolysis will release free K1 and K2 (inhibitors of kinases 1and 2 respectively). In addition, the molecule is designed to possess an intrinsicdual K1/K2 targeting property as an intact molecule, thereby behaving as a type IImolecule (Fig. 1C, steps 2 and3). The expected advantage of the latter property lies in the fact that in the eventthat the hydrolysis of K1-K2 is slow inside the tumour cell, the intact structurecan still induce a tandem blockade of the oncogenic targets Kin-1 and Kin-2.Overall, this novel targeting approach, which is now designated as type IIIcombi-targeting, is designed to induce multispecies dynamic inside the cells withthe dual tyrosine kinase Kin-1 and Kin-2 inhibition as a constant. Here we challengethis concept using an optimized molecule AL776, which was designed to block areceptor tyrosine kinase, EGFR, as Kin-1, and a non-receptor tyrosine kinase, c-Src,as Kin-2.

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