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Sustained antiproliferative mechanisms by RB24, a targeted precursor of multiple inhibitors of epidermal growth factor receptor and a DNA alkylating agent in the A431 epidermal carcinoma of the vulva cell line.

Banerjee R, Rachid Z, Qiu Q, McNamee JP, Tari AM, Jean-Claude BJ - Br. J. Cancer (2004)

Bottom Line: Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion.The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion.Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene.

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. M 7.15, Montreal, Quebec, Canada H3A 1A1.

ABSTRACT
Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity.

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Mentions: The design of our combi-molecule, RB24, was based on the premise that acetoxymethyltriazenes are known to be hydrolysed to a hydroxymethyltriazene intermediate that rapidly degrades into the corresponding monoalkyltriazene (Hemens et al, 1984; Cameron et al, 1985; Hemens and Vaughan, 1986). The latter further heterolyses to an aromatic amine+a DNA-damaging species. In a recent publication (Banerjee et al, 2003), we demonstrated that indeed RB24 was capable of generating the ultimate amino compound RB10, in a symmetrically inversed relationship. As depicted in Scheme 1Scheme 1, decomposition through the intermediates, RB14 and ZR08, is the sole mechanism by which RB24 can be converted to RB10. Thus, the significant EGFR inhibitory potency of the putative intermediates RB14, ZR08 and RB10 indicates that RB24 would maintain its TK inhibitory potency throughout the multistep degradation cascade. More importantly, RB24 and its two derivatives (RB14, ZR08) induced irreversible inhibition of EGFR TK when tested alone, a protracted inhibition that may be rationalised in the light of recent data on the mechanism of irreversible acrylamide-based inhibitors. It is now known that 4-anilinoquinazolines bearing a 6-acrylamido group react with the cysteine 773 of EGFR, thereby irreversibly blocking the receptor (Fry et al, 1998; Smaill et al, 1999, 2000). Based on the mechanism of decomposition of RB24 (Banerjee et al, 2003), we did not expect a direct reaction between the cysteine thiol group and the acetoxy moiety. Perhaps, if the iminium ion is formed in the active site of EGFR, it may react with the cysteine as depicted in Scheme 2Scheme 2. Addition of thiol group to the iminium ions of acetoxymethyltriazene has already been reported by Iley et al (1991) who developed this type of triazenes as lyase inhibitors. Moreover, the ultimate triazene metabolite of RB24, ZR08, may also directly alkylate the cysteine residue. Quinazolinotriazenes of the same class, BJ2000 and SMA41, have already been demonstrated to induce irreversible inhibition (Brahimi et al, 2002; Matheson et al, 2003b, 2004b).


Sustained antiproliferative mechanisms by RB24, a targeted precursor of multiple inhibitors of epidermal growth factor receptor and a DNA alkylating agent in the A431 epidermal carcinoma of the vulva cell line.

Banerjee R, Rachid Z, Qiu Q, McNamee JP, Tari AM, Jean-Claude BJ - Br. J. Cancer (2004)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747684&req=5

Mentions: The design of our combi-molecule, RB24, was based on the premise that acetoxymethyltriazenes are known to be hydrolysed to a hydroxymethyltriazene intermediate that rapidly degrades into the corresponding monoalkyltriazene (Hemens et al, 1984; Cameron et al, 1985; Hemens and Vaughan, 1986). The latter further heterolyses to an aromatic amine+a DNA-damaging species. In a recent publication (Banerjee et al, 2003), we demonstrated that indeed RB24 was capable of generating the ultimate amino compound RB10, in a symmetrically inversed relationship. As depicted in Scheme 1Scheme 1, decomposition through the intermediates, RB14 and ZR08, is the sole mechanism by which RB24 can be converted to RB10. Thus, the significant EGFR inhibitory potency of the putative intermediates RB14, ZR08 and RB10 indicates that RB24 would maintain its TK inhibitory potency throughout the multistep degradation cascade. More importantly, RB24 and its two derivatives (RB14, ZR08) induced irreversible inhibition of EGFR TK when tested alone, a protracted inhibition that may be rationalised in the light of recent data on the mechanism of irreversible acrylamide-based inhibitors. It is now known that 4-anilinoquinazolines bearing a 6-acrylamido group react with the cysteine 773 of EGFR, thereby irreversibly blocking the receptor (Fry et al, 1998; Smaill et al, 1999, 2000). Based on the mechanism of decomposition of RB24 (Banerjee et al, 2003), we did not expect a direct reaction between the cysteine thiol group and the acetoxy moiety. Perhaps, if the iminium ion is formed in the active site of EGFR, it may react with the cysteine as depicted in Scheme 2Scheme 2. Addition of thiol group to the iminium ions of acetoxymethyltriazene has already been reported by Iley et al (1991) who developed this type of triazenes as lyase inhibitors. Moreover, the ultimate triazene metabolite of RB24, ZR08, may also directly alkylate the cysteine residue. Quinazolinotriazenes of the same class, BJ2000 and SMA41, have already been demonstrated to induce irreversible inhibition (Brahimi et al, 2002; Matheson et al, 2003b, 2004b).

Bottom Line: Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion.The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion.Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene.

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. M 7.15, Montreal, Quebec, Canada H3A 1A1.

ABSTRACT
Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity.

Show MeSH
Related in: MedlinePlus