The combi-targeting concept: dissection of the binary mechanism of action of the combi-triazene SMA41 in vitro and antitumor activity in vivo.

We have previously reported the synthesis of SMA41, a unimolecular combination of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of the quinazoline class and a DNA-damaging monomethyltriazene termed "combimolecule". Hydrolysis of 1-[4-(m-tolylamino)-6-quinazolinyl]-3-methyltriazene (SMA41) gives rise to an intact TKI [6-amino-4-(3-methylanilino)quinazoline; SMA52] capable of inhibiting epidermal growth factor (EGF)-induced EGFR autophosphorylation and a DNA-targeting methyldiazonium species. Herein, we showed that SMA41 blocked EGF-induced EGFR autophosphorylation by an irreversible mechanism, suggesting that it may covalently damage the receptor in these cells. More importantly, this was associated with significant inhibition of mitogen-activated protein kinase activation in A431 cells. In cells treated with [14C]SMA41, radio-high-performance liquid chromatography detection of both N7- and O6-methylguanine revealed an almost complete repair of the O6-methylguanine lesions and a greater tolerance of the N7-methylguanine adducts 24 h post-treatment. In contrast to temozolomide (a cyclic triazene used in the clinic) and the reversible inhibitor SMA52, SMA41 induced significant cell cycle arrest in S, G2, and M phases 24 h after a 2-h drug exposure. Furthermore, in vivo studies demonstrated that SMA41 was well tolerated. At 200 mg/kg, it showed approximately 2-fold greater antiproliferative activity than SMA52 in A431 cells implanted in immunocompromised SCID mice. These results suggest that the binary targeting properties of SMA41 are associated with a binary cascade of events in the cells that seem to culminate into significant growth inhibition in vitro and in vivo.

The combi-targetinG concept: intracellular fragmentation of the binary epidermal growth factor (EGFR)/DNA targeting "combi-triazene" SMA41.

We have designed a novel tumor targeting strategy that consists of designing molecules termed "combi-molecules" or TZ-I to be masked forms of multiple antitumor agents. One such molecule SMA41, the TZ-I prototype, has been shown to target the epidermal growth factor receptor (EGFR) and to degrade under physiological conditions to give SMA52 (I) (an inhibitor of EGFR) and methyldiazonium (TZ) (a DNA alkylating species). While the antiproliferative advantages of this novel binary targeting strategy have now been demonstrated, the exact subcellular localization of the degradation products released from SMA41 remained elusive. Here we exploited the fluorescence properties of SMA52 to study its release from SMA41 and its subcellular distribution. Further, using 14C-labeled SMA41, we determined the distribution of the methydiazonium within subcellular macromolecules (DNA, RNA, protein). The results showed that SMA41 degraded to SMA52 in the carcinoma of the vulva cell line A431 with a half-life of 11min. The latter compound was primarily distributed in the perinuclear region. At equimolar concentrations, higher levels of SMA52 were observed when released from SM41 than when the cells were directly exposed to SMA52, indicating that the combi-molecular approach may offer a transport advantage to the released bioactive species. Radioactivity associated with SMA41 3-[ 14C]-methyl group was distributed throughout DNA, RNA, and protein, the latter macromolecule being the most alkylated. The results suggest that SMA41 (TZ-I) may diffuse into the cells, break down into two species: SMA52 (I) concentrated in the perinuclear region and methyldiazonium (TZ) that diffuses in all intracellular organelles and unspecifically alkylates RNA, protein, and nuclear DNA.

The combi-targeting concept: a novel 3,3-disubstituted nitrosourea with EGFR tyrosine kinase inhibitory properties.

PURPOSE: To study the dual mechanism of action of FD137, a 3,3-disubstituted nitrosourea designed to block signaling mediated by the epidermal growth factor receptor (EGFR) on its own and to be hydrolyzed to an inhibitor of EGFR plus a DNA-damaging species. MATERIALS AND METHODS: HPLC was used to determine the half-life (t(1/2)) of FD137 and to characterize its derived metabolite FD110. The dual mechanisms of DNA damaging and EGFR tyrosine kinase (TK) targeting were ascertained by the comet assay for DNA damage and by inmunodetection of phosphotyrosine in an ELISA and a whole-cell assay for EGFR-mediated signaling. The antiproliferative effects of the different drugs and their combinations were determined by the sulforhodamine B (SRB) assay. RESULTS: In contrast to BCNU, FD137 significantly blocked EGF-induced EGFR autophosphorylation (IC(50) 4 micro M) in the human solid tumor cell line A431. DNA damage induced by FD137 could only be observed after 24 h exposure, but the level of DNA damage remained 3.6-fold lower than that induced by BCNU. This difference was rationalized by the 160-fold greater stability of FD137 when compared with BCNU in serum-containing medium. Further, degradation of FD137 was accompanied by the slow release of FD110, an extremely potent inhibitor of EGFR TK [IC(50) (EGFR autophosphorylation) <0.3 micro M]. The complex properties of FD137 translated into a 55-fold greater antiproliferative activity than BCNU against the EGFR-overexpressing A431 cells that coexpresses the O(6)-alkylguanine transferase (AGT). Depletion of AGT in these cells by the use of O(6)-benzylguanine (O(6)-BG) enhanced their sensitivity to BCNU by 8-fold, but only by 3-fold to FD137. CONCLUSIONS: The results overall suggest that the superior antiproliferative activity of FD137 when compared with BCNU may be associated with its ability to behave as a combination of many species with different mechanisms of action. However, the enhancement of its potency by O(6)-BG suggests that its antiproliferative effect was at least partially mitigated by AGT and perhaps it may be largely dominated by its signal transduction inhibitory component.

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PURPOSE: Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. METHODS: Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. RESULTS: Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G(2)/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. CONCLUSIONS: These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

Inhibition of epidermal growth factor receptor-mediated signaling by "Combi-triazene" BJ2000, a new probe for Combi-Targeting postulates.

The Combi-Targeting concept postulates that a molecule termed combi-molecule (C-molecule) with binary epidermal growth factor receptor (EGFR) targeting/DNA-damaging properties and with the ability to be hydrolyzed to another EGFR inhibitor should induce sustained antiproliferative activity in cells overexpressing EGFR. Because we postulate that the EGFR affinity of the C-molecule and that of its hydrolytic metabolites are critical parameters for sustained potency against EGFR-overexpressing cells, we synthesized BJ2000 (IC(50) = 0.1 microM, competitive binding at ATP site), a novel C-molecule that can decompose into a 6-amino-4-anilinoquinazoline FD105 (IC(50) = 0.2 microM). Studies using the EGFR-overexpressing A431 cells revealed that BJ2000 could damage DNA and block epidermal growth factor-stimulated EGFR autophosphorylation by a partially irreversible mechanism. Blockade of EGFR autophosphorylation subsequently induced inhibition of mitogen-activated protein kinase activation and c-fos gene expression. Enzyme-linked immunosorbent assay and growth factor-mediated stimulation of proliferation assays in the EGFR-expressing NIH3T3HER14 demonstrated the preferential EGFR-targeting properties of BJ2000, and more importantly suggest that blockade of EGFR phosphorylation by this drug translate into significant growth inhibitory effects. These properties culminated into irreversible antiproliferative effects as confirmed by a sulforhodamine B assay. Five days after a 2-h treatment, BJ2000 retained significant antiproliferative effect in A431 cells, whereas its reversible metabolite FD105 almost completely lost its activity. This result in toto lend support to the Combi-Targeting concept according to which a molecular conjugate kept small enough to interact with EGFR and designed to degrade into another inhibitor of the same target plus a DNA-damaging species may induce sustained growth inhibitory effect in EGFR-overexpressing cells.