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

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”.

Predicting therapeutic nanomedicine efficacy using a companion magnetic resonance imaging nanoparticle

“…With the EGFR-expressing 4T1 tumor model (26), we stratified excised tumors into “low,” “medium,” and “high” antibody groups. Co-injected TNP-encapsulated docetaxel was then quantified.…”

Dual-faced SH3BGRL: oncogenic in mice, tumor suppressive in humans

“… 1 c-Src, a prometastasis oncogene, is well characterized to promote cellular invasion, migration and has been justly proposed as a therapeutic target in cancers. 2 , 3 , 4 , 5 However, the biochemical regulation of c-Src activation is not fully understood. The best-characterized Src regulator is phosphorylation, with the two major sites of phosphorylation on murine and chicken c-Src being Tyr416 (Tyr419 in humans) and Tyr527 (Tyr530 in humans).…”

Three-dimensional organotypic matrices from alternative collagen sources as pre-clinical models for cell biology

“…It has previously been shown that ROCK inhibition by Y-27632 can inhibit the infiltration of MDA-MB-231 cells using 2D in vitro assays 102 – 104 . Conversely, the EGFR inhibitor Gefitinib has been shown to have potent anti-proliferative effects on MDA-MB-231 cells 105 – 107 , while the anti-invasive effect is less well established 108 – 110 . To quantify the changes in invasion depth upon inhibition, invaded MDA-MB-231 cells at each 100 μm increment were divided by the number of cells on the surface of the collagen matrix, in a similar manner to the invasive index calculated in Fig.…”