Changes in cellular signaling proteins in extracts from A549, H460, and U2OS cells treated with cisplatin or docetaxel.

Cell extracts from A549, H460, and U2OS human cancer cell lines treated with cisplatin and docetaxel were analyzed by mass spectrometry (MS) proteomic analysis. The extracts were enriched for cellular signaling proteins using a mix of three different immobilized kinase inhibitors (Purvalanol B, Bisindolylmaleimide X, and (R)-3-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-yl)benzoic acid (SB6-060-05)) on sepharose bead columns. Raw data is deposited in the PRIDE database [1], project number PXD005286. Data presented () shows changes relative to untreated control for each biological replicate for the three cell lines.

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling.

Extended structure-activity study of thienopyrimidine-based EGFR inhibitors with evaluation of drug-like properties.

Thieno[2,3-d]pyrimidines are attractive derivatives for cancer treatment, among others through regulation of the epidermal growth factor receptor tyrosine kinase (EGFR-TK). In an extended SAR study, 44 new compounds of this class have been evaluated as inhibitors, while simultaneously focussing on ADME properties. Through the application of bioisosters, hybrid structures, solubilizing tails, and a combination approach several successful alterations in terms of activity and physiochemical properties were accomplished. Compounds based on benzylamines were found superior to aniline hybrid structures with respect to activity and ADME profile. Exploration of the former class revealed meta- and para amides as favourable 6-aryl substituents, contributing to an increase in activity and acting as a linker for solubilizing tails. Next, combinations of activity-inducing groups on the same scaffold resulted in new drug candidates. Compounds containing 6-aryls with the (2-(dimethylamino)ethyl)carbamoyl substituent were found equipotent to Erlotinib. Compared to this commercial drug, improved solubility and metabolic stability were observed. However, the thieno[2,3-d]pyrimidines with a solubilizing tail was by Caco-2 experiments found to have permeability issues, making further drug development difficult. Selected compounds were further analysed for toxicity and teratogenicity in zebrafish embryos. Two thienopyrimidines were both found to be less lethal than Erlotinib and to perform as well in terms of teratogenicity. Finally, the most promising thienopyrimidine drug was evaluated in a panel of human cancer cell lines, showing a clear potential for thienopyrimidines as anti-cancer agents.

Truncated structures used in search for new lead compounds and in a retrospective analysis of thienopyrimidine-based EGFR inhibitors.

An approach for optimization of epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors using truncated thienopyrimidine structures combined with enzymatic assay has been evaluated. This was done by synthesis and EGFR activity measurement of a series of fragment structures and their corresponding drug-model analogues. On average, the activity of the drug-like structures increased a ten-fold as compared to the fragments. However, the potency of the drug-model compound could not be precisely predicted, visualising the typical challenge with linking substructures. Additionally, the activity data provided useful SAR information with respect to stereochemical preference and the structure requirements of the 4-amino group. A retrospective evaluation of binding efficiency in previously discovered thieno[2,3-d]pyrimidines, suggests a high probability of obtaining potent EGFR inhibitors if based on the 3-chloro-4-fluoroaniline moiety. Compounds derived from (S)-2-hydroxy-1-phenylethylamine also have resulted in highly active EGFR-TK inhibitors. In contrast the 3-ethynylaniline containing structures appears more difficult to develop. Thieno[2,3-d]pyrimidin-4-amines have also been used for construction of irreversible EGFR-TK inhibitors. The data indicate these compounds to possess sub-optimal non-covalent interactions.

Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines as highly potent EGFR-TK inhibitors with Src-family activity.

The epidermal growth factor receptor is an important target in molecular cancer therapy. Herein, the enzymatic inhibition potential of a series of chiral and non chiral pyrrolopyrimidine based derivatives have been investigated and optimised. Overall, seven new compounds were identified having enzymatic IC50 values comparable to or better than the commercial drug Erlotinib. High activity was also confirmed towards the epidermal growth factor receptor L858R and L861Q mutants. Based on calculated druglike properties, eight compounds were further evaluated towards a panel of 52 other kinases revealing interesting Src-family kinase and colony stimulating factor 1 receptor kinase inhibitory activity. Cell proliferation studies with the cell lines A431, C-33A, AU-565, K-562 and genetically engineered Ba/F3-EGFR(L858R) cells also showed several molecules to be more active than Erlotinib, and thus confirming these pyrrolopyrimidines as attractive drug candidates or lead structures.

Structure-activity study leading to identification of a highly active thienopyrimidine based EGFR inhibitor.

Based on the thieno[2,3-d]pyrimidine scaffold, a series of new 4-amino-6-aryl thienopyrimidines have been prepared and evaluated as EGFR tyrosine kinase inhibitors. The in vitro activity was found to depend strongly on the substitution pattern in the 6-aryl ring, the stereochemistry, and the basicity at the secondary 4-amino group. A stepwise optimization by combination of active fragments led to the discovery of three structures with EGFR IC50 < 1 nM. The most potent drug candidate had an IC50 of 0.3 nM towards EGFR and its mutants L858R and L861Q. Studies using human cancer cell lines and an EGFR-L858R reporter cell system revealed good cellular potency, verifying the identified thienopyrimidines as promising lead structures.