Anilinoquinazoline inhibitors of the RET kinase domain-Elaboration of the 7-position.

We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging.

A fluorescence-based assay for the apurinic/apyrimidinic-site cleavage activity of human tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of phosphodiester bonds between the DNA 3'-phosphate and tyrosine residues and plays a major role in the repair of stalled topoisomerase I-DNA covalent complexes. Given this role, Tdp1 is of interest as a potential target for anticancer therapy. Inhibiting Tdp1 in combination with clinically used Top1 inhibitors may potentiate the effects of the latter and help to overcome some of the chemoresistance issues currently observed. In addition, Tdp1 can function during DNA repair to remove a variety of other 3' adducts from DNA such as phosphoglycolates and abasic or apurinic/apyrimidinic (AP) sites. Here we describe a new mix-and-read homogeneous fluorogenic assay for the measurement of the AP-site cleavage activity of Tdp1 that is compatible with high-throughput screening. The application of such an assay will open up further avenues for the discovery of novel Tdp1 inhibitors.

IncucyteDRC: An R package for the dose response analysis of live cell imaging data.

We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM.

Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade.

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series.  Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity.

Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.

Toxoflavins and deazaflavins as the first reported selective small molecule inhibitors of tyrosyl-DNA phosphodiesterase II.

The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.