The Division of Cancer Therapeutics (which includes the Cancer Research UK Cancer Therapeutics Unit) at the Institute of Cancer Research (ICR) is a unique, academic, multidisciplinary organisation dedicated to the discovery of mechanism-based drugs for the treatment of cancer.
Within the Division of Cancer Therapeutics, the Hit Discovery & Structural Design (HDSD) Team, led by Dr. Rob van Montfort, is responsible for hit generation for drug discovery projects using high-throughput and fragment screening approaches. A variety of cancer targets are investigated, including those involved in reversible protein phosphorylation, stress response, chromatin modification and targets involving specific protein-protein interactions. In addition, the team supports the design and improvement of hits to potent and selective inhibitors with a range of assay and biophysical techniques as well as through iterative protein-ligand crystallography.
Much of the group’s research is core-funded by Cancer Research UK and scientific activities take a bench-to-bedside approach. Throughout this process, emphasis is placed on ensuring that mechanistic assays are used to inform the decisions that drive each drug discovery project forward. Researchers work closely with clinical and nursing colleagues in The Royal Marsden Hospital to evaluate novel agents developed in-house and elsewhere. Cancer Therapeutics has been highly successful over many years and several drugs have been registered for the treatment of cancer patients, e.g. melphalan, chlorambucil, busulphan, carboplatin and raltitrexed, whilst abiraterone is in last stage Phase III clinical trials for prostate cancer. On average, two preclinical development candidates per year have been nominated over the last 5 years, illustrating the group’s fulfilment of their mission to discover novel therapeutics of potential benefit to patients.
The HDSD team consists of the Structure-Based Drug Design Group and the Analytical Technology and Screening (ATS) Group, the latter of which was until recently led by Dr. Wynne Aherne, now retired. Much of Dr. Aherne’s research career focused on the development of analytical methods for pharmacokinetic and pharmacodynamic measurements, especially the use of different immunoassay formats. Dr. Aherne joined the ICR in 1991 and studied the pharmacology of several anticancer agents, notably as part of the team involved in the successful development of inhibitors of thymidylate synthase. Dr. Aherne established the high-throughput screening facilities in the Cancer Research UK Cancer Therapeutics Unit and was responsible for managing several drug discovery projects in the Centre, including those in the chromatin field. In addition, she supervised more than a dozen PhD students and clinical fellows; and published over 190 papers, scientific reviews and book chapters.
Today, the ATS Group is led by Dr. Rosemary Burke who is responsible for primary screening, hit validation and supporting medicinal chemistry as hits progress through lead optimisation towards the selection of a preclinical candidate. Dr. Burke’s team has significant expertise in assay development, both biochemical and cell-based, and has also developed effective mechanistic assays for several projects using ELISA and IF endpoints.
The ATS Group utilises a wide range of PerkinElmer’s assay technologies, including AlphaScreen®, LANCE®, easylite-Kinase™, FlashPlate® assays and DELFIA®. Primary screens and most secondary assays are run in 384-well plates and throughput is facilitated by automated liquid handling equipment and stackable plate readers. The group has recently used Alpha assays in primary screens for inhibitors of the checkpoint (CHK1) kinase, and DELFIA® assays to determine the potency of selected validated hits.
Hit generation through high-throughput screening (HTS) of the group’s compound collection (~80,000 chemically diverse chemicals) was recently augmented by the acquisition of a ~2000 fragment (200-320 MW) library selected using calculated physicochemical properties and in-house derived structural filters. The impact of fragment screening on drug discovery was reviewed by Chessari and Woodhead, 2009.
The group’s present approach is to screen the fragment library against their target of choice in a biochemical assay at high concentration (200-400 mM) and to validate the weakly active but ligand efficient hits using X-ray crystallography or another biophysical method e.g. thermal shift binding assays. Confidence in this approach was gained following a successful screen of a set of 361 ‘template’ compounds (MW <350) against the checkpoint kinase CHK1 (Matthews et al., 2009). The compounds were screened in triplicate at a concentration of 250 mM using an AlphaScreen assay format (Figure 1). AlphaScreen assays have been successfully used for HTS screens for inhibitors of CHK1 and other kinases using final compound concentrations of 20-30 mM.
Figure 1.AlphaScreen CHK1 assay format.
Each plate was also screened in the presence of Triton X100, to eliminate aggregating compounds, and in an ‘interference’ assay using a biotinylated phosphorylated peptide as a positive control (Burns et al., 2006). The reproducibility of the controls (total enzyme activity and no enzyme blanks) in the primary screen is shown in Figure 2.
Figure 2. Reproducibility of the controls for the AlphaScreen CHK1 assay.
Twenty hits were identified (>25 % inhibition at 250 mM) and nine of these were validated by X-ray crystallography (Figure 3). A DELFIA assay was used to determine the potency of selected validated hits. One of these template hits has since been progressed by iterative structure-based design to give potent (<1 mM) inhibitors of CHK1 with appropriate CHK-1 mediated effects in colon cancer cells. This work was carried out in collaboration with Dr. Ian Collins and Dr. Michelle Garrett at the ICR, and Dr. John Reader at Sareum Ltd.
Figure 3. Representative molecular modeling fits generated from X-ray crystallography data showing two distinct hits (template compounds A and B) bound to the catalytic pocket of CHK1 (Left). For both compounds A and B, IC50 values measured using DELFIA closely matched the percent inhibition observed during the screening campaign performed with AlphaScreen (Right).
These investigations provided confidence in high concentration fragment biochemical screening and the approach, using a variety of assay formats, has now been successfully integrated into the hit generation strategies within the Division of Cancer Therapeutics at the ICR.
References
- Chessari G and Woodhead AJ (2009). From fragment to clinical candidate—a historical perspective. Drug Discovery Today; 14 (13-14): 668-675.
- Matthews TP, Klair S, Burns S, Boxall K, Cherry M, Fisher M, Westwood IM, Walton MI, McHardy T, Cheung K-MJ, Van Montfort R, Williams D, Aherne W, Garrett MD, Reader J and Collins I (2009). Identification of Inhibitors of Checkpoint Kinase 1 through Template Screening. J. Med. Chem.; 52 (15): 4810-4819.
- Burns S, Travers J, Collins I, Rowlands MG, Newbatt Y, Thompson N, Garrett MD, Workman P and Aherne W (2006). Identification of Small-Molecule Inhibitors of Protein Kinase B (PKB/AKT) in an AlphaScreen™ High-Throughput Screen. J. Biomol. Screening; 11 (7): 822-827.
The researchers gratefully acknowledge the financial support of Cancer Research UK (CUK grant numbers C309/A8274 and A8365) and NHS funding to the NIHR Biomedical Research Centre.
To find out more, please visit the websites of The Division of Cancer Therapeutics and The Institute of Cancer Research.
