Affinity Selection

Label Free Affinity Selection Platform (LFAP)

The Label Free Affinity Selection Platform (LFAP) aims at the identification of small molecular binders to target proteins in a very broad range of affinities from very low to very high without any fluorescent labels on either ligand or protein. The 3 key advantages of this assay platform are: (a) No possible interference from any tracers, (b) speed and efficiency in the iterative identification binders and the subsequent sorting out of unsuitable compounds and too low affinity binders, (c) the opportunity to find more chemical starting points for difficult targets like protein-protein interactions, (d) no assay development if it is not known whether the project will see any hits identified, (e) suitability for integration into academic chemical biology workflows, (f) complete flexibility of where to start, end and which methods to use or leave out in a target screen.

Quantitative micro-dialysis (qµD) and qµD-ThermD (Data evaluation software for quantitative microdialysis at equilibrium)

Microdialysis is a powerful label free technique for the determination of binding affinities to target proteins, binding specificity and selectivity to targets, their protein family members and control proteins like HSA and BSA, and oligomerization and unspecific interactions of small molecules. CTB have developed a new thermodynamic equilibrium simulation software which enables Kd determination from quantification of compound concentrations in the dialysis chambers by HPLC, mass spectrometry or other analysis methods with Kd ranges up to ~ 500 µM dissociation constants.

Plate-based size exclusion chromatography assay, pbSEC

The method which is often tried first to test quickly whether there are any binders contained in a library to be screened against a target protein is our plate-based size exclusion chromatography assay technique(pbSEC). It is a robust, miniaturised and high throughput screening method utilised for homogenous solution based affinity selection. pbSEC is routinely used in CTB for primary screening of small molecule compound libraries against a great variety of important protein and enzyme drug targets and can deliver low affinity binders with Kds as low as 7 mM. In this method, target proteins are first pre-incubated with compound pools (usually between 10 and 50 compounds, selected based on clearly distinguishable molecular mass) in a microplate format. Protein-compound complexes and free unbound compound are then separated by centrifuge induced size exclusion chromatography and the eluted fraction analysed by LC-MS/MS for detection of compounds showing affinity for the target protein or enzyme. Very importantly, pbSEC can also be run in a “kinetic” mode. With the assumption that most small molecules will bind to a target with a diffusion controlled on-rate, the Kd defines the off-rate and therefore the half-life of the complex. Therefore, it is very straightforward to identify high affinity binders in a pool of binders found in a first run. The sample of protein and binders found in the collection plate is re-analysed in the same process, a certain time after the first run, for example a day later. Only higher affinity binders are remaining bound to the protein and can then be selected for further analysis. This rapid, label-free assay technique offers significant benefit since it has been configured for screening compound pools and can be applied to screening of chemically diverse molecules. pbSEC can also be utilised for screening poorly characterised protein targets since structural information is not required by this method. pbSEC is the principal method applied in our lab for primary screening and hit identification and is routinely applied for screening large compound libraries from industry collaborators.

MassFinder, a proprietory software for automated pbSEC analysis

To compliment our plate-based size exclusion chromatography assay set-up(pbSEC), we have developed a fully automated software pipeline for mass spec analysis of pbSEC runs. With compound pools defined, proprietary mass spectrometry file formats are read and cumulative ion counts for masses present within compound screening pools calculated. This automated first pass is then followed up by manual inspection of the top hits within each pool before progression to hit confirmation assays.

Fragment Based Screening

Despite the many successes from traditional high throughput screening (HTS) approaches, the high attrition rate of chemical compounds contained in complex, drug-like molecular libraries underpin the need for an alternative approach to lead discovery. Fragment based screening (FBS) is one such method to identify chemical leads and is an industry wide utilised technology as a complimentary approach to traditional HTS approaches. The concept of the FBS approach is that large drug-like molecules are built up of two, or more, smaller, lower complexity components (fragments) that bind with a correspondingly weaker affinity. Screening for the smaller fragments using: low molecular weight, fewer compound libraries (in the range of hundreds to a few thousand) and highly sensitive biophysical screening methods, such as NMR or X-ray crystallography, will yield a core structural starting point for hit-to-lead optimisation. The first fragment-led drug developed and approved for use is the B-Raf enzyme inhibitor Vemurafenib, for the treatment of late-stage melanoma. Currently, there are numerous compounds in clinical development that originated from FBS approaches.

[1] Doak, B. C., Norton, R. S. & Scanlon, M. J. The ways and means of fragment-based drug design. Pharmacol. Ther. 167, 28–37 (2016).

[2] Erlanson, D. a, Wells, J. a & Braisted, a C. Tethering: Fragment-based drug discovery. Annu. Rev. Biophys. Biomol. Struct. 33, 199–223 (2004).

[3] Silvestre, H. L., Blundell, T. L., Abell, C. & Ciulli, a. Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery. Proc. Natl. Acad. Sci. 110, 12984–12989 (2013).

At CTB we develop new ways to combine label free affinity selection methods, suitable for high throughput detection of small molecule protein interactions with up to 10 mM K_D with “traditionally” successful fragment based screening methods like NMR and X-ray crystallography. These workflows allow to focus the time consuming and expensive techniques on compounds which have already been identified as binders, although, without structural information and/or exactly determined affinities.

magB Assay

The magnetic bead-based ligand binding assay (magB) is an affinity selection method which is utilised for assaying recombinant target proteins which cannot be isolated by purification. A target protein is assayed in a crude lysate and is isolated on magnetic beads via affinity for a corresponding antibody which is coupled to the beads. The bead-antibody-protein complexes are then incubated with compound before the protein is specifically cleaved and subjected to analysis by LC-MS/MS to identify binding compounds. Difficulties associated with recombinant protein production and enzyme handling are reduced in this assay system as there is no requirement for purified protein. This assay is utilised as a rapid, qualitative primary screen for the identification of novel compounds with specific binding affinity for target proteins or as a secondary validation screen to show direct binding of hit compounds.

See publication:

Wilson K, Mole DJ, Homer NJ, Iredale JP, Auer M, and Webster SP (2014). A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery. Journal of Biomolecular Screening, 1-7, Online October 8, DOI: https://doi.org/10.1177/1087057114554171, PMID: 25296660.

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