Screening


Figure 1 The % inhibition of


phosphofructokinase activity in a biochemical assay in the


absence of reducing agent (left


hand panel), with 2mM DTT as a reducing agent (middle


panel) and with 5mM cysteine


as a reducing agent (right hand panel) by a library of


compounds with known assay


interference mechanisms (blue – aggregators, green –


chelators, yellow – coloured,


orange – fluorescent, brown – redox cyclers, red – luciferase binders, grey – reactive)


changes in the drug discovery landscape have led to the rise of the ‘virtual biotech’, where a nucleus of individuals, typically a combination of disease specialists and ex-pharma project managers backed by private investment, access all of the req- uisite discovery and development infrastructure via outsourcing to CROs7. However, these small or micro organisations, often originating from academia, inevitably have gaps in their capacities, capabilities and/or institutional knowledge base and one area where this is evident is in high throughput screening (HTS).


Lead generation and high throughput screening HTS is a key strategy for finding chemical matter as a starting point for small molecule drug discov- ery programmes. It comprises screening large libraries of compounds (typically hundreds of thousands) in one or more biological assays fol- lowed by a series of triaging activities aimed at pri- oritising one or more validated hit series. A validat- ed hit series can be defined as a series of com- pounds which possess a progressive structure- activity relationship with strong evidence of target engagement, an indication that selectivity over close target orthologues is achievable and with physicochemical properties that are appropriate for preparation of a clinical candidate8. While HTS is an effective method, it requires sig-


nificant infrastructure in the form of chemical libraries, compound storage and logistics9 and, vitally, expertise in assay development and execu- tion10. In many cases this expertise has been accu- mulated over many years and resides within major pharma companies, although increasing invest- ment in academia and major initiatives such as the European Lead Factory11 has increased access to the required infrastructure and expertise for small companies and academic groups.


54


False positives While the ‘quality’ of the compound collection is sometimes considered a synonym for success in a high throughput screening campaign, in our opin- ion the careful design and execution of the primary assay and the subsequent confirmation of hit authenticity is as important, if not more so. The concept of Pan-Assay Interference Compounds (PAINS) is now widely accepted, whereby some compounds, or trace contaminants from chemical synthesis, can act as inhibitors or activators of mul- tiple targets via unproductive or non-specific mechanisms12. The key qualifier in this concept is the term ‘Pan-Assay’, indicating that these com- pounds appear as hits time and time again, which any professional screening organisation will quick- ly recognise and can then annotate as frequent hit- ters or remove them altogether from the library. Unfortunately, not all unproductive mechanisms of inhibition can/will be flagged as PAINS as it is important to recognise that the nature of the assay/target interference mechanisms is context- dependent, eg some compounds will only act as aggregators under certain buffer conditions and over particular time periods. Indeed, some interfer- ence mechanisms can be incredibly subtle, elegant even, and appear exquisitely selective for the target of interest. So much so that even the most consci- entious drug hunter can and will end up wasting time and resources unwittingly following these up. This is where careful assay development and the considered sequencing of testing helps minimise the frequency and therefore long-term cost of these frustrating dead-end forays. Two recent reports illustrate some of the issues


and subtleties of false positives. Ciulli et al13 reported on hits with 20µM potency in a biochem- ical screen and confirmed a molecular interaction with the target using orthogonal techniques (isothermal calorimetry and NMR), although the


Drug Discovery World Fall 2019


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