Screening and beyond in drug discovery

Over the past few decades the reductionist approach has been extensively employed to feed the industry’s small molecule drug pipelines.¹ The adoption of this approach was logical at its inception as it enabled the identification of hit compounds that specifically bind and modulate the activity of their respective drug targets. However, this approach yields compounds that are weakly active, lack selectivity and possess sub-optimal properties. These hit compounds therefore require significant optimisation in order to yield a lead compound² that is potent with optimised selectivity, physicochemical and ADMET properties.^3-4

Hit-to-lead

In order to successfully progress compounds to a lead compound, numerous properties must be determined – including adequate biological, chemical, pharmacological and pharmacokinetic characterisation – and each are summarised in Table 1. Most of these studies are in vitro and involve complex processes including cell viability assays. Cell viability relies upon the integrity of the cell membrane, key cellular biochemical reactions and specific cellular markers.⁵ Knowledge regarding the extent of cytotoxicity that is induced by a compound is a parameter that must be determined prior to progressing it in the drug discovery value chain. A variety of microtitre plate-based cytotoxicity assays in colourimetric, uorometric and luminescence detection technologies are available. The colourimetric-based methods have historically been the most widely employed and are well validated. These include quantification of the following:

1. Mitochondrial succinate dehydrogenase activity of cells using the tetrazoles XTT and MTT⁶
2. Extracellular lactate dehydrogenase activity by measuring NADH consumption⁷
3. Acid phosphatase activity as a marker of lysosomal activity⁸
4. Remaining glucose in cell culture medium using a glucose oxidase-peroxidase assay⁹
5. Cell proliferation using crystal violet dye accumulation in the nucleus¹⁰
6. Lysosomal accumulation of a cationic dye neutral red¹¹
7. Protein synthesis using sulforhodamine B binding to proteins.¹²

Fluorometric-based cytotoxicity assays are also available and include those that quantify the following:

1. Lactate dehydrogenase activity using a coupled reaction that results in the conversion of resazurin into resorufin (CytoTox-ONE Assay)¹³
2. Protease activity that is released from cells with a compromised cell membrane and a non-cell permeable fluorogenic peptide substrate (CytoTox-Fluor Cytotoxicity Assay)¹²
3. The activity of two proteases using cell permeable (giving the live cell measurement) and non-cell permeable (giving the dead cell measurement) substrates (MultiTox-Fluor Multiplex Cytotoxicity Assay).¹⁴

Luminescence-based assays are also available and include those that quantify the following:

1. The activity of a protease that is released from cells that no longer retain an intact cell membrane using a luminogenic peptide substrate (CytoTox-Glo Cytotoxicity Assay)¹⁵
2. Glutathione-5-transferase activity using a luciferin-derived substrate, the product of the reaction being a substrate of firefly luciferase¹⁶
3. Intracellular ATP using a luciferase reaction (CellTiter-Glo Luminescent Cell Viability Assay).¹⁷

There is also a multiplex assay that uses a fluorescence and luminescence readout essentially the same as the MultiTox-Fluor Multiplex Cytotoxicity Assay mentioned above, except the substrate for dead cells is luminogenic rather than being fluuorogenic.¹⁸

Lead-to-candidate

The optimisation of a lead molecule into a candidate is a very challenging, multiparameter process.^19-20 It should lead to identification of a molecule that satisfies pre-defined criteria with regard to in vitro and in vivo activity, pharmacokinetics, toxicological and pharmaceutical properties, and complexity of chemical synthesis (Table 2). It will typically have nano-molar potency against its primary biological target and will have undergone significant medicinal chemistry optimisation, comprehensive in vitro selectivity screening, determination of physicochemical and pharmaceutical properties and initial formulation studies, detailed in vitro ADMET studies, mutagenicity testing, detailed secondary pharmacology studies, detailed in vivo pharmacodynamics and pharmacokinetic studies and comprehensive toxicological profiling.

Although drug discovery is fraught with challenges, as highlighted above, the prospect of discovering a new drug is highly rewarding, as it offers the potential for mankind to live healthier and longer lives.

References

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