A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…chelating compounds, aggregating compounds, redox cycling compounds and compounds acting through redox-mediated covalent modification in DSF and SPR, has been experimentally analyzed and compared with the effect expected for stoichiometric inhibitors. [46] For these, the two methods yield equivalent results. Nevertheless, it is recommended to test at least the most promising hits with both DSF and SPR, if technically and logistically feasible, to unambiguously verify stoichiometric binding.…”

“…However, formation of particles measured by differential light scattering alone is not sufficient to definitely exclude a compound. [46] This is illustrated by a self-assembling LMW compound that forms nanofibrils and in this conformation seems to promote activation of procaspase-3 [47] and thus induce cell death. [48] However, it should be noted that the authors emphasize differences compared to standard globular protein aggregators.…”

“…[106] A recent comparison of various techniques for characterization of HTS hits [107] points to some pitfalls of individual methods and also discusses how they are best combined. Arguably, stabilization of the target against thermal melting as measured by differential scanning fluorimetry [108,109] (DSF), also known as fluorescent thermal shift assay, [46] is most reliable. The reason is that this method does not involve any labeling or immobilization and therefore provides positive proof of stoichiometric compound binding to the target in solution.…”

“…The reason is that this method does not involve any labeling or immobilization and therefore provides positive proof of stoichiometric compound binding to the target in solution. On the other hand, resulting data should not be fitted by the Hill equation [46] and negative results with these methods do not rule out stoichiometric binding. Therefore, DSF should not be used as the only criterion for definite decisions but rather for relative prioritization in conjunction with other methods.…”

“…[110,111] As an alternative, surface plasmon resonance (SPR) is also widely applied to measure interaction of compounds with their targets. While the immobilization of the target on a gold surface allows for washing and reuse, thus reducing the amount of protein that is required, it also requires thorough controls from blank, non-treated surfaces [46] and diligent optimization of immobilization protocols to exclude or avoid undesirable influences on the protein, the ligand or their interaction. However, protocols to immobilize even challenging protein species such as GPCRs [112] or the monomeric, oligomeric and fibrillar conformational variants of the Abeta protein fragment [113] have recently been published, indicating that the approach is now quite universally applicable.…”