Advances in Fragment-Based Drug Discovery : studies of cAMP-dependent protein-kinase A using X-ray-crystallography, surface-plasmon-resonance and high compound concentration assays

Development of a new, or candidate, therapeutic drug is a challenging process that must ensure that favorable target selectivity, potency, pharmacokinetics, and pharmacodynamics, as well as lack of toxicity, all fall within the therapeutic window. During the hit-optimiza­tion stage, the focus shifts toward optimizing potency and target selectivity. Fragment-based methods have recently been developed to the point where they represent a promising strategy in drug discov­ery, where a variety of biophysical techniques may be employed for fragment library screen­ing and characterizing hit-fragments. Hit-fragments deduced from fragment-based screenings typically have ligand efficiencies (LE) exceeding those of average HTS‑hits. Structure data on the complexes formed by fragment-target-protein structures yield a much-better starting point for hit optimization and lead discovery. This dissertation presents two fragment-screening studies. Under the first, surface-plasmon-resonance (SPR) analyses and biochemical assays at high compound concentrations (HCA) were employed in primary screenings of protein-kinase A (PKA) that were followed by X-ray crystallographic determinations of the structures of the PKA‑frag­ments involved. The aim of that study was testing the characteristics, outcomes, and limits of both SPR and HCA as fragment-screening methods, as well as estimating hit rates that could be confirmed by X‑ray crystallographic analyses. Under the second, in-house, biochemi­cal-assay data were used for selecting the fragment-like inhibitors of PKA to be subjected to X‑ray crystallo­graphic structure determina­tions. The biochemical-assay data involved were taken from screening cam­paigns, such as high-through­put screenings (HTS), or other, available, in-house, biochemi­cal-assay runs. The goal there was estimating the extent to which existing HTS‑data might be util­ized for obtaining three-dimensional, fragment-target, protein structure data, without need for conducting any additional fragment-screening runs. Following screening a library of 257 fragment-like compounds using SPR and HCA, a total of 26 hit-fragments were chosen for X-ray structure determinations, which yielded the structures of nine frag­ment-PKA-struc­tures. Under the second approach, 67 fragments exhibiting > 50 % inhibi­tions taken from the avail­able, in-house, biochemical-assay data were selected for structure deter­min­a­tions, which yielded the structures of 21 fragment-PKA-complexes. Both approaches yielded respectable hit rates and descriptions of the characteristics of numerous fragment-pro­tein interactions. The structural information and data on fragment-target-protein complexes gained from those two setups might well accelerate the drug-discovery process throughout the pharmaceutical industry