Utilizing SPR as a follow-up to a high-throughput screening campaign provides an easy and reliable means to identify false-positive hits such those showing “promiscuous” binding behavior as well as nonbinders, suggested Jörg Bomke, principal scientist, molecular interactions and biophysics, NCE lead discovery technologies, Merck Serono Research & Development.
He discussed how small molecule interaction studies with SPR are utilized for the new chemical entities (NCE) drug discovery process at his company.
“Quite often the kinetics can explain discrepancies between the biochemical and cellular activities of diverse chemical scaffolds. Further, using SPR for fragment screening not only delivers high quality (well-behaving) hits, but also determines their affinities up to the millimolar range.
“We are titrating our fragments from 2 millimolar downward to assess their properties at this concentration and to sort out the misbehaving compounds. I believe this is very important for obtaining good success rates in crystallization trials with millimolar and higher micromolar fragment hits since a co-crystal structure determination needs a high occupancy of the binding site with the fragment.
“Ideally, the fragment concentration should be five to ten times higher than its affinity to the target.”
Bomke also explained how generating a kinetic profile helps with decision making. “Starting after a high-throughput screening run with scaffolds that exhibit already slow kinetics makes life easier for the medicinal chemists. Our experience shows that in some cases you cannot implement such a profile to your hit series even after prolonged chemical synthesis and optimization processes.
“In this respect, not only long residence times on the target are of interest, but also slow association rate constants and deviations from simple 1:1 models. Both of the latter can provide much information about the nature and mechanism of the drug-target interaction.”
For example, the importance of obtaining kinetic profiles at later stages of NCE drug development is the ability to identify lead candidates having a high affinity, but low “on” rates as well as low exposure levels or a high clearance.
“These candidates might not reach sufficiently high plasma levels over a longer period of time to induce an effect on their target protein, and therefore fail to show in vivo efficacy in animals or humans."
It is also important to work with full-length proteins when possible. “One key observation over the last few years is that studying just one protein construct may lead to incorrect conclusions.
“By working with different protein constructs, mutants, post-translationally modified protein, and isoforms as well as using additional assays such as competition setups or co-injections (i.e., with co-substrates), one should gain a deeper insight to help reduce the gap between the in vitro world and the in vivo target physiology.”