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Feb 15, 2012 (Vol. 32, No. 4)

Coupling SPR Increases Value

  • NCE Decisions

    Click Image To Enlarge +
    HTS hit series prioritization in drug discovery: NCE titrations on target protein surfaces: Hit series B shows a more desired kinetic profile; with its lower “off-rate”, this series might represent the better starting point (scaffold) for a drug development program. [Merck Serono Research & Development]

    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.”

  • SPR and GPCRs

    G protein-coupled receptors (GPCRs) are one of the most important classes of targets for drug discovery. They are linked to many disorders, yet the inability to apply structure-based drug design has hampered identification of novel therapeutic compounds, according to Andrei Zhukov, Ph.D., senior scientist, Heptares Therapeutics.

    “Since GPCRs are very unstable when isolated from their native membrane environment, it is very difficult to crystallize them and reveal their structures by x-ray analysis. Without knowing the structure, rational drug design is very difficult. GPCRs also cannot be studied using biophysical methods such as SPR.”

    Dr. Zhukov says that Heptares employs its StaR® (stabilized receptor) technology to introduce a small number of point mutations into GPCRs that increase their thermal stability in complex with a ligand to maintain the receptor in a specific physiological conformation. Amino acids are systematically mutated, and the small number of mutations having the highest stabilizing effect are selected and combined.

    Stabilized receptors can be isolated from cells, purified without loss of ligand binding activity, and used for ligand interaction analysis by SPR.

    “At the initial stage of drug design, SPR is used for hit identification by fragment screening. Later, during lead optimization, SPR is used for affinity ranking of the leads as well as to characterize the kinetics of drug-target interaction, which is known to have a strong impact on drug efficacy.”

    Dr. Zhukov reported on a study in which several receptors were attached to Biacore (GE Healthcare) chips while a series of known binders and novel drug candidates were screened to obtain kinetic and affinity parameters.

    “Another approach we use is Biophysical Mapping™ whereby binders from different chemical series are screened by SPR against binding-site mutants to define the role of active site residues in ligand binding and to refine binding models obtained using computational chemistry when the crystal structure is not yet available.

    “Using this approach, important GPCRs, for the first time, have become tractable targets for drug discovery. For example, a highly selective agonist against the M1 receptor is advancing to the clinic as a potential treatment for Alzheimer disease and for improving cognition.

    “Similarly, Heptares has identified a range of small molecule antagonist chemotypes to orexin 1/2 for treating sleep and potentially addiction disorders. Heptares also has ongoing lead discovery programs using the same approach to find GLP-1 agonists/allosteric modulators for diabetes and mGluR5 negative allosteric modulators for psychiatric disorders.”


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