Fragment-based screening is an efficient and attractive approach in the identification of new chemical starting points against a range of biological targets, but in particular for challenging targets that are considered undruggable. Fragments are smaller, basic building blocks that carry fewer functional groups. Due to their less complex nature, fragments have lower binding affinities to the target with equilibrium dissociation constants (Kd) ranging from the low micromolar to the millimolar concentration. However, provide increased likelihood of identifying interactions with the protein partner and for this reason generally provide higher hit rates and with the potential to improve binding affinities more rapidly and efficiently.

o2h utilizes Biacore T200 Surface Plasmon Resonance (SPR) as a primary screening modality to efficiently evaluate fragments for direct binding at high micromolar concentration (~100uM); along with competition studies to characterize specific protein-fragment interactions. Subsequently, fragments with confirmed activity are validated in orthogonal experiments such as thermal stability (TS) to confirm assay traction.

Our SPR assay development team will generate experimental design strategies with stage-wise progression prior to initiation. We address multiple aspects during the development phase and some of which are listed below –

• Target immobilization based on literature precedence (if available)
• Buffer scouting and direct binding of immobilized target protein with preferred natural peptide or control compounds
• Competitive displacement or allosteric interaction with immobilized target protein
• Sensorgrams with graphical illustrations of K_d, K_on and K_off and with saturable interactions
• Injection time window for maximal signal response and data extraction
• Residence time of immobilized protein, stability and target responsiveness
• Evaluation of any known target protein binders (i.e. peptides, cofactors, including small molecule tool compounds)

Initial fragment screening is conducted at a single, high micromolar concentration (specific concentration to be agreed with collaborator) to identify suitable hits and the output is then grouped based on their binding affinities. Typically fragments with activities >50% of the theoretical Rmax are shortlisted and re-evaluated for a full kinetic characterization. Inclusion of suitable controls allow us to discriminate between specific and non-specific interactions, and a counter screen against an unrelated control or mutant protein also enable us to identify hits that are selective towards the target protein. Identity and purity of the final hits will be confirmed by LC-MS.

o2h can support in orthogonal assay validation experiments for better confidence in hit identification such as fluorescence based thermal stability experiments as indicated above. In case of additional requirement such as X-ray Crystallography and NMR, these services can be provided with our established 3^rd party collaborators.

Eventually, shortlisted fragment can be efficiently evolved either through purchase of related molecules (“SAR by catalogue”) using our procurement team or our dedicated chemistry team can provide support to design and synthesise analogues with improved potency together with our integrated drug discovery services to profile across multiple activity-based assay platforms.  Using structural and computational information, we can assist in understanding the binding mode of lead molecules. Site-directed mutagenesis studies can be undertaken, with the option to evaluate both mutants and their wildtype aiding in SAR development and lead optimization.

Case Study

Dual-specificity tyrosine-regulated kinase 1A, DYRK1a Kinase target – Catalytic domain

Figure 1.

Figure 1. Fragment library and primary screening platform using SPR to evaluate binding affinities to target protein, DYRK1A. A. o2h designed library by medicinal chemists in conjunction with academic computational chemists comprising of ~600 fragments with measured solubility and purity; B. Assay validation determining DYRK1A thermal stability in the presence of 1% and 5% DMSO (left panel); full range kinetics of DYRK1A interaction with controls – DYRKtide and AZDYRK1B-33 (middle panel); and their corresponding dose response curves (right panel); C. single point screen at 100uM and grouping based on their binding affinities to DYRK1A; D. Fragments run in forward and reverse direction with data collected in duplicates to check for interference (if any) from the previous run onto the next fragment screen. Pink bar indicates positive control which is run intermittently to check for immobilized target responsiveness. E. representative three-point screening of shortlisted fragments and, F. their corresponding orthogonal validation in thermal shift assay