On-bead screening
One-Bead One-Compounds Confocal Nanoscanning and Post-Synthesis Post-Screening labelling of compounds on single microbeads
OBOC-CONA and PSPS Technology
In the need to develop a chemical biology screening platform which holds promise to test a larger number of potential proteins, rather than just industry standard validated drug targets we developed the One-Bead – One-Compound Confocal Nanoscanning technique, (OBOC-CONA). This screening platform is characterized by high resource efficiency and allows simple and cost effective testing of virtually any target protein, is connected to fast discovery cycle times for higher throughput. Importantly, with limited resources for acquiring large small molecular libraries in academic contexts, the cost effective production of libraries directly on the solid support and the possibility to screen these libraries without bead cleavage and purification of individual compounds is ultimately important. The difficulty here lies in setting up a process which links chemical synthesis, high-throughput high sensitivity screening with hit compound identification, isolation and analysis of the three most important, in fact business critical, parameters: Purity, target binding affinity in homogenous solution, and structure. This was achieved in CTB by a combination of physical and chemical methods. We developed Confocal Scanning (CONA), performed on the bead picker instruments (PS02, PS04), which allow confocal fluorescence imaging of ~ 180,000 100 micro meter sized beads in 7 hours. After screening, the bead picker device on PS02 and PS04, enables ranking and then isolating single micro-beads as function of the fluorescence emission intensity of the dye conjugated to the protein bound to a single small molecule, peptide, or peptidomimetic. From single isolated hit beads, the post-synthesis post-screening labelling technique (PSPS) results in purity, structure, and solution binding affinity of the hit compound to the unlabelled target. OBOC-CONA, the PS instruments and the PSPS process made on-bead screening a technique fulfilling industry quality standards. With the link between on-bead binding and solution binding on a single bead level we were able to address most, if not all of the business critical problems of screening combinatorial chemical libraries directly on the site of synthesis, i.e. on the solid support. After the OBOC-CONA + PSPS process had been established not one single hit compound from on-bead screening was resynthesized in larger amounts for downstream profiling, e.g. in cellular assays or for co-crystallization. The difference between target binding affinities on the solid matrix and in solution is often as high as one log-step. In other words, a target might bind to a compound linked to a microbead with low nanomolar KD but only with high micromolar KD in solution. Such a compound would not fulfil expectations of either medicinal chemist who invest in producing the hit compound or profiling labs aiming to establish functional cellular activity. However, if it is proven that a reagents binds to the target with sufficient affinity to make functional highly likely, often this means > 10 µM KD, from ~ 50 picomole substance no downstream issues will arise.
The 3 key publications describing these techniques are:
Hintersteiner M, Kimmerlin T, Kalthoff F, Stoeckli M, Garavel G, Seifert JM, Meisner NC, Uhl V, Buehler C, Weidemann T, Auer M, (2009) A single bead labelling method for combining confocal fluorescence on-bead screening and solution validation of tagged one-bead one-compound libraries. Chemistry & Biology 16(7), 724-35. doi:10.1016/j.chembiol.2009.06.011, PMID: 19635409.
Hintersteiner M, Buehler C, Uhl V, Schmied M, Müller J, Kottig K, Auer M, (2009) Confocal Nanoscanning, Bead Picking (CONA) - PickoScreen microscopes for automated and quantitative screening of one-bead one compound libraries. Journal of Combinatorial Chemistry 11(5), 886-894; DOI:10.1021/cc900059q, PMID: 19603813.
Hintersteiner M, Buehler C, Auer M (2012). On-Bead Screens Sample Narrower Affinity Ranges of Protein-Ligand Interactions Compared to Equivalent Solution Assays. ChemPhysChem. 13(15), 3472-80, doi: 10.1002/cphc.201200117, PMID: 22829563.
bead_pick_short_MH.m4vUPS-CONA novel on-bead assay for interrogation of interactions and enzymatic activities in the ubiquitin system
To address the need for a versatile and accessible assay technology in the ubiquitination field, we developed a fluorescence-based on-bead confocal method for monitoring various reactions within the ubiquitin-proteasome system (UPS) in real time (Auer, Koszela and Tyers, 2016). The assay employs a fluorescently labelled ubiquitin or ubiquitin-like protein modifier, and a substrate or enzyme of interest, which is immobilised on a micro-bead. Upon ubiquitin conjugation to the on-bead substrate or enzyme, the fluorescence of conjugates can be detected on the bead surface using confocal microscopy, distinguishably from the background fluorescence in solution (Hintersteiner et al., 2009, 2012). After acquisition of images on a confocal plane, the fluorescence of the ‘rings’, corresponding to cross-section of the beads and correlated to the amount of the fluorescent conjugate, is analysed and quantified. Due to the small size of beads (typically less than 120 µm in diameter), hundreds to thousands of micro-beads can be placed in a well of a multi-well plate, providing an impressive number of repetitions per well and ensuring detailed quantification. We successfully applied UPS-CONA to a range of ubiquitination reactions, including monitoring a multi-step enzymatic cascade in one well. Currently, chemical and biosimilars libraries are screened for molecules which specifically modulate activity and interactions of human E2 enzymes. Identification of such molecules would be of interest as research tools and potentially as novel therapeutics in many disease states caused by aberrant ubiquitination, such as cancer or neurodegeneration.
Figure:
A: Schematic of the assay. An enzyme of interest or substrate for ubiquitination (here Ube2C as a eGFP-fusion, green) was immobilised on microbeads and subjected to ubiquitination reaction in the presence of a fluorescent Cy5 ubiquitin (red), an E1 enzyme Ube1, Mg2+ and ATP. B: Images were acquired on a confocal fluorescence microscope in eGFP (for Ube2C) and Cy5 (ubiquitin) channels. In the presence of ATP, ubiquitination reaction took place, resulting in formation of two-colour ‘rings’ in the cross-section of microbeads. C: Ring intensities were analysed using proprietary software and revealed conjugation of Cy5-Ub to GFP-Ube2C over time.
Combining Phage Display and Confocal Nanoscanning to identify high affinity binders (Phage-CONA)
Phage display is a well-established and powerful method for the detection of peptide and biosimilar ligands to target proteins. Peptides and biosimilars are presented on coat proteins of phages, such as the pIII coat protein of the M13 phage. Panning, rounds of incubating the phage library with target proteins and stringent washes, allow elimination of low affinity binders. However, the method typically delivers too large a pool of medium affinity hit binders often obscuring high affinity but low-abundancy binders, requiring costly and time consuming analysis, such as DNA sequencing, Surface Plasmon Resonance (SPR) or fluorescence anisotropy to determine the binders with the highest binding affinity. In 2008 González-Techera et al. [1] developed a method to overcome this bottleneck. Panning products can be ‘switched’ to form fusions with bacterial alkaline phosphatase (mBAP), allowing detection of the stronget binders by affinity ELISA. The “switch” is done by using restriction enzyme to excise the pIII gene and then re-ligate the phagemid to bring mBAP into frame with the library. Such a method is workable but still time consuming. The Auer group is further developing the method using site-specific DNA recombination to switch out the pIII gene in vivo leading to the creation of binder-HaloTag® (HT) fusions. The resulting fusion proteins can then be used to screen against bead-bound target protein or bead- bound target protein-fluorescent protein conjugates (FP), using the CONA (Confocal Nanoscanning) method established by the Auer lab at the University of Edinburgh [2-6]. Ratiometric image analysis on the PE Opera High Content Imager between the peptide-HT hit and the target-FP emissions allows determining a score representative of the relative affinity of the peptide with the high statistical accuracy of the CONA technology. The group’s proprietary BREAD software is used to perform the analysis. Only clones corresponding to peptides with the highest affinity score are sequenced to determine their primary structure. By this means, even if they are underrepresented, the most valuable peptide ligands can be rapidly identified for accurate affinity measurements by internal single molecule spectroscopy, for crystallography and as starting point for library generation and on-bead and solution based screening.
1. González-Techera A, Umpiérrez-Failache M, Cardozo S, Obal G, Pritsch O, Last JA, Gee SJ, Hammock BD and González-Sapienza G (2008) High-Throughput Method for Ranking the Affinity of Peptide Ligands Selected from Phage Display Libraries. Bioconjugate Chemistry 19: 993-1000.
2. Meisner NC, Hintersteiner M, Seifert JM, Bauer R, Benoit RM, Widmer A, Schindler T, Uhl V, Lang M, Gstach H, Auer M (2009) Terminal adenosyl transferase of posttranscriptional regulator HuR revealed by confocal on-bead screening. Journal of Molecular Biology 386: 435–450.
3. Hintersteiner M, Kimmerlin T, Kalthoff F, Stoeckli M, Garavel G, Seifert JM, Meisner NC, Uhl Volker, Buehler C, Weidemann T, Auer M (2009) Single Bead Labeling Method for Combining Confocal Fluorescence On-Bead Screening and Solution Validation of Tagged One-Bead One-Compound Libraries. Chemistr & Biology 16: 724-735.
4. Hintersteiner M, Ambrus G, Bednenko J, Schmied M, Knox AJS, Meisner N, Gstach H, Seifert J, Singer EL, Gerace L and Auer M (2010) Identification of a Small Molecule Inhibitor of Importin β Mediated Nuclear Import by Confocal On-Bead Screening of Tagged One-Bead One-Compound Libraries. ACS Chemical Biology 5: 967-979.
5. Hintersteiner M, Knox A, Mudd G, Auer M (2012) Journal of Chemical Biology 5: 63–79.
6. Hintersteiner M, Buehler C, Auer M, (2012) On-bead Screens Sample Narrower Affinity Ranges of Protein-Ligand Interactions compared to Equivalent Solution Assays. ChemphysChem 13: 3472-3480.
