Traditional drug discovery and drug development targets enzymes and other proteins with well-defined ligand-binding pockets. However signalling pathways are often mediated by protein-protein interactions involving shallow grooves on protein surfaces. We have experience with a wide variety of protein interaction assays for either library screens in high-throughput (singly or multiplexed followed by deconvolution) or more limited sample sets.

These assays use either recombinant purified proteins, cell-free extracts or they can be based on protein interactions in intact cells. Fragment library screens based on Differential Scanning Fluorimetry will also soon be available.

Screening compounds or specific reagent series/concentration series are handled via acoustic dispenser for nanolitre precision and minimal wastage or traditional pipetting robotics and reagent dispensers for larger volume transfers, parallel library manipulations and high-throughput cell-culture setup.

An example of a small cell-free screen is shown in Figure A, leading through hit expansion to the compound series shown in Figure B. Figure C illustrates one of the cell-based protein interaction assays in used (described in ref 2). While users typically keep small molecule data and even some assay methodologies confidential for a period of time, references below illustrate some of the public data illustrating the approaches used and their application. We can provide libraries, assist in library handling, assay set-up and data acquisition and interpretation according to your needs.

Ultra-high sensitivity assays are available – for example, in a side-by-side comparision with a conventional assay (ref 3) with the same target, ligand and competitor, the assay in (A) uses up to 10000 x less protein per assay sample, is measured up to 20 faster and provides a far superior z’-factor. This sensitivity increase facilitates the use of native expression systems and difficult-to-express proteins

Contact: Michael Courtney (michael.courtney [at] utu.fi)

References

1. Li LL, Ginet V, Liu X, Vergun O, Tuittila M, Mathieu M, Bonny C, Puyal J, Truttmann AC, Courtney MJ (2013) The nNOS-p38MAPK pathway is mediated by NOS1AP during neuronal death. J Neurosci. 33, 8185-8201. doi: 10.1523/JNEUROSCI.4578-12.2013 PMID: 23658158
2. Li LL, Melero-Fernandez de Mera RM, Chen J, Ba W, Nadif Kasri N, Zhang M, Courtney MJ (2015) Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models. J Neurosci. 35, 7349-7364. doi: 10.1523/JNEUROSCI.0037-15.2015 PMID: 25972165
3. Li LL, Cisek K, Courtney MJ (2017) Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element. Front. Mol. Neurosci. 10:58. doi: 10.3389/fnmol.2017.00058. PMID: 28360833.
4. Melero-Fernandez de Mera RM*, Li LL*, Popinigis A, Cisek K, Tuittila M, Yadav L, Serva A, Courtney MJ (2017) A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs. (*equal contribution) Nat. Commun. 8, 15017. doi: 10.1038/ncomms15017. PMID: 28497795.
5. Lee WH, Li LL, Chawla A, Hudmon A, Lai YY, Courtney MJ, Hohmann AG. Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice. Pain. 2018 May;159(5):849-863. doi: 10.1097/j.pain.0000000000001152. PubMed PMID: 29319606; PubMed Central PMCID: PMC6085107.
6. Lee WH, Carey LM, Li LL, Xu Z, Lai YY, Courtney MJ, Hohmann A. [EXPRESS] ZLc002, a putative small molecule inhibitor of nNOS interaction with NOS1AP, suppresses inflammatory nociception and chemotherapy-induced neuropathic pain and synergizes with paclitaxel to reduce tumor cell viability. Mol Pain. 2018 Aug 29:1744806918801224. doi: 10.1177/1744806918801224. [Epub ahead of print] PubMed PMID: 30157705.
7. Lee WH, Carey LM, Li LL, Xu Z, Lai YY, Courtney MJ, Hohmann A. [EXPRESS]