Oxidative stress plays a crucial role in numerous diseases and pathological processes such as inflammation and neurotoxicity. As targets, we focus on the adaptor protein Keap1, which regulates the endogenous antioxidant response, and the superoxide-generating multi-subunit enzyme complex NADPH oxidase 2 (NOX2).

We screen our libraries of fragments (small substructures of drug like molecules) by biochemical and sensitive biophysical methods (FP, SPR, ligand-based NMR). Promising hits are optimized into lead compounds by medicinal chemistry, X-ray crystallography, and pharmacological assays.

1.  For Keap1, we have analysed current literature inhibitors for their ability to cross the blood-brain barrier (Pallesen et al, 2018, J Med Chem). This was followed by a comparative assessment study, where the known compounds were tested in a range of assays to validate and compare their activity towards Keap1. Interestingly, about half of the reported Keap1 inhibitors were found to be false positives (Tran and Pallesen et al, 2019, J Med Chem and Derek Lowe’s blog).
   

   

2. We have also addressed Keap1 by FBDD pursuing three strategies: First, we deconstructed the known compounds into a target-biased library of 77 fragments and tested them for Keap1 binding using four orthogonal biophysical assays. The binding modes of key fragment hits where determined by X-ray crystallography, which allowed us to merge two fragments into novel compounds with high affinities to Keap1 (Pallesen et al, 2021, J. Med. Chem and the Practical Fragments blog). Secondly, we have screened our commercial library of 2,500 fragments and optimized the compounds by X-ray-crystallography and medicinal chemistry (on-going). Thirdly, we screened the Diamond Light Source’s XChem library by X-ray crystallography and are currently optimizing the hits.
   
   

   

3. Recently, we applied FBDD to develop novel protein-protein interaction inhibitors of the p47phox subunit of NOX2. We screened our 2,500 fragments by fluorescence polarization (FP) and thermal shift assay (TSA) followed by surface plasmon resonance (SPR) validation. Biostructural studies by NMR and SAXS indicated that two fragments bound to two separate binding sites in the elongated conformation of p47phox, and the design of dimeric inhibitors thereby resulted in potent compounds (Solbak et al, 2020, J Med Chem). Since then, the compounds have been further optimized and tested in various biological assays (on-going work)
   
   

   

4. PDZ domains are intriguing but challenging drug targets. Recently, we investigated the druggability of PSD-95’s PDZ1-2 domain by fragment-based screening and a computational method. This resulted in new fragments shown to bind the PDZ domains of PSD-95 (Zang et al, 2020, ChemMedChem).

5. We have also used SPR to characterize protein-protein interactions for our collaborators related to KDM5B (Dorosz et al, 2019, Sci Rep) and NEMO (Jussupow et al, 2020, Sci Adv); and recently, we contributed with LC-MS pharmacokinetic data to a small-molecule obesity study (Grunddal, 2021, Mol Metab).

Medicinal chemistry, SPR* and biochemical/biophysical assays are core methods of our group. X-ray crystallography is performed in close collaboration with internal colleague Prof. Michael Gajhede using various beamlines in Europe, e.g. ESRF (Grenoble), EMBL (Hamburg), MAX IV (Lund)**, and Diamond Light Source (Oxfordshire). NMR for studying ligand-protein interactions is done with collaborators in Germany (Helmholtz Zentrum München) and Denmark (Technical University of Denmark), and testing in disease models by collaborators in Denmark (University of Copenhagen and University of Southern Denmark), Sweden (Lund University and Karolinska Institute), and USA (University of Miami).

- *See SelectScience interview about our Pioneer system and how we use it
- **See HALOS interview about X-ray experiments at MAXIV related to p67phox/NOX2

We are also engaged in collaborative projects within brain cancer (with Asc. Prof. Petra Hamerlik, Danish Cancer Society Research Center) and bacterial targets (with Prof. Morten O. A. Sommer, Technical University of Denmark).

We thank the following foundations for their generous support of our research:

• BioInnovation Institute (BII)
• China Scholarship Council
• HALOS Cross Border Research Grant
• Hørslev Foundation
• Augustinus Foundation
• Lundbeck Foundation
• A.P. Møller Foundation