Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein–protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein–protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

Characterization of protein-ligand interactions is essential for the pre-clinical development of drug candidates and Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) has emerged as a valuable tool in this process. HDX-MS has predominantly been employed with high affinity compounds with only a few examples of its application for weaker binders such as fragments. Nevertheless, HDX-MS usage could be instrumental in Fragment-Based Drug Discovery (FBDD) programs. In this work, the drug-target protein Cyclophilin D (CypD) was used as a model to explore the boundaries of fragments binding characterization by HDX-MS (fHDX-MS). We performed a systematic study on the optimal conditions for fHDX-MS execution and found that fragments with binding affinities in the double-digit mM range are still amenable to fHDX-MS. We observed that, despite the intrinsic low resolution of HDX-MS, fragments binding sites that partially overlap can still be distinguished. Overall, this study shows that fHDX-MS can be a useful method for FBDD.

Mitochondrial permeability transition pore (MPTP) dysregulation can be correlated with a variety of human diseases including multiple sclerosis, cardiovascular and neurological diseases. Human Cyclophilin D is a known MPTP regulator and although devoid of deep orthosteric sites, it is believed it can be targeted to develop clinically relevant candidates. Fragment-based drug discovery is a powerful approach for such drug-targets and we revisited a panel of 52 fragment hits by X-ray crystallography, which remained structurally elusive from a previous fragment screening campaign. Using two CypD mutants in a systematic parallel approach combining co-crystallization and soaking techniques (FragInc approach - from Fragment Incubation), new high-resolution crystal structures of CypD-fragment complexes were obtained. We identified a pyrazolo[1,5-a]pyrimidin-2(1H)-one fragment binding to a cleft in-between the known CypD S1' and S2 pockets. These structural insights guided the chemical synthesis of four new molecules to address simultaneously the novel pyrazolo binding pocket and the S2 site, and their crystal structures in complex with CypD were determined. This work opens new perspectives for structure-based drug design of novel tri-vector inhibitors targeting human CypD and proposes a parallel multi-technique methodology to address low-affinity fragments by X-ray crystallography.