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Intrinsically disordered proteins (IDPs) play important roles in disease pathologies; however, their lack of defined stable 3D structures make traditional drug design strategies typically less effective against these targets. Based on promising results of targeted covalent inhibitors (TCIs) on challenging targets, we have developed a covalent design strategy targeting IDPs. As a model system we chose tau, an endogenous IDP of the central nervous system that is associated with severe neurodegenerative diseases via its aggregation. First, we mapped the tractability of available cysteines in tau and prioritized suitable warheads. Next, we introduced the selected vinylsulfone warhead to the non-covalent scaffolds of potential tau aggregation inhibitors. The designed covalent tau binders were synthesized and tested in aggregation models, and inhibited tau aggregation effectively. Our results revealed the usefulness of the covalent design strategy against therapeutically relevant IDP targets and provided promising candidates for the treatment of tauopathies.

Surface plasmon resonance biosensor technology (SPR) is ideally suited for fragment-based lead discovery. However, generally suitable experimental procedures or detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are lacking. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. By adopting a multiplexed strategy, the range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded. We here illustrate innovative strategies using five challenging targets and variants thereof. Two libraries of 90 and 1056 fragments were screened using two different flow-based SPR biosensor systems, allowing different experimental approaches. Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted.

Over the past decade intrinsically disordered proteins (IDPs) have emerged as a biologically important class of proteins, many of which are of therapeutic relevance. Here, we investigated the interactions between a model IDP system, tau K18, and nine literature compounds that have been reported as having an effect on tau in order to identify a robust IDP&ndash, ligand system for the optimization of a range of biophysical methods. We used NMR, surface plasmon resonance (SPR) and microscale thermophoresis (MST) methods to investigate the binding of these compounds to tau K18, only one showed unambiguous interaction with tau K18. Several near neighbors of this compound were synthesized and their interactions with tau K18 characterized using additional NMR methods, including 1D ligand-observed NMR, diffusion-ordered spectroscopy (DOSY) and 19F NMR. This study demonstrates that it is possible to detect and characterize IDP&ndash, ligand interactions using biophysical methods. However, care must be taken to account for possible artefacts, particularly the impact of compound solubility and where the protein has to be immobilized.