Logo: to the web site of Uppsala University

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery.  However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. 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. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B  (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau  (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.

Even though they account for 10% of the global disease burden and besides the new record of funding in 2018, tropical diseases are still neglected by the majority of pharmaceutical companies and public funding. The reason can be mostly found in the fact that for these diseases a conventional drug discovery process is often too expensive. The development of new approaches in the early stage of drug discovery has therefore a key role in fighting Neglected Tropical Diseases (NTD). AEGIS is a European network which relates on collaborations for a multidisciplinary approach, in order to “accelerate” drugs development – hence reducing the costs. As part of the network, this project is focused on a rational exploration of the chemical space, together with an in depth-analysis of molecular interactions for a better characterization of targets involved in NTD.

One cost-efficient way for exploring pharmacologically relevant chemical space is fragment based lead discovery (FBLD). This approach requires an extensive understanding of the target properties; therefore, a pipeline of orthogonal methods was developed to validate the suitability of the target for FBLD.

The choice of a fragment library has a significant impact on the experimental strategy. Not only the validation of the target, but also practical issues concerning technology, orthogonal validation and applicability have to be considered when initiating a fragment screening campaign.

Another rational approach for the discovery of new drugs is looking at the target structure, especially when considering protein complexes interaction. Through the structural analysis of the protein-protein interface, several short peptides derived from the binding partner were analysed in their interaction with the target both in vitro and in cell. This allowed to identify the key sequence for the binding to the target and the internalization of the complex, both crucial information for a structure-based approach.  

The internalization process of the target was characterized by a real-time cell binding assay (RT-CBA), revealing a higher level of complexity than what was previously described.

Implementing RT-CBA in the early stage is a strategy that might improve the success rate of drug development. The possibility to develop an intracellular time resolved molecular interaction assay between small molecules and a model target fused to a fluorescent protein was therefore explored.

From a fragment screening campaign to a structure-based approach, culminating with a real time intracellular validation of hits, all the assays developed address the different possibilities for accelerating the drug discovery process in the early stages.