In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment,i.e.small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure-based hit-to-lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow-up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof-of-concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow-up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single-digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X-ray crystallography.

Fragment screening directly on protein crystals has been applied using AnalytiCon’s collection of intermediates that have been utilized to generate libraries of larger synthetic natural product-like molecules. The fragments with well-balanced physicochemical properties show an impressively high hit rate for a screen using the aspartic protease endothiapepsin. The subsequent validation and expansion of the discovered fragment hits benefits from AnalytiCon’s comprehensive library design. Since the screened fragments are intermediates that share a common core with larger and closely related analogs with modulated substitution patterns, they allow for the retrieval of off-the-shelf follow-up compounds, which enable the development of design strategies for fragment optimization. A promising bicyclic core scaffold found in several fragment hits could be validated by selecting a set of enlarged follow-up compounds. Due to unexpected changes in binding mode and no significant improvement in ligand efficiency, this series was quickly deemed unsuitable and therefore discontinued. The structures of follow-up compounds of two other fragments helped to evaluate a putative fusion of two overlapping fragment hits. A design concept on how to fuse the two fragments could be proposed and helps to plan a suitable substitution pattern and promising central bridging element.

The conversion of hits to leads in drug discovery involves the elaboration of chemical core structures to increase their potency. In fragment-based drug discovery, low-molecular-weight compounds are tested for protein binding and are subsequently modified, with the tacit assumption that the binding mode of the original hit will be conserved among the derivatives. However, deviations from binding mode conservation are rather frequently observed, but potential causes of these alterations remain incompletely understood. Here, two crystal forms of the spliceosomal RNA helicase BRR2 were employed as a test case to explore the consequences of conformational changes in the target protein on the binding behaviour of fragment derivatives. The initial fragment, sulfaguanidine, bound at the interface between the two helicase cassettes of BRR2 in one crystal form. Second-generation compounds devised by structure-guided docking were probed for their binding to BRR2 in a second crystal form, in which the original fragment-binding site was altered due to a conformational change. While some of the second-generation compounds retained binding to parts of the original site, others changed to different binding pockets of the protein. A structural bioinformatics analysis revealed that the fragment-binding sites correspond to predicted binding hot spots, which strongly depend on the protein conformation. This case study offers an example of extensive binding-mode changes during hit derivatization, which are likely to occur as a consequence of multiple binding hot spots, some of which are sensitive to the flexibility of the protein.