Fragment-based drug discovery (FBDD) has become a standard approach for generating starting points in medicinal chemistry. Small fragments bind weakly but can be chemically elaborated into stronger ligands. The difficulty is that moving from weak binders to measurable leads usually involves multiple design-make-test-analyse (DMTA) cycles: each analogue must be synthesised, purified, tested biochemically, and crystallised. This is slow and leaves much chemical space unexplored.
Researchers developed a new technique called Binding-Site Purification of Actives (B-SPA) to bypass this bottleneck. Instead of purifying every product, they test crude reaction mixtures directly on protein crystals. Their study, published in Angewandte Chemie International Edition, shows how this works in practice. High-throughput macromolecular crystallography on Diamond’s I04-1 beamline enabled the team to detect which molecules bind, even if they were a minor product in a mixture. This structural “filtering” step dramatically accelerates hit-to-lead workflows.
Expanding fragments into thousands of analogues without purification
The team focused on the second bromodomain of PHIP ( disPHIP(2)), a protein implicated in epigenetic regulation and linked to cancers. A fragment hit (compound F709) had been identified in earlier crystallographic screens, but like most fragments, its binding was weak and undetectable in solution assays. Researchers wanted to explore chemical space around this initial fragment to see which modifications improve binding.
They designed up to six independent synthetic routes, each involving multi-step reactions (up to five synthetic steps), exploring different vectors of substitution (i.e. different parts of the fragment to substitute, such as replacing a ring, modifying substituents, adding functional groups). The designs were guided by synthetic tractability: only routes that are feasible with reliable chemistry were chosen. Using a low-cost robotic liquid handler, the group performed 1,876 reactions, generating diverse libraries of potential binders.
Each crude reaction mixture was checked by LC–MS using an automated tool (MSCheck) that flags the presence of the expected molecular ion. Out of 1,876 attempted syntheses, 1,108 mixtures (59%) contained the intended product. Rather than purify, the team directly soaked PHIP(2) crystals with these crude mixtures and collected data at Diamond’s I04-1 beamline, which is optimised for high-throughput macromolecular crystallography.
Read more on the Diamond website