Efficient large-scale exploration of fragment hit progression by exploiting binding-site purification of actives (B-SPA) through combining multi-step array synthesis and HT crystallography.

Fragment approaches are long-established in target-based ligand discovery. Nevertheless, their full transformative potential lies dormant, because progressing hits to potency remains difficult and underserved by methodology developments, which mostly focus on screening. The only credible progression paradigm is conventional design-make-test analyse (DMTA) medicinal chemistry, which is costly and thus necessitates picking winners early, thereby effectively discarding all the other hits. We here demonstrate the workability of an alternative strategy, namely immediate large-scale exploration of diverse hit-inspired compounds. The key insight is that it is effective to cheaply parallelize large numbers of non-uniform multi-step reactions, because even without compound purification, a high-quality readout of binding is available, namely crystallography of fragment screening. This has the sensitivity to detect even low-level binding of slightly active compounds, which the targeted binding site extracts directly from crude reaction mixtures (CRMs). In this proof-of-concept study, we expand a fragment hit from a crystal-based screen of the second bromodomain of human PHIP, using array synthesis on low-cost robotics to implement 6 independent multi-step reaction routes of up to 5 steps, attempting the synthesis of 1876 diverse expansions; designs were entirely driven by synthetic tractability. Expected product was present in 1108 CRMs, as detected by automated mass spectrometry; and 22 individual products were resolved in crystal structures of CRMs added to crystals. These provided an initial SAR map, revealed pose stability in 19 and instability in 3 products, and resolved stereochemical preference. Unexpectedly, in view of the naïve design approach, one resolved compound even showed on-scale biochemical potency (IC50=34 μM) and biophysical affinity (Kd=50 μM) after resynthesis. This binding-site purification of actives (B-SPA) process is formulaic and engineerable, here yielding the output of >25 person-years in ~20 days, with solvent use reduced from >4,500L to <20L. Thus, this approach, coupled with algorithmically guided compound and reaction design and new formalisms for data analysis, lends itself to routine fragment progression.