The starting template fragment, F709 did not yield any detectable signal in solution assays and elaborating fragments aims at increasing the compound's affinity for the target. Hence, once identified, crystallographic hits must be confirmed via orthogonal methods. For this purpose, we synthesised in-house, or ordered the pure and independently synthesised compounds via Enamine Ltd. These were then evaluated with Amplified Luminescence Proximity Homogeneous Assay (AlphaScreen™) testing for biochemical activity. A timeresolved grating-coupled interferometry-based biosensor assay, using a pulsed injection scheme (waveRAPID®) , was used to estimate affinity and interaction kinetic rate constants (Fig. ). Compound PHIP-AM1-20, product of Iter 1 (Fig. ), had a measurable effect in both assays with a Kd and a IC50 of 50.03 µM and 31.15 µM corresponding to ligand efficacies of 2.00 and 1.25 µM/heavy atom for the kinetic analysis and alpha-screen, respectively (Fig. ). The binding event appears to be a reversible 1-step 1:1 interaction. This represents a 2-to 3-fold increase in binding affinity when compared to the best binder obtained by Cox et al., (2016) that were elaborations around the first DSI-poised fragment binders identified against PHIP(2). Overall, at scale potency was achieved starting from an undetectable fragment.