In order to identify hit fragments in the development of therapeutics, the contribution of the photoreactive warhead to binding affinity was investigated. A primary goal of the PhABit platform was to identify hits that can subsequently be developed without the photoaffinity label. A key consideration, therefore, was the contribution of the photoreactive warhead to the binding affinity. Analogues of hit PhABits were synthesised with acetyl-and butanoyl-amides in place of the photoreactive group to explore the impact this had on potency (10-19, Supplementary Figure ). A decrease of less than 0.6 log units in the TR-FRET assay was observed upon truncating to the acetylamide analogues, and potency was typically recovered in the butanoylamide analogues (Figure ). The retention of potency upon removal of the photoreactive moiety confirmed binding was driven by reversible fragment-protein interactions.