X-ray crystallographic fragment screening (XCFS) uses fragment-sized molecules (~60 - 300 Da) to access binding sites on proteins that may be inaccessible to larger drug-like molecules (>300 Da). Previous studies from our lab and others have shown that fragments containing halogen atoms have a higher binding occurrence compared to non-halogenated fragments. Specifically, we showed that 4-halopyrazoles hold potential for predicting the likelihood of success of a XCFS campaign. Here, we designed the Halo Library containing 46 halogenated fragments (including the 'universal fragment' 4-bromopyrazole). The basis of fragment selection was presence of (at least) one halogen atom, and binding to or inhibitory activity against (at least) two targets in literature. The library was screened against crystals of HIV-1 reverse transcriptase with drug rilpivirine, yielding an overall hit rate of 26%. Two new binding sites were discovered in addition to previously reported sites, and several hot spots were identified (i.e., sites with multiple fragment hits). This small library may thus provide a convenient tool for assessing feasibility of a target for XCFS, mapping hot spots and cryptic sites, as well as finding fragment binders that can be useful for developing drug leads.

X-ray crystallographic fragment screening (XCFS) uses fragment-sized molecules (∼60 to 300 Da) to access binding sites on proteins that may be inaccessible to larger drug-like molecules (>300 Da). Previous studies have shown that fragments containing halogen atoms bind more often to proteins than non-halogenated fragments. Here, we designed the Halo Library containing 46 halogenated fragments (including the "universal fragment" 4bromopyrazole), a majority of which have been reported to bind to or inhibit one or more targets. The library was screened against the crystals of HIV-1 reverse transcriptase with the drug rilpivirine, yielding an overall hit rate of 26%. Two new binding sites were discovered, and several hot spots were identified. This small library may thus provide a convenient tool for rapidly assessing the feasibility of a target for XCFS, mapping hot spots and cryptic sites, as well as finding fragment binders that can be useful for developing drug leads.