The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase)/IMP cyclohydrolase (ATIC), catalyzes the penultimate and final steps in the de novo purine biosynthesis pathway that produces IMP, and hence, is a potential target for antineoplastic intervention.We have focused on the identification of novel, non-folate based inhibitors that selectively inhibit AICAR Tfase activity, which may cause less severe cytotoxicity than traditional anti-folates, such as methotrexate.Here, we present a fragmentbased approach for ATIC inhibition combining X-ray crystallography and molecular modeling.A systematic search for novel inhibitors of AICAR Tfase utilizing molecular docking coupled with in vitro enzymatic assay enabled discovery of several novel non-folate based inhibitors.Through detailed structural and computational analysis of previous sulfonyl-containing inhibitor complex structures, several low molecular weight fragments were identified that bound to discrete regions of AICAR Tfase active sites.Novel inhibitors were then designed through fragment tethering using different covalent linker groups and their binding modes were predicted by molecular docking.Enzymatic inhibition assays on selected potential leads and their complexed crystal structures will facilitate the next round of inhibition elaboration.