Menin　functions　as　an　oncogenic　cofactor　of　mixed　lineage　leukaemia　(MLL)　fusion　proteins　in　leukaemogenesis.　The　menin-MLL　interface　is　a　potential　therapeutic　target　in　acute　leukaemia　cases.　In　this　study,　approximately　900　clinical　compounds　were　evaluated　and　ranked　using　pharmacophore-based　virtual　screening,　the　top　29　hits　were　further　evaluated　by　biochemical　analysis　to　discover　the　inhibitors　that　target　the　menin-MLL　interface.　Two　aminoglycoside　antibiotics,　neomycin　and　tobramycin,　were　identified　as　menin-MLL　inhibitors　with　binding　affinities　of　18.8　and　59.9　mu　M,　respectively.　The　results　of　thermal　shift　assay　validated　the　direct　interactions　between　the　two　antibiotics　and　menin.　The　results　of　isothermal　titration　calorimetry　showed　that　the　equilibrium　dissociation　constant　between　menin　and　neomycin　was　approximately　15.6　mu　M.　We　also　predicted　the　binding　modes　of　inhibitors　at　the　menin-MLL　interface　through　molecular　docking　analysis.　The　results　indicated　that　neomycin　and　tobramycin　competitively　occupy　the　binding　site　of　MLL.　This　study　has　shed　light　on　the　development　of　powerful　probes　and　new　therapies　for　MLL-mediated　leukaemogenesis.　(C)　2014　Elsevier　Ltd.　All　rights　reserved.