The　MUS81-EME1/2　structure-specific　endonucleases　play　a　crucial　role　in　the　processing　of　stalled　replication　forks　and　recombination　intermediates,　and　have　been　recognized　as　an　attractive　drug　target　to　potentiate　the　anti-cancer　efficacy　of　DNA-damaging　agents.　Currently,　no　bioactive　small-molecule　inhibitors　of　MUS81　are　available.　Here,　we　performed　a　high-throughput　small-molecule　inhibitors　screening,　using　the　FRET-based　DNA　cleavage　assay.　From　7920　compounds,　we　identified　dyngo-4a　as　a　potent　inhibitor　of　MUS81　complexes.　Dyngo-4a　effectively　inhibits　the　endonuclease　activities　of　both　MUS81-EME1　and　MUS81-EME2　complexes,　with　IC50　values　of　0.57　μM　and　2.90　μM,　respectively.　Surface　plasmon　resonance　(SPR)　and　electrophoretic　mobility　shift　assay　(EMSA)　assays　reveal　that　dyngo-4a　directly　binds　to　MUS81　complexes　(KD　∼　0.61　μM)　and　prevents　them　from　binding　to　DNA　substrates.　In　HeLa　cells,　dyngo-4a　significantly　suppresses　bleomycin-triggered　H2AX　serine　139　phosphorylation　(γH2AX).　Together,　our　results　demonstrate　that　dyngo-4a　is　a　potent　MUS81　inhibitor,　which　could　be　further　developed　as　a　potentially　valuable　chemical　tool　to　explore　more　physiological　roles　of　MUS81　in　the　cells.　©　2023　Elsevier　Ltd