Ribosomal　protein　S1　(RpsA)　has　been　identified　as　a　novel　target　of　pyrazinoic　acid　(POA),　which　is　the　active　form　of　pyrazinamide　(PZA),　in　vivo.　RpsA　plays　a　crucial　role　in　trans-translation,　which　is　widespread　in　microbes.　In　our　investigation,　we　first　described　the　discovery　of　promising　RpsA　antagonists　for　drug-resistant　mycobacterium　(MtRpsAd438A)　and　M.　smegmatis,　as　well　as　wild-type　M.　tuberculosis.　These　antagonists　were　discovered　via　structure/ligand-based　virtual　screening　approaches.　A　total　of　21　targeted　compounds　were　selected　by　virtual　screening,　combined　scores,　affinity,　similarities　and　rules　for　potential　as　drugs.　Next,　the　affinities　of　these　compounds　for　three　targeted　proteins　were　tested　in　vitro　by　applying　various　technologies,　including　fluorescence　quenching　titration　(FQT),　saturation　transfer　difference　(STD),　and　chemical　shift　perturbation　(CSP)　assays.　The　results　showed　that　seven　compounds　had　a　high　affinity　for　the　targeted　proteins.　Our　discovery　set　the　stage　for　discovering　new　chemical　entities　(NCEs)　for　PZA-resistant　tuberculosis　and　providing　key　residues　for　rational　drug　design　to　target　RpsA.　©　2018　Elsevier　Inc.