Towards New Scaffolds for Antimicrobial Activity—In Silico/In Vitro Workflow Introducing New Lead Compounds

<b>Background/Objectives</b>: The rapid evolution of bacterial resistance and the high cost of drug development have attributed greatly to the dearth in drug design. Computational approaches and natural product exploitation offer potential solutions to accelerate drug discovery. <b>Methods</b>: In this research article, we aimed to identify novel antibacterial hits. For the in silico studies, molecular scaffolds from the in-house chemical library of the Department of Pharmacy of Athens (Pharmalab) and the National Cancer Institute (NCI) were screened and selected for further experimental procedures. Compounds from both libraries that were not previously screened for their antimicrobial properties were tested in vitro against Gram-positive and Gram-negative bacterial strains. The microdilution method was used to determine the minimum inhibitory concentrations (MICs). <b>Results</b>: In silico screening identified twenty promising molecules from the NCI and seven from the Pharmalab databases. The unexplored compounds for their antibacterial activity can be characterized as weak strain-specific antimicrobials. The <b>NSC 610491</b> and <b>NSC 610493</b> were active against <i>Staphylococcus aureus</i> (MIC: 25 and 12.5 µg/mL, respectively) and methicillin-resistant <i>S. aureus</i> (MRSA) (MIC: 50 and 12.5 µg/mL, respectively). Six out of seven hydroxytyrosol (HTy) compounds were moderately active (MIC: 25-50 µg/mL) against <i>S. aureus</i>, MRSA and <i>Enterococcus faecalis</i>. For the Gram-negative bacteria, no activity was detected (≥100 µg/mL). <b>Conclusions</b>: The tested scaffolds could be considered as promising candidates for novel antimicrobials with improvements. Further experimentation is required to assess mechanisms of action and evaluate the efficacy and safety.