Cheminformatic analysis of protein surfaces provides binding site insights andinforms identification strategiesMilisavljević, Andrej (Avtor) Pražnikar, Jure (Avtor) Bren, Urban (Avtor) Jukič, Marko (Avtor) protein surface analysissmall-molecule binding site detectionmachine learningcheminformaticsamino acidindexbinding sitemall-molecule–protein interactionsin-silico drug designAims: Understanding protein–ligand binding site behavior is central to structure-based drug design. Weanalyzed amino acid composition and interactions in protein–ligand small-molecule binding sites anddeveloped a novel method for binding site prediction.Materials and methods: We analyzed the PDBBind+ database, which contains the largest protein–ligand binding site dataset known to us, using existing cheminformatics packages and in-house code.We used the resulting data to train a binding site prediction model.Results: Within solvent-accessible binding regions, tryptophan, phenylalanine, tyrosine, methionine,and glycine, were enriched. Interaction analysis revealed hydrophobic contacts as the most frequent,followed by hydrogen bonds, water-bridged hydrogen bonds, salt bridges, π–π, π–cation, and occa-sional halogen interactions. We introduced the amino acid binding site enrichment index (ABSE), tosupport small-molecule binding site detection, and developed a model that discriminates binding sitesequences from protein surface patches with 0.91 accuracy.Conclusions: This work offers interpretable composition–interaction relationships and practical tool forbinding site characterization. To facilitate application, we provide a free, open-source, fast, bindingsiteidentification tool (AABS), available at https://gitlab.com/Jukic/aabs. We anticipate that these findingsand tool will advance binding site prediction and accelerate computationally intensive drug discoverywithin medicinal chemistry.Taylor&Francis20252025-12-08 12:39:05Članek v reviji96180UDK: 543COBISS_ID: 259712771DOI: 10.1080/17568919.2025.2592531ISSN pri članku: 1756-8927sl© 2025 The Author(s).