Structural determinants underlying the supramolecular binding between carborane and proteins in water

Carboranes are chemically and biologically stable boron‑carbon clusters with promising applications in medicinal chemistry. While their use in boron neutron capture therapy (BNCT) has been extensively explored, recent attention has shifted toward understanding their interactions with biological macromolecules, particularly proteins. Here, we characterize the interaction between closo-ortho-carborane and lysozyme (LSZ) using NMR spectroscopy, molecular docking and molecular dynamics simulations, and enzymatic assays. Experimental data demonstrate that carborane forms a stable 1:1 complex with LSZ (Carborane@LSZ), retaining the monomeric state and the protein fold, with only a limited number of amino acids involved in the interaction. In particular, NMR chemical shift perturbations revealed specific binding near the substrate-binding pocket, a result corroborated by molecular docking and molecular dynamic simulations. Carborane fits into a hydrophobic pocket near the substrate-binding site, where the recognition process is driven by hydrophobic interactions complemented by classical hydrogen and non-standard dihydrogen bonding. Carborane-@LSZ complex partially inhibits enzymatic activity (∼33 %). Extending this approach to bovine serum albumin (BSA) revealed similar binding principles, underscoring the generality of carborane-protein supramolecular interactions. These findings provide fundamental insights into pristine carboranes recognition by proteins and establish a foundation for designing carborane-based therapeutics and delivery platforms in nanomedicine.