Biological profiling of structurally diverse LEGO-LPPOs reveals differences in antibacterial activity, cytocompatibility, and in vivo tolerability.

Lipophosphonoxins represent a promising class of membrane-active antimicrobial agents with potential relevance for skin-targeted antimicrobial applications. Antibacterial activity of a panel of LEGO-lipophosphonoxin (LEGO-LPPO) compounds was evaluated by minimum inhibitory concentration (MIC) profiling against selected Gram-positive and Gram-negative bacterial strains. Cellular responses were subsequently assessed in human keratinocytes (HaCaT) and primary human dermal fibroblasts using MTS-based viability assays after 3 and 7 days of exposure across a broad concentration range. The tested LEGO-LPPOs exhibited pronounced variability in antibacterial activity and cytotoxicity profiles. Cell-based screening revealed marked heterogeneity in cellular responses, with fibroblasts consistently showing higher sensitivity than keratinocytes, particularly under prolonged exposure. Based on integrated MIC and IC₅₀ profiles, four representative compounds (DR_527P1, DR_556P1, DR_34P1, and DR_33P1) were selected for further functional characterization using keratinocyte migration assays, cytoskeletal organization analysis, and protein expression profiling. These compounds exerted distinct effects on cell migration, cytoskeletal organization and protein expression. In addition, in vivo tolerability was evaluated by maximal tolerated dose assessment in mice to provide a translational perspective on compound safety. Collectively, these findings show that selected structurally diverse LEGO-LPPO derivatives display divergent biological profiles that are not predicted by antibacterial potency alone. Rather than establishing a comprehensive structure-activity relationship (SAR), this study provides integrated biological profiling of representative LEGO-LPPO compounds and identifies structural diversity points and biological liabilities that should be considered during further optimization for local anti-infective applications.