Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. However, many AMPs fail to progress into clinics due to unexpected difficulties found in preclinical in vivo phases. Our research has focused on crotalicidin (Ctn), an AMP from snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work, we evaluate the in vivo efficacy of both the native-sequence AMPs and their retroenantio counterparts in a murine infection model with Acinetobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogs either exhibit toxicity or lack efficacy when administered to mice. Our findings underscore the critical importance of in vivo infection model evaluation to fully calibrate the therapeutic potential of AMPs.

RNase 2 is the most abundant human RNase A member in macrophages, and its expression is activated upon virus exposure. Here, we explored the protein role by co-transcriptomics of wild-type (WT) and RNase 2-knock-out (KO) macrophages in absence/presence of a virus-derived single-stranded RNA (ssRNA40). Results revealed that RNase 2 is key for maintaining cell homeostasis. RNase 2 deletion induced the expression of stress-response markers and abolished the antiviral response. In contrast, the up-regulated genes in WT macrophages participate in pro-inflammatory response through TLR8-dependent pathways and antiviral immunity, with activation of MAPK and JAK-STAT pathways. We identified five top tRNA-derived small RNAs (tDRs) in response to ssRNA40 in presence of RNase 2, showing preferential cleavage at CA and uridine rich regions of anticodon loops. Another conserved target in all RNase2-generated 3’tDRs was found at the CCA end, releasing truncated 3’-CC. Among tDRs unique for WT exposed to ssRNA40, tRNA-LysTTT 3’-half stands out with the highest significant sequence coverage difference. Last, the immune response profile of RNase 2-KO cells is mostly recovered by addition of tRNA-LysTTT digestion products generated in vitro by recombinant RNase 2, suggesting an activation mechanism mediated by tDRs. Results highlight the essential role of RNase 2 selective tRNA cleavage in antiviral response and inflammation. Graphical Abstract

Antimicrobial peptides (AMPs) are regarded as a promising alternative to traditional antibiotics in the face of ever-increasing resistance. Our research has focused on crotalicidin (Ctn), an AMP from a snake venom, and a fragment thereof, Ctn[15-34], with improved in vitro antimicrobial and anticancer activities and remarkable serum stability. As the retroenantio versions of both AMPs maintained favorable profiles, in this work we evaluate the in vivo efficacy of both the na-tive-sequence AMPs and their retroenantio counterparts in a murine infection model with Aci-netobacter baumannii. A significant reduction in bacterial levels is found in the mice treated with Ctn[15-34]. However, contrary to expectations, the retroenantio analogues either exhibit toxicity or lack efficacy when administered to mice.

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role.