Design and Synthesis of Lipopolysaccharide-Binding Antimicrobial Peptides Based on Truncated Rabbit and Human CAP18 Peptides and Evaluation of Their Action Mechanism.

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4-128 µg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.

Anticalins small engineered binding proteins based on the lipocalin scaffold.

Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel ß-barrel, which is open to the solvent at one end. There, four loops connect the ß-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.