# The conserved N-terminal histidine in an engineered peptide mediates sepsis treatment efficacy via dual binding to CD14 and LPS

## TL;DR

A modified peptide called sHVF18 treats sepsis by binding to both LPS and CD14, and changing its N-terminal histidine affects its effectiveness.

## Contribution

The study identifies the conserved N-terminal histidine as critical for dual binding to CD14 and LPS, enabling effective sepsis treatment.

## Key findings

- Substituting the N-terminal histidine with lysine or arginine improves LPS binding but impairs CD14 interaction.
- sHVF18 with conserved histidine effectively reduces inflammation and improves survival in polymicrobial sepsis.
- Balancing LPS and CD14 binding is essential for therapeutic efficacy in sepsis treatment.

## Abstract

Sepsis remains a major clinical challenge due to the limited efficacy of existing therapies in controlling excessive inflammation. The engineered stapled peptide sHVF18, derived from an evolutionarily conserved thrombin innate fold, binds both lipopolysaccharide (LPS) and the LPS-binding groove of CD14, enabling dual targeting of bacterial components and host immune signaling. To define structural prerequisites for this dual action, we combined evolutionary analysis, in silico modeling, and experimental methods. Substituting the N-terminal histidine with lysine (K) or arginine (R) improved solubility, reduced aggregation, and enhanced interactions with LPS. However, unexpectedly, K substitutions impaired CD14 binding, whereas R variants retained weaker affinity, possibly through cation-π interactions. The essential role of the evolutionarily conserved N-terminal histidine for CD14 interactions and therapeutic efficacy was demonstrated using LPS-induced shock and polymicrobial sepsis models. While the K variant exhibited superior efficacy in LPS-induced shock, its disrupted CD14 interactions rendered it ineffective in polymicrobial sepsis. In contrast, sHVF18, by engaging both LPS and CD14, effectively reduced inflammation and improved survival in polymicrobial sepsis. These findings highlight that targeting of both LPS and CD14 is essential for therapeutic efficacy, underscoring multivalency as a key principle for future sHVF18-based sepsis therapeutics.

This study reveals that balancing LPS and CD14 binding is critical for the efficacy of the stapled peptide sHVF18 in treating infections. Substitution of N-terminal histidine with arginine or lysine enhances LPS affinity but reduces CD14 interaction, highlighting a novel dual-targeting mechanism for anti-infective peptide therapeutics.