Structure-function-guided exploration of the antimicrobial peptide polybia-CP identifies activity determinants and generates synthetic therapeutic candidates

Antimicrobial peptides (AMPs) constitute promising alternatives to classical antibiotics for the treatment of drug-resistant infections, which are a rapidly emerging global health challenge. However, our understanding of the structure-function relationships of AMPs is limited, and we are just beginning to rationally engineer peptides in order to develop them as therapeutics. Here, we leverage a physicochemical-guided peptide design strategy to identify specific functional hotspots in the wasp-derived AMP polybia-CP and turn this toxic peptide into a viable antimicrobial. Helical fraction, hydrophobicity, and hydrophobic moment are identified as key structural and physicochemical determinants of antimicrobial activity, utilized in combination with rational engineering to generate synthetic AMPs with therapeutic activity in a mouse model. We demonstrate that, by tuning these physicochemical parameters, it is possible to design nontoxic synthetic peptides with enhanced sub-micromolar antimicrobial potency in vitro and anti-infective activity in vivo. We present a physicochemical-guided rational design strategy to generate peptide antibiotics.


Supplementary Figure 4.
Helical wheel representations of the second generation of Pol-CP-NH2 analogs generated using the Heliquest server considering theoretical helical structure and physicochemical properties derived from the amphipathic distribution. Yellow circles indicate hydrophobic/aliphatic residues, blue circles represent positively charged residues, purple circles indicate polar uncharged residues, red circles represent negatively charged residues, and gray circles represent residues with hydrophobicity close to zero. The black arrows inside the helical wheel projection of each peptide represent their hydrophobic moment vector, whose magnitude is indicated by the size of the arrows. Figure 5. In vitro antimicrobial activity of the lead peptides from the second generation of Pol-CP-NH2 derived agents. Serially diluted (0 -128 µmol L -1 ) peptides were added to a 96-well plate containing 10 4 bacterial cells in each well and incubated at 37 o C for 24 h. After the exposure, the solution in each well was measured in a microplate reader (600 nm) to check inhibition of bacteria compared to the untreated controls and presented as heat maps of antimicrobial activities (µmol L -1 ) against four bacteria strains: Escherichia coli strain BL21, S. aureus strain ATCC12600 and P. aeruginosa strains PA01 and PA14. Assays were performed in independent triplicates.  .0 a LC/ESI-MS data were obtained on a Model 6130 Infinity mass spectrometer coupled to a Model 1260 HPLC system (Agilent), using a Phenomenex Gemini C18 column (2.0 mm × 150 mm, 3.0 µm particles, 110 Å pores). Solvent A was 0.1% TFA in water, and solvent B was 90% ACN in solvent A. Elution with a 5-95% B gradient was performed over 20 min, 0,2 mL min -1 flow and peptides were detected at 220 nm. Mass measurements were performed in a positive mode with the following conditions: mass range between 100 to 2500 m/z, ion energy of 5.0 V, nitrogen gas flow of 12 L min -1 , solvent heater of 250 o C, multiplier of 1.0, capillary of 3.0 kV and cone voltage of 35 V.  Table 2. Considerations for each one of the second library analogs designed synthesized in this work to check the importance of different kinds of substitutions and how well can the optimal hotspots describe activity propensities.

Peptide
Design Considerations

ILGTLLGLKKSL-NH2
To check if a subtle change in the side chain of the residue at position 5 would alter the peptide biological behavior even with an enhanced net positive charge

ILGTKLGLLKSL-NH2
To check the importance of Ile presence at position 5 even when substituted by a residue with an additional charge since it decreased when it was substituted by an Ala residue

ILGKILGLLKSL-NH2
To check effect of charge introduction at the interface of hydrophobic and hydrophilic faces

ILGTILKLLKSL-NH2
Lys is common residue in the 7th position of small wasp venom peptides and Gly substitution by an Ala residue improved activity

ILGTILGLFKSL-NH2
To check the effect of increased hydrophobicity

ILGTILGLLKS-NH2
To check the effect of increasing hydrophilic/hydrophobic ratio

ILETKLGLLKSE-NH2
To check the effect of the introduction of negatively charged residues on the hydrophilic face

GLGTILGLLKSL-NH2
Gly is the most common residue in the 1st residue of small cationic amphipathic peptides