QSAR Reveals Decreased Lipophilicity of Polar Residues Determines the Selectivity of Antimicrobial Peptide Activity

Antimicrobial resistance has increased rapidly, causing daunting morbidity and mortality rates worldwide. Antimicrobial peptides (AMPs) have emerged as promising alternatives to traditional antibiotics due to their broad range of targets and low tendency to elicit resistance. However, potent antimicrobial activity is often accompanied by excessive cytotoxicity toward host cells, leading to a halt in AMP therapeutic development. Here, we present multivariate analyses that correlate 28 peptide properties to the activity and toxicity of 46 diverse African-derived AMPs and identify the negative lipophilicity of polar residues as an essential physiochemical property for selective antimicrobial activity. Twenty-seven active AMPs are identified, of which the majority are of scorpion or frog origin. Of these, thirteen are novel with no previously reported activities. Principal component analysis and quantitative structure–activity relationships (QSAR) reveal that overall hydrophobicity, lipophilicity, and residue side chain surface area affect the antimicrobial and cytotoxic activity of an AMP. This has been well documented previously, but the present QSAR analysis additionally reveals that a decrease in the lipophilicity, contributed by those amino acids classified as polar, confers selectivity for a peptide to pathogen over mammalian cells. Furthermore, an increase in overall peptide charge aids selectivity toward Gram-negative bacteria and fungi, while selectivity toward Gram-positive bacteria is obtained through an increased number of small lipophilic residues. Finally, a conservative increase in peptide size in terms of sequence length and molecular weight also contributes to improved activity without affecting toxicity. Our findings suggest a novel approach for the rational design or modification of existing AMPs to increase pathogen selectivity and enhance therapeutic potential.

a The lipophilicity of polar or nonpolar amino acids at pH 7.0 taken from Frecer (28) and Fauchere & Pliska (54) calculated using π FP = log P o/w (a.a.) − log P o/w (Gly) b The lipophilicity of polar or nonpolar amino acids at pH 7.0 according to the Wimley-white bilayer/water scale (74) c The side chain surface areas (SCSA) of amino acids taken from Frecer (28) as calculated by Connolly surfaces method (46) d Count of small lipophilic (CSL) or aromatic (CAR) residues taken from Frecer (28) e The number of rotatable bonds in an amino acid side chain: R.B.i = Rotatable Bonds (a.a.) -Rotatable Bonds (Gly) S3

Figure S1 :
Figure S1: The percentage human erythrocyte viability after respective treatment with 46 novel AMPs.Erythrocytes were treated with 16 scorpion, 15 frog, 11 tick and 4 primate derived AMPs at a screening concentration of 256 μg/mL each at 37 °C for 24h.Percentage viability is relative to the growth control; PBS treated cells.Error bars show the mean ± SE.Significant difference indicated by *, **, *** and **** represents p < 0.05, p < 0.01, p <0.001 and p < 0.0001 values.Results are of three independent biological repeats each performed in triplicate

Figure S2 :
Figure S2: The percentage HaCat cell viability after respective treatment with 46 novel AMPs.Hacat cells were treated with 16 scorpion, 15 frog, 11 tick and 4 primate derived AMPs at a screening concentration of 256 μg/mL each at 37 °C for 24h.Percentage viability is relative to the growth control; PBS treated HaCat cells.Error bars show the mean ± SE.Significant difference indicated by *, **, *** and **** represents p < 0.05, p < 0.01, p <0.001 and p < 0.0001 values.Results are of three independent biological repeats each performed in triplicate

Figure S7 :
Figure S7: Scree plot for Gram-negative PCA showing the number of PCs with eigenvalues greater than 1.5.PC-1, PC-2, PC-3, and PC-4 show eigenvalues greater than 1.5 and thus qualify to be used for the interpretation of results.

Figure S8 :
Figure S8: Scree plot for Gram-positive PCA showing the number of PCs with eigenvalues greater than 1.5.PC-1, PC-2, PC-3, and PC-4 show eigenvalues greater than 1.5 and thus qualify to be used for the interpretation of results.

Table S1 :
Sidechain descriptors of amino acids used in PCA and QSAR analyses

Table S2 :
Antibiogram employed in the AMP susceptibility testing for susceptible and resistant (A) Gram-negative and -positive bacteria and (B) fungi.

Table S3 :
Inhibitory activity of 15 frog derived AMPs against a panel of susceptible and resistant bacteria

Table S3 B
: Inhibitory activity of 15 frog derived AMPs against a panel of susceptible and resistant bacteria shown

Table S4 :
Inhibitory activity of 16 scorpion derived AMPs against a panel of susceptible and resistant bacteria

Table S4 B
: Inhibitory activity of 16 scorpion derived AMPs against a panel of susceptible and resistant bacteria shown

Table S5 :
Inhibitory activity of 11 tick derived AMPs against a panel of susceptible and resistant bacteria S. aureus (MRSA)

Table S5 B
: Inhibitory activity of 11 tick derived AMPs against a panel of susceptible and resistant bacteria shown

Table S6 :
Inhibitory activity of 4 primate derived AMPs against a panel of susceptible and resistant bacteria

Table S6 B
: Inhibitory activity of 4 primate derived AMPs against a panel of susceptible and resistant bacteria shown

Table S7 :
Inhibitory activity of 15 frog derived AMPs against a panel of fungal species

Table S7 B
: Inhibitory activity of 15 frog derived AMPs against a panel of fungal species shown

Table S8 :
Inhibitory activity of 16 scorpion derived AMPs against a panel of fungal species

Table S8 B
: Inhibitory activity of 16 scorpion derived AMPs against a panel of fungal species shown

Table S9 :
Inhibitory activity of 11 tick derived AMPs against a panel of fungal species

Table S9 B
: Inhibitory activity of 11 tick derived AMPs against a panel of fungal species shown

Table S10 :
Inhibitory activity of 4 primate derived AMPs against a panel of fungal species

Table S10 B
: Inhibitory activity of 4 primate derived AMPs against a panel of fungal species shown

Table S11 :
Haemolytic AMPs with an HC10 or HC50 below 256 µg/mL *Most active antimicrobial peptides identified in the screens **AMPs with an HC10 or HC50 above 256 µg/mL are regarded as non-haemolytic and the data not shown

Table S12 :
LC 50 determined in the HaCat cell line of the 12 most toxic AMPs.
* indicates peptides with the highest toxicity

Table S13 :
Results of Bartlett's test calculated for all 46 peptides against Gram-positive and -negative bacteria

Table S14 :
Parameters determining the no. of PCs selected for Gram-negative, -positive and fungal data interpretation