Design and characterization of a new hybrid peptide from LL-37 and BMAP-27

Figures & data

Table 1 Predicted percentages of secondary structure for the parent peptides (LL-37 and BMAP-27) and B1 using HNN

Peptide	Natural sequence	Amino acid (n)	Alpha helix (%)	Extended strand (%)	Random coil (%)
LL-37	LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES	37	78.38	0.00	21.62
BMAP-27	GRFKRFRKKFKKLFKKLSPVIPLLHL	26	53.85	15.38	30.77
B1	KFKKLFKKLSPVFKRIVQRIKDFLR	25	88.00	0.00	12.00

Notes: The software used to generate the percentages in this table was from Guermeur Y.¹⁶ The bold letters represent the parts that were taken from LL-37 sequence to produce the hybrid peptide.

Abbreviation: HNN, Hierarchical Neural Network.

Table 2 The prediction of the physiochemical properties of LL-37, BMAP-27, and B1.

Peptide	Amino acid (n)	Molecular weight	Theoretical pI	Instability index	Aliphatic index	GRAVY#	Hydrophobic ratio	Total net charge
LL-37	37	4,493.32	10.61	23.34	89.49	−0.724	35%	+6
BMAP-27	26	3,226.10	12.32	21.48	101.15	−0.365	42%	+10
B1	25	3,163.98	11.77	20.99	101.20	−0.408	44%	+9

Note: The parameters were calculated using ProtParam from ExPASy¹⁷ and APD.¹⁸Abbreviations: pI, isoelectric point; GRAVY, The Grand Average hydropathy value of the peptide.

Figure 1 Three-dimensional structure of B1. Note: The structure was generated by homology modeling using MODELLER²⁰ and the figure was prepared using PyMol.³⁵

Table 3 MICs of B1 against different bacterial strains

Bacterial strains	ATCC	MIC (μM)
Gram positive
Standard strains MS S. aureus	29,213	20
S. epidermidis	12,228	20
Resistant strains
MR S. aureus	33,591	20
MR S. aureus	43,300	20
MR S. aureus	BAA-41	20
E. faecium	BAA-2316	20
Gram negative
Standard strains
E. coli	25,922	20
Resistant strains
E. coli	35,218	20
MDR P. aeruginosa	BAA-2114	10

Abbreviations: ATCC, American Type Culture Collection; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MS, methicillin sensitive; MR, methicillin resistant; MDR, multidrug resistant.

Table 4 The antibiofilm activity of B1 against MR S. aureus ATCC 33951 and MDR P. aeruginosa ATCC BAA-2114

Peptide concentration (μM)	Viable biofilm cells (%) – MR S. aureus ATCC 33951	Viable biofilm cells (%) – MDR P. aeruginosa ATCC BAA-2114
200	0.0%	0.0%
140	0.0%	0.0%
80	3.5%	4.5%
60	4.0%	8.5%
40	6.5%	21.0%
20	14.0%	45.0%
10	55.0%	62.0%

Abbreviations: MR, methicillin resistant; MDR, multidrug resistant.

Table 5 Correlation between FIC and the synergistic effect upon combination with antibacterial agents

Bacterial strains ATCC	Antibiotics	MIC of B1 alone (μM)	MIC of B1 combined (μM)	MIC of antibiotic alone (μM)	MIC of antibiotic combined (μM)	(%) Reduction in antibiotic MIC	FIC index	Synergism
MR	LVX	20	2.5	10	0.75	92.50%	0.20	Synergistic
S. aureus	CHL	20	2.5	130	65	50.00%	0.63	Additive
33591	RIF	20	2.5	0.04	0.00125	96.88%	0.16	Synergistic
	AMP	20	5	85	37.5	55.88%	0.69	Additive
	ERY	20	2.5	125	3.5	97.20%	0.15	Synergistic
MR	LVX	20	5	10	4	60.00%	0.65	Additive
S. aureus	CHL	20	2	15	4	73.33%	0.37	Synergistic
43300	RIF	20	5	1	0.3	70.00%	0.55	Additive
	AMP	20	7.5	20	7.5	62.50%	0.55	Additive
	ERY	20	2.5	100	20	80.00%	0.75	Additive
MR	LVX	20	7.5	10	7.5	25.00%	0.33	Synergistic
S. aureus	CHL	20	0.25	25	5	80.00%	1.13	Indifferent
BAA-41	RIF	20	0.5	0.005	0.00075	85.00%	0.21	Synergistic
	AMP	20	3.75	40	15	62.50%	0.18	Synergistic
	ERY	20	7.5	350	250	28.57%	0.56	Additive
MDR	LVX	10	2.5	12	5	58.33%	1.09	Indifferent
P. aeruginosa	CHL	10	1.25	200	50	75.00%	0.67	Additive
BAA-2114	RIF	10	0.25	50	37.5	25.00%	0.38	Synergistic
	AMP	10	7.5	>500	375	–	–	–
	ERY	10	2.5	125	37.5	70.00%	0.55	Additive

Abbreviations: MR, methicillin resistant; MDR, Multidrug resistant; LVX, levofloxacin; CHL, chloramphenicol; RIF, rifampicin; AMP, ampicillin; ERY, erythromycin; MIC, minimum inhibitory concentration; FIC, fractional inhibitory concentration.

Figure 2 Hemolytic activity of B1 against human erythrocytes after an exposure time of 1 hr.

Figure 3 Cytotoxicity assay of B1 on HEK293 mammalian cell line using MTT assay.

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