Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

H + /Na + transport activity normalized against the value of 1 (1.00) and antibacterial activity are also listed.
H + /Na + transport activity normalized against the value of 1 (1.00) and antibacterial activity are also listed.
Antibacterial activity against S. pyogenes was evaluated using the three concentrations. +++: inhibition by the 640-fold diluted peptide solution, ++: inhibition by the 160-fold diluted solution, +: inhibition by the 40-fold diluted solution, -: no inhibition. , where a is the weight of Fmoc-protected resin (mg), and b is absorbance at 301 nm. Procedures for microwave-assisted solid-phase peptide synthesis (SPPS). Peptide 1 was prepared on a peptide synthesizer MWS-1000A. Standard operation was shown as follows:
Step Step 4: The activated N-Fmoc-amino acid was coupled with the peptide on the resin (40 °C, 200 W; 20 min) and the 5 mL LibraTube containing the resin was washed with NMP (2.00 mL, 30 sec × 6).
Steps 1−4 were repeated and amino acids were condensed on the solid support.
Step Step 5: The activated N-Fmoc-amino acid was coupled with the peptide on the resin (60 °C, 200 W; 20 min) and the 5 mL LibraTube containing the resin was washed with NMP (2.00 mL, 30 sec × 6).
Step 6: The four resin batches suspended in CH2Cl2 were mixed in one LibraTube.
The N-Fmoc group of the above bead-linked pentadecapeptide was removed by steps 2 and 3 of the standard split-and-mix synthesis protocol. The beads in the 5 mL LibraTube was washed with CH2Cl2 (2.00 mL, 30 sec × 6), MeOH (2.00 mL, 30 sec × 6), and Et2O (2.00 mL, 30 sec × 6), and dried under vacuum to give the bead-linked amine (306 mg).
To the above bead-linked amine (306 mg) were added p-nitrophenyl formate (6,   at room temperature. The reaction mixture was stirred at room temperature for 1 min. The resultant mixture was added to the above HMBA-ChemMatrix resin S2 in the 20 mL LibraTube at room temperature. After being stirred at room temperature for 12 h, the reaction mixture was filtered, and washed with DMF Procedure for automated solid-phase peptide synthesis (automated SPPS). Peptides A1, B01-B04, B11-B13, B21, and B22 were prepared on an automated peptide synthesizer Initiator + Alstra. Standard operation was shown as follows: Step 1: The solid supported N-Fmoc peptide was deprotected with piperidine/NMP (1/4, 60 °C, 5 min).
Step     H + /Na + transport assay of bead-derived peptides. 1 The data were transformed to the relative values against mean value of the two control wells for the plate.
The mean value of the two control wells was set to 1.00. The H + /Na + transport activities of the purified peptides were evaluated as EC50 (nM) by means of three replicates. Sigmoidal curve fittings were performed on R 4 with drc package. 5 Four-parameter logistic model was applied for the fitting (Supplementary Figure 5).
The hemolytic activities of the purified peptides were evaluated as the concentrations causing 10% hemolysis (HC10, nM) by means of three replicates. Sigmoidal curve fittings were performed on GraphPad Prism (GraphPad software, Supplementary Figure 7).