An intrinsically disordered antimicrobial peptide dendrimer from stereorandomized virtual screening

Inverse Polyamidoamine (i‐PAMAM) Dendrimer Antimicrobials

“…Time‐kill assays, which assess the number of remaining colony forming units (cfu) over time upon exposure to the compounds, confirmed the bactericidal effect of 3 and 4 ( Figure 2 ,a and 2,b ). However, the rate of killing was comparable to that of polymyxin B and somewhat slower than for membrane disruptive compounds such as our peptide dendrimers, which typically completely kill bacteria within 30 minutes [24, 29, 37] . Indeed, both dendrimers were entirely inactive in a vesicle leakage assay measuring the release of 5(6)‐carboxyfluorescein from synthetic vesicles consisting of egg white phosphatidyl glycerol (EYPG) which mimics bacterial membranes ( Figure 2 ,b ) [40] .…”

“…However, the rate of killing was comparable to that of polymyxin B and somewhat slower than for membrane disruptive compounds such as our peptide dendrimers, which typically completely kill bacteria within 30 minutes. [24,29,37] Indeed, both dendrimers were entirely inactive in a vesicle leakage assay measuring the release of 5( 6)carboxyfluorescein from synthetic vesicles consisting of egg white phosphatidyl glycerol (EYPG) which mimics bacterial membranes (Figure 2,b). [40] Both dendrimers were also entirely inactive on vesicle consisting of egg white phosphatidyl choline (EYPC) that mimics eukaryotic membranes, in line with the fact that they were non-hemolytic.…”

“…Here we built on our own experience of obtaining peptide dendrimers [18–21] as pure and stable products from solid‐phase peptide synthesis (SPPS) [22–24] . We envisioned that a stable PAMAM analog should result from simply moving the carbonyl group of β‐alanine across the amide bond.…”

“…[16,17] Here we built on our own experience of obtaining peptide dendrimers [18][19][20][21] as pure and stable products from solid-phase peptide synthesis (SPPS). [22][23][24] We envisioned that a stable PAMAM analog should result from simply moving the carbonyl group of β-alanine across the amide bond. Indeed, this simple change transforms the β-alanine unit into 1,3-diaminopropane and the ethylene diamine unit into glycine, thereby eliminating the possibility of a retro-Michael reaction (Figure 1).…”

To Fold or Not to Fold: Diastereomeric Optimization of an α-Helical Antimicrobial Peptide

“…To further probe if α-helical folding was required for activity, we prepared the fully stereorandomized sequence sr - ln65 , a racemic mixture of the 1024 possible diastereomers, as well as sr - ln65L 6 with pure l -leucine at position 6 of the sequence, containing all 1024 diastereomers with single chirality at position 6 such as to make a possible folding detectable by CD. Remarkably, both sr - ln65 and sr - ln65L 6 were as antibacterial as ln65 but much less hemolytic, an effect comparable to our previous observation with AMPDs and sr -AMDPs, suggesting that the antibacterial bioactive conformation of ln65 might be disordered while the hemolytic bioactive conformation would be α-helical. , However, while CD spectra of sr - ln65 were nearly flat as expected because the stereorandomized sequence is racemic, those of sr - ln65L 6 showed approximately 17% α-helix content in 5 mM DPC or with TFE, suggesting that a significant fraction of the 1024 possible diastereomers of ln65 might be α-helical (Figure c). Therefore, the activity of sr - ln65 might also be explained by the presence of some highly active and α-helical diastereomers, such as ln69 , mixed with inactive and possibly disordered diastereomers.…”