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

Abstract

Membrane disruptive α-helical antimicrobial peptides (AMPs) offer an opportunity to address multidrug resistance; however, most AMPs are toxic and unstable in serum. These limitations can be partly overcome by introducing D-residues, which often confers protease resistance and reduces toxicity without affecting antibacterial activity, presumably due to lowered α-helicity. Here, we investigated 31 diastereomers of the α-helical AMP KKLLKLLKLLL. Three diastereomers containing two, three, and four D-residues showed increased antibacterial effects, comparable hemolysis, reduced toxicity against HEK293 cells, and excellent serum stability, while another diastereomer with four D-residues additionally displayed lower hemolysis. X-ray crystallography confirmed that high or low α-helicity as measured by circular dichroism indicated α-helical or disordered structures independently of the number of chirality switched residues. In contrast to previous reports, α-helicity across diastereomers correlated with both antibacterial activity and hemolysis and revealed a complex relationship between stereochemistry, activity, and toxicity, highlighting the potential of diastereomers for property optimization.

Antimicrobial Peptide–Peptoid Hybrids with and without Membrane Disruption

“…Peptoid units can be introduced during standard Fmoc solid-phase peptide synthesis (Fmoc-SPPS) using the so-called submonomer strategy, which consists in bromoacetylation of the N -terminus of the growing chain followed by substitution of the bromide using an excess of a primary amine, here isobutylamine for a leucine peptoid unit ( N Leu) or tert -butyl (4-aminobutyl)­carbamate for a lysine peptoid unit ( N Lys). We prepared the eleven selected peptide–peptoid hybrids together with undecapeptides ln65 and ln69 , as well as the PrAMP oncocin, to be used as positive controls, using high-temperature (60 °C) semiautomated Fmoc-SPPS on Rink amide resin in DMF with di-isopropyl carbodiimide (DIC) and OxymaPure as coupling reagents as described previously. , Addition of amino acids was repeated twice, while acylation with bromoacetic acid and displacement with the primary amine were performed only once. All products were obtained in pure form by acidic cleavage and deprotection followed by preparative reversed-phase HPLC (Scheme ).…”

The amphipathic design in helical antimicrobial peptides

“…Remarkably, for those AMPs in both models 1 and 2, the impact of “helix breakers” on the structural and biological properties varies depending on each case. For example, stereorandomized derivative 23 of peptide 21 and its all‐D‐enantiomeric 22 ( both in model 1A and 2B) , the antibacterial activity was only slightly decreased whereas the minimum hemolysis concentration (MHC) was notably increased, thus indicating a significant improvement in selectivity [4a] . This interesting data suggested that many mutants among all the 1024 possible diastereometers that have better activity than both peptides 21 and 22 .…”

“…This interesting data suggested that many mutants among all the 1024 possible diastereometers that have better activity than both peptides 21 and 22 . Then, a diastereomeric series of 21 was prepared to further examine the structure‐activity relationships [4a] . In terms of secondary structure, there are two directions observed.…”

“…Additionally, diastereomeric optimization are demonstrated to improve the pharmacological properties of helical AMPs, including proteolytic resistance, antimicrobial potency and reduced toxicity [4a,b] . Nevertheless, a major drawback arises from the enormous number of possible mutant sequences.…”

One chiral fingerprint to find them all

“…In the context of developing computational tools for new modalities including beyond-Ro5 molecules [ 24 , 25 ], in our case for peptides with variable chain topology and stereochemistry [ 26 – 28 ], we have adapted molecular fingerprints based on atom-pairs [ 29 – 32 ] for large molecules such as peptides and proteins [ 33 – 35 ]. In particular, we combined atom-pair analysis and circular substructures as encoded by the Morgan fingerprint ECFP4 [ 36 , 37 ], with the principle of data compression using MinHashing [ 38 – 41 ], to design MAP4, a MinHashed Atom-Pair fingerprint.…”