Sequence-based prediction of the intrinsic solubility of peptides containing non-natural amino acids.

Non-natural amino acids are increasingly used as building blocks in the development of peptide-based drugs as they expand the available chemical space to tailor function, half-life and other key properties. However, while the chemical space of modified amino acids (mAAs) such as residues containing post-translational modifications (PTMs) is potentially vast, experimental methods for measuring the developability properties of mAA-containing peptides are expensive and time consuming. To facilitate developability programs through computational methods, we present CamSol-PTM, a method that enables the fast and reliable sequence-based prediction of the intrinsic solubility of mAA-containing peptides in aqueous solution at room temperature. From a computational screening of 50,000 mAA-containing variants of three peptides, we selected five different small-size mAAs for a total number of 37 peptide variants for experimental validation. We demonstrate the accuracy of the predictions by comparing the calculated and experimental solubility values. Our results indicate that the computational screening of mAA-containing peptides can extend by over four orders of magnitude the ability to explore the solubility chemical space of peptides and confirm that our method can accurately assess the solubility of peptides containing mAAs. This method is available as a web server at https://www-cohsoftware.ch.cam.ac.uk/index.php/camsolptm .

Peptoids with Antibiofilm Activity against the Gram Negative Obligate Anaerobe, Fusobacterium nucleatum.

Peptoids (oligo N-substituted glycines) are peptide analogues, which can be designed to mimic host antimicrobial peptides, with the advantage that they are resistant to proteolytic degradation. Few studies on the antimicrobial efficacy of peptoids have focused on Gram negative anaerobic microbes associated with clinical infections, which are commonly recalcitrant to antibiotic treatment. We therefore studied the cytotoxicity and antibiofilm activity of a family of peptoids against the Gram negative obligate anaerobe Fusobacterium nucleatum, which is associated with infections in the oral cavity. Two peptoids, peptoid 4 (NaeNpheNphe)4 and peptoid 9 (NahNspeNspe)3 were shown to be efficacious against F. nucleatum biofilms at a concentration of 1 µM. At this concentration, peptoids 4 and 9 were not cytotoxic to human erythrocytes or primary human gingival fibroblast cells. Peptoids 4 and 9 therefore have merit as future therapeutics for the treatment of oral infections.

Synthesis of Antibacterial Nisin⁻Peptoid Hybrids Using Click Methodology.

Antimicrobial peptides and structurally related peptoids offer potential for the development of new antibiotics. However, progress has been hindered by challenges presented by poor in vivo stability (peptides) or lack of selectivity (peptoids). Herein, we have developed a process to prepare novel hybrid antibacterial agents that combine both linear peptoids (increased in vivo stability compared to peptides) and a nisin fragment (lipid II targeting domain). The hybrid nisin⁻peptoids prepared were shown to have low micromolar activity (comparable to natural nisin) against methicillin-resistant Staphylococcus aureus.

Ions Modulate Stress-Induced Nanotexture in Supported Fluid Lipid Bilayers.

Most plasma membranes comprise a large number of different molecules including lipids and proteins. In the standard fluid mosaic model, the membrane function is effected by proteins whereas lipids are largely passive and serve solely in the membrane cohesion. Here we show, using supported 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers in different saline solutions, that ions can locally induce ordering of the lipid molecules within the otherwise fluid bilayer when the latter is supported. This nanoordering exhibits a characteristic length scale of ∼20 nm, and manifests itself clearly when mechanical stress is applied to the membrane. Atomic force microscopy (AFM) measurements in aqueous solutions containing NaCl, KCl, CaCl2, and Tris buffer show that the magnitude of the effect is strongly ion-specific, with Ca2+ and Tris, respectively, promoting and reducing stress-induced nanotexturing of the membrane. The AFM results are complemented by fluorescence recovery after photobleaching experiments, which reveal an inverse correlation between the tendency for molecular nanoordering and the diffusion coefficient within the bilayer. Control AFM experiments on other lipids and at different temperatures support the hypothesis that the nanotexturing is induced by reversible, localized gel-like solidification of the membrane. These results suggest that supported fluid phospholipid bilayers are not homogenous at the nanoscale, but specific ions are able to locally alter molecular organization and mobility, and spatially modulate the membrane's properties on a length scale of ∼20 nm. To illustrate this point, AFM was used to follow the adsorption of the membrane-penetrating antimicrobial peptide Temporin L in different solutions. The results confirm that the peptides do not absorb randomly, but follow the ion-induced spatial modulation of the membrane. Our results suggest that ionic effects have a significant impact for passively modulating the local properties of biological membranes, when in contact with a support such as the cytoskeleton.

Peptoid Efficacy against Polymicrobial Biofilms Determined by Using Propidium Monoazide-Modified Quantitative PCR.

Biofilms containing Candida albicans are responsible for a wide variety of clinical infections. The protective effects of the biofilm matrix, the low metabolic activity of microorganisms within a biofilm and their high mutation rate, significantly enhance the resistance of biofilms to conventional antimicrobial treatments. Peptoids are peptide-mimics that share many features of host defence antimicrobial peptides but have increased resistance to proteases and therefore have better stability in vivo. The activity of a library of peptoids was tested against monospecies and polymicrobial bacterial/fungal biofilms. Selected peptoids showed significant bactericidal and fungicidal activity against the polymicrobial biofilms. This coupled with low cytotoxicity suggests that peptoids could offer a new option for the treatment of clinically relevant polymicrobial infections.

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A procedure for the preparation and isolation of nucleoside-5'-diphosphates.

Tris[bis(triphenylphosphoranylidene)ammonium] pyrophosphate (PPN pyrophosphate) was used in the SN2 displacements of the tosylate ion from 5'-tosylnucleosides to afford nucleoside-5'-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5'-diphosphates from crude reaction mixtures.

The role of phosphoglycans in the susceptibility of Leishmania mexicana to the temporin family of anti-microbial peptides.

Natural product antimicrobial peptides (AMPs) have been proposed as promising agents against the Leishmania species, insect vector borne protozoan parasites causing the neglected tropical disease leishmaniasis. However, recent studies have shown that the mammalian pathogenic amastigote form of L. mexicana, a causative agent of cutaneous leishmaniasis, is resistant to the amphibian-derived temporin family of AMPs when compared to the insect stage promastigote form. The mode of resistance is unknown, however the insect and mammalian stages of Leishmania possess radically different cell surface coats, with amastigotes displaying low (or zero) quantities of lipophosphoglycan (LPG) and proteophosphoglycan (PPG), macromolecules which form thick a glycocalyx in promastigotes. It has been predicted that negatively charged LPG and PPG influence the sensitivity/resistance of promastigote forms to cationic temporins. Using LPG and PPG mutant L. mexicana, and an extended range of temporins, in this study we demonstrated that whilst LPG has little role, PPG is a major factor in promastigote sensitivity to the temporin family of AMPs, possibly due to the conferred anionic charge. Therefore, the lack of PPG seen on the surface of pathogenic amastigote L. mexicana may be implicated in their resistance to these peptides.

Investigating the anti-leishmanial effects of linear peptoids.

Peptoids, a class of peptide mimetics, have emerged as promising anti-infective agents against a range of bacterial infections. Herein we present the first study of the antiparasitic and specifically the anti-leishmanial properties of linear peptoids. Peptoids were identified as having promising activity against Leishmania mexicana axenic amastigotes, a causative agent of cutaneous leishmaniasis.