Cationic ultrashort lipopeptides (USLPs) are promising antimicrobial candidates to combat multidrug-resistant bacteria. Using DICAMs, a newly synthesized family of tripeptides with net charges from −2 to +1 and a fatty amine conjugated to the C-terminus, we demonstrate that anionic and neutral zwitterionic USLPs can possess potent antimicrobial and membrane-disrupting activities against prevalent human pathogens such as Streptococcus pneumoniae and Streptococcus pyogenes. The strongest antimicrobials completely halt bacterial growth at low micromolar concentrations, reduce bacterial survival by several orders of magnitude, and may kill planktonic cells and biofilms. All of them comprise either an anionic or neutral zwitterionic peptide attached to a long fatty amine (16–18 carbon atoms) and show a preference for anionic lipid membranes enriched in phosphatidylglycerol (PG), which excludes electrostatic interactions as the main driving force for DICAM action. Hence, the hydrophobic contacts provided by the long aliphatic chains of their fatty amines are needed for DICAM’s membrane insertion, while negative-charge shielding by salt counterions would reduce electrostatic repulsions. Additionally, we show that other components of the bacterial envelope, including the capsular polysaccharide, can influence the microbicidal activity of DICAMs. Several promising candidates with good-to-tolerable therapeutic ratios are identified as potential agents against S. pneumoniae and S. pyogenes. Structural characteristics that determine the preference for a specific pathogen or decrease DICAM toxicity have also been investigated.
Bacterial resistance to the most commonly used antibiotics is a major social and economic challenge worldwide. This is largely a consequence of antibiotic overuse, either by misprescribing or overprescribing, causing pathogens to adapt and evolve to drug resistance. In this scenario, engineered USLPs have appeared as a new class of antibacterials ranked among the most promising alternatives to fight resistant pathogenic bacteria both as planktonic cells and biofilms, including ESKAPE group pathogens, and even fungi. Typically, USLPs comprise one fatty acid chain attached to the N-terminus of a short cationic peptide (2–6 amino acids long) whose C-terminus is in the amide form. Both components favor the binding and insertion of USLPs into the negatively charged bacterial membrane and determine their antimicrobial activity and mode of action. The net charge and the type and position of basic amino acids (frequently arginine or lysine) and fatty acid chain(s) are all relevant for antimicrobial activity.
The bacterial cytoplasmic membrane is crucial for bacterial survival in all cell metabolic conditions, serving as a selective permeability barrier and site for critical cellular processes. USLPs can permeate and, eventually destroy, the cell membrane of bacteria causing damages that are difficult to recover from, and additional modes of action have been found in certain cases. Compared to classical antibiotics, their fast bacteria-killing action reduces the likelihood of microbial resistance, and little evidence of resistance to USLPs has been found. In addition, their small size makes them more druggable, simplifies their synthesis, facilitates structural optimization, and may reduce the immunogenic response. As with cationic antimicrobial peptides and larger lipopeptides, limitations of USLPs as antimicrobials include systemic toxicity (disruption of lipid bilayers), degradation, and low bioavailability. These shortcomings may be circumvented by chemical modification of peptides using natural and non-natural amino acids or peptidomimetics, rational substitution of amino acids, and varying the mode of lipidation (type and number of acyl chains or the attachment mode).
Experimental screening of various components of our diverse in-house library of small compounds revealed that lipotripeptides DICAMs 1–4 displayed a marked antibacterial effect against S. pneumoniae, a human pathogen of paramount clinical relevance. Remarkably, DICAMs 1–4 bear either a net negative or a zero charge and a fatty amine chain conjugated to the C-terminal amino acid, instead of being cationic peptides lipidated through the N-terminal amino acid or the side-chain of lysine moieties. Specifically, our prototype compounds contain a C-terminal Glu residue bound to a stearyl-amine chain, a non-natural D-Pro (Pro) central amino acid, and a polar (Asn) or neutral (Ala) N-terminal residue substituted or not with a benzyloxycarbonyl (Cbz) group. So far, only one study including two negatively charged USLPs that were active on Streptococcus agalactiae has been published. Of note, anionic nonribosomally synthesized lipopeptides like daptomycin or surfactin are active against Gram-positive pathogens in a Ca 2+ and/or salt concentration-dependent way.
The potent anti-pneumococcal activity and structural novelty of our USLP prototype prompted us to investigate the antimicrobial potential of these readily synthetically-available small lipotripeptides. We herein report the solid phase synthesis (SPPS), biological evaluation and SAR studies of a library of USLPs based on the hit compounds 1–4. Specifically, our study assessed the effect of varying amino acid sequence, fatty amine length, and N-terminal protecting groups’ addition to the activity against selected pathogens, cytotoxicity and hemolysis. DICAM’s ability to permeate large unilamellar vesicles (LUVs) composed of anionic and zwitterionic lipids or disrupt (permeation and depolarization) the bacterial membrane was also investigated. Several membrane-disrupting DICAMs have been identified with high microbicidal activity against S. pneumoniae and S. pyogenes—another prominent human pathogen—and selectivity for pathogens over human cells. Remarkably, most lethal compounds contain a tripeptide that is either anionic or neutral zwitterionic and preferentially interact with and disrupt anionic lipid membranes with a high percentage of PG. This rules out electrostatic interactions as the main driving force for DICAM attachment to bacterial membranes and opens the possibility of additional modes of action besides membrane permeation and depolarization.
