Synthetic Antimicrobial Peptoids: Next Generation Anti-Infectives

Posted on June 11th, 2019

Maxwell Biosciences is developing a new class of potent, safe and biostable synthetic peptide-like small molecules currently in toxicology testing to treat common infections in humans.

Antimicrobial Peptoid

Maxwell’s new drug class – Synthetic antimicrobial peptoids (SAMPs) – are short, sequence- and length-specific oligopeptides: they are based on a oligoglycine backbone (similar to naturally occurring peptides), yet with a difference in their molecular structure side chains are appended to backbone nitrogens. Due to the stronger nitrogen bond, this novel peptoid (meaning “peptide-like”) synthetic antimicrobials overcome bacterial protease degradation, evolution of bacterial resistance and have been shown by multiple labs to be efficacious at low doses. Maxwell’s lead antimicrobial peptoids are sequence- and chain-length specific, 6- to 13mer oligo-N-substituted glycines, designed to serve as structural, functional, and mechanistic mimics of natural antimicrobial peptides used by the human immune system to defend against all kinds of pathogens. This antimicrobial peptoid class is protected through a granted patent assigned to Maxwell Biosciences by the US Department of Energy and US National Institutes of Health.

We have published the design, characterization, antimicrobial activity, and biomimetic mechanisms of synthetic antimicrobial peptoids in major peer-reviewed journals. Synthetic antimicrobial peptoids are synthesized at low cost on a robotic synthesizer and can be scaled up easily, with facile access to high chemical diversity. In their ease of synthesis, peptoids are unique among peptide mimetics. To identify our lead candidates (6mer-13mers), 70 different short synthetic antimicrobial peptoids were synthesized, purified, and tested against viruses, 47 different bacterial microorganisms, including wild-type and drug-resistant variants. Many of our antimicrobial peptoid drug candidates are as potent as highly potent, well-known antimicrobial drugs currently approved by the FDA (0.4-6.5 µM minimum inhibitory concentrations, MICs). The minimum inhibitory concentration is the smallest amount of a drug necessary to prevent visible growth of the pathogen. 

Our published SAMP (Peptoid 1) and a comparator antimicrobial peptide, Pexiganan, are both effective against multi-drug resistant P. aeruginosa (PA) strains that are resistant to fluoroquinolone, ciprofloxacin, carbapenem, and imipenem. Peptoid 1 has been tested and is potently active against the following PA strains: ATCC 27853 (MIC 5.5 µM), H103 (MIC 1.1 µM), PA14 (MIC 12.5 µM), #9 (MIC 8.8 µM), #198 (MIC 2.2 µM), #213 (MIC 2.2 µM), LES400 (MIC 1.1 µM), H1027 (MIC 1.1 µM), and H1030 (MIC 2.2 µM). Among these PA strains, PA14, #9, #198, and #213 are resistant to piperacillin / tazobactam, meropenem, ceftazidime, ciprofloxacin and cefepime, while #9 is also polymyxin B resistant. PA strains LES400, H1027, and H1030 are the Liverpool Epidemic Strain (LES), and LES400 is resistant to gentamicin and tobramycin, while H1030 is resistant to colistin, amikacin, gentamicin and tobramycin. Our lead SAMP is potently active against these PA strains.

Our investigations of mechanisms of action for our lead SAMP, Peptoid 1, demonstrated that SAMPs kill bacteria primarily by passing through bacterial membranes and acting on intracellular targets, by binding irreversibly to free-floating DNA, RNA, and ribosomes, and causing rapid flocculation of bacterial intracellular biomass—within intact bacteria. By scanning electron microscopy, treated bacteria look shriveled, while remaining intact. Higher resolution methods including transmission electron microscopy and soft X-ray tomography of treated bacteria show that bacterial ribosomes and DNA form a dense, compact mass inside dead bacteria. Bacteria are killed in under 10 minutes, and do not recover.

SAMPs offer good selectivity for bacteria over mammalian cells (selectivity ratios of 8-35, defined as SAMP concentration lethal to 10% of mammalian cells, LD10, divided by MIC). We evaluated cytotoxicity against A549 lung cells, human red blood cells, and murine J774A.1 macrophages. The SAMPs’ selectivity originates from the greater attraction to negatively charged bacterial surfaces than to zwitterionic mammalian cell membranes. SAMP mechanism of action is effective in the bacterial cytosol (bacteria are a “bag of ribosomes”) and have less toxicity in the mammalian cytosol, with its mitochondria, endoplasmic reticulum, and Golgi apparatus structures, which all scaffold, spool, organize and segregate ribosomes and DNA so as to prevent SAMPs from exerting flocculation mechanisms of action. Current studies are focused on maintaining compound potency while increasing selectivity via novel modifications of SAMPs. We have filed new intellectual property on these enhancements.

The biomimetic mechanism of action of SAMPs was shown using a wide range of biophysical tools, including studies of intact bacteria, as well as scanning electron microscopy, transmission electron microscopy, and soft X-ray tomography of untreated vs. treated bacteria. We used super-resolution fluorescence microscopy to show that our lead SAMP causes rapid-onset solidification of the internal contents of bacterial cells; this mechanism is identical to that of the human AMP, the cathelicidin LL-37. Due to the mechanism of action, the likelihood of bacterial resistance emerging to AMPs may be less than that of conventional antibiotics, which have more specific molecular targets. Additionally, bacteria have not developed significant resistance to natural host defense peptides after over 500 million years of evolution.

SAMP antimicrobial activity does not diminish at high bacterial loads compared to AMPs. We conducted a simple assay in E. coli to compare SAMP activity to an AMP at varying concentrations of inoculum. As bacterial load (CFU/mL) increases, the MIC of Peptoid 1 remains relatively unchanged, while MIC values noticeably increase for the optimized AMP pexiganan. We hypothesize that proteases released from dying bacteria may reduce the bioavailability of AMPs but do not affect SAMPs, which have a N-substituted chemical backbone.

Some AMPs, such as the pleiotropic human host defense peptide LL-37, are active against biofilms at concentrations less than or equal to the minimal inhibitory concentration (MIC) determined for planktonic bacteria. Our SAMP Peptoid 1 has been shown to prevent the formation of PA biofilms, kill existing PA biofilms, and detach existing biofilms.

Delivered intraperitoneally, SAMP Peptoid 1 is effective in a Staph. aureus infection mouse model, showing a 2-log order reduction in bacterial counts compared to saline controls, and was well tolerated. Peptoid 1 was tested for efficacy in an in vivo model of chronic suppurative otitis media, and after treatment in a single mouse, no PAO1 bacteria were cultured, suggesting complete eradication. A recent pilot study, showed that Peptoid 1 is non-toxic to aural neurons (non-ototoxic) even at 10X the compound’s minimum inhibitory concentration (MIC) after single administration. Given that the neurons in the middle ear are exquisitely sensitive to antimicrobial compound toxicity, we consider this to be an excellent indication that Peptoid 1 may be well tolerated in the ear, nose, throat, and lung of human patients.