High-Throughput Screening Identifies Synthetic Peptides with Antibacterial Activity against Mycobacterium abscessus and Serum Stability.

The rise in antibiotic resistance in bacteria has spawned new technological approaches for identifying novel antimicrobials with narrow specificity. Current antibiotic treatment regimens and antituberculosis drugs are not effective in treating Mycobacterium abscessus. Meanwhile, antimicrobial peptides are gaining prominence as alternative antimicrobials due to their specificity toward anionic bacterial membranes, rapid action, and limited development of resistance. To rapidly identify antimicrobial peptide candidates, our group has developed a high-density peptide microarray consisting of 125,000 random synthetic peptides screened for interaction with the mycobacterial cell surface of M. abscessus morphotypes. From the array screening, peptides positive for interaction were synthesized and their antimicrobial activity was validated. Overall, six peptides inhibited the M. abscessus smooth morphotype (IC50 = 1.7 µM for all peptides) and had reduced activity against the M. abscessus rough morphotype (IC50 range: 13-82 µM). Peptides ASU2056 and ASU2060 had minimum inhibitory concentration values of 32 and 8 µM, respectively, against the M. abscessus smooth morphotype. Additionally, ASU2060 (8 µM) was active against Escherichia coli, including multidrug-resistant E. coli clinical isolates, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. ASU2056 and ASU2060 exhibited no significant hemolytic activity at biologically relevant concentrations, further supporting these peptides as promising therapeutic candidates. Moreover, ASU2060 retained antibacterial activity after preincubation in human serum for 24 h. With antimicrobial resistance on the rise, methods such as those presented here will streamline the peptide discovery process for targeted antimicrobial peptides.

Production of high-complexity frameshift neoantigen peptide microarrays.

Parallel measurement of large numbers of antigen-antibody interactions are increasingly enabled by peptide microarray technologies. Our group has developed an in situ synthesized peptide microarray of >400 000 frameshift neoantigens using mask-based photolithographic peptide synthesis, to profile patient specific neoantigen reactive antibodies in a single assay. The system produces 208 replicate mircoarrays per wafer and is capable of producing multiple wafers per synthetic lot to routinely synthesize over 300 million peptides simultaneously. In this report, we demonstrate the feasibility of the system for detecting peripheral-blood antibody binding to frameshift neoantigens across multiple synthetic lots.

Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

A Simple Platform for the Rapid Development of Antimicrobials.

Recent infectious outbreaks highlight the need for platform technologies that can be quickly deployed to develop therapeutics needed to contain the outbreak. We present a simple concept for rapid development of new antimicrobials. The goal was to produce in as little as one week thousands of doses of an intervention for a new pathogen. We tested the feasibility of a system based on antimicrobial synbodies. The system involves creating an array of 100 peptides that have been selected for broad capability to bind and/or kill viruses and bacteria. The peptides are pre-screened for low cell toxicity prior to large scale synthesis. Any pathogen is then assayed on the chip to find peptides that bind or kill it. Peptides are combined in pairs as synbodies and further screened for activity and toxicity. The lead synbody can be quickly produced in large scale, with completion of the entire process in one week.

Synthetic Antibacterial Peptide Exhibits Synergy with Oxacillin against MRSA.

One proposed solution to the crisis of antimicrobial resistant (AMR) infections is the development of molecules that potentiate the activity of antibiotics for AMR bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Rather than develop broad spectrum compounds, we developed a peptide that could potentiate the activity of a narrow spectrum antibiotic, oxacillin. In this way, the combination treatment could narrowly target the resistant pathogen and limit impact on host flora. We developed a peptide, ASU014, composed of a S. aureus binding peptide and a S. aureus inhibitory peptide conjugated to a branched peptide scaffold, which has modest activity against S. aureus but exhibits synergy with oxacillin for MRSA both in vitro and in a MRSA skin infection model. The low concentration of ASU014 and sub-MIC concentration of oxacillin necessary for activity suggest that this molecule is a candidate for future medicinal chemistry optimization.

Cross-Reactive Synbody Affinity Ligands for Capturing Diverse Noroviruses.

Noroviruses are the most common cause of acute gastroenteritis in the developed world. Noroviruses are a diverse group of nonenveloped RNA viruses that are continuously evolving. This leads to the rise of immunologically distinct strains of the same genotype on a frequent basis. This diversity presents a unique challenge for detection and tracking of new strains, with the continuous need for new norovirus affinity ligands. Our group developed a family of bivalent synbody affinity ligands using a virus-like particle (VLP) from the 2006 GII.4 Minerva strain of norovirus. We produced more than 20 synbodies with low nanomolar dissociation constants (KD < 10 nM) for GII.4 VLP. We measured binding affinity for four synbodies against VLPs from multiple GI and GII genotypes and found that the synbodies were broadly cross-reactive with affinities that ranged from 0.5 to 8 nM. We tested the ability of these synbodies to capture norovirus from dilute solutions and found that one synbody could capture GII.4 from a 200 000-fold dilution from a norovirus positive stool sample. When these synbodies were tested for the ability to capture of multiple genotypes, we found that four different genotypes were recognized. These data demonstrate that the synbody approach can generate multiple affinity ligands for future use in norovirus detection and possible therapeutic development.

Conjugation Approach To Produce a Staphylococcus aureus Synbody with Activity in Serum.

Synbodies show promise as a new class of synthetic antibiotics. Here, we explore improvements in their activity and production through conjugation chemistry. Maleimide conjugation is a widely used conjugation strategy due to its high yield, selectivity, and low cost. We used this strategy to conjugate two antibacterial peptides to produce a bivalent antibacterial peptide, called a synbody that has bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA). The synbody was prepared by conjugation of a partially d-amino acid substituted synthetic antibacterial peptide to a bis-maleimide scaffold. The synbody slowly degrades in serum, but also undergoes exchange reactions with other serum proteins, such as albumin. Therefore, we hydrolyzed the thiosuccinimide ring using a mild hydrolysis protocol to produce a new synbody with similar bactericidal activity. The synbody was now resistant to exchange reactions and maintained bactericidal activity in serum for 2 h. This work demonstrates that low-cost maleimide coupling can be used to produce antibacterial peptide conjugates with activity in serum.