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

Abstract

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.

A Simple Platform for the Rapid Development of Antimicrobials

“…Seven to 10 peptides are selected and the 100 peptide stock used to synthesize all combinations of the peptides into synbodies. We developed a modular and combinatorial approach using maleimide-thiol conjugation to rapidly produce synbody libraries [19][20][21] where selected peptides are conjugated to different peptide scaffolds, each with two conjugation sites, in a combinatorial manner and in parallel. With this approach, dozens or even hundreds of synbodies can be produced in a day.…”

“…We had previously demonstrated that synbodies could be developed with antibiotic activity 18 and shown the value of using D amino acid-containing peptides 19,20 . The challenge we addressed here was whether the process could be done very quickly.…”

“…Based on HAI screening, peptides p227, p149, and p125 were used to prepare synbodies through pairwise conjugation to a previously developed synbody scaffold, Sc2 20 . Each synbody was hydrolyzed to prevent thiol-exchange reactions that occur when maleimide conjugated peptides are exposed to excess thiols, such as in albumin 19 . Synbodies were tested in the HAI assay and p227-p227, p125-p149, p125-p125, and p149-p149 each exhibited inhibition at 0.781 µM (Fig.…”

“…These peptides were tested by MIC and they did not inhibit S. epidermidis growth at 50 uM (not shown). Therefore, we selected two positives binders (p52 and p104) and one negative control peptide (p42) and conjugated to an S. aureus lytic peptide 19 , called Ly, to produce a small synbody library that was then tested for inhibitory activity. We found that synbodies p42-Ly did not inhibit S. epidermidis growth at 50 uM, as expected, while p107-Ly, and p104-Ly inhibited bacterial growth with MICs of 12.5 µM, and 6.25 µM, respectively (Fig.…”

“…We used the synbody technology [14][15][16][17][18][19][20][21] as the starting point for developing a platform to meet these specifications. Synbodies are bivalent peptides with antibody like features that are chemically synthesized.…”