Development of Broad-Spectrum Cyclic Amphiphilic Peptides against Multidrug-Resistant Bacteria

ABSTRACT

The emergence of antibacterial resistance to common frontline antibiotics, such as methicillin, vancomycin, cephalosporins,

and carbapenem, have created a global public health challenge for millions of patients. It is therefore critical to discover and

commercialize new antimicrobial agents that can successfully neutralize multidrug-resistant bacteria (MDRB) with minimal

toxicity. The objective of this proposal is to develop unique first-in-class amphiphilic cyclic antimicrobial peptides (AMPs) that are active against clinically relevant pathogens like Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE pathogens). We

propose to develop AMPs containing natural and/or unnatural hydrophobic and positively charged residues for their broad-

spectrum activity and efficacy against specific MDR pathogens, using in vitro and in vivo assays. We have discovered that a cyclic amphipathic peptide [R4W4], which comprises tryptophan (W) and arginine (R) amino acids was effective against diverse bacterial pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) (MIC = 2.7 µg/mL),

Pseudomonas aeruginosa (MIC = 42.8 µg/mL), Klebsiella pneumoniae (MIC = 16.0 µg/mL), and Escherichia coli (MIC = 16.0 µg/mL) and showed synergistic activity with tetracycline against MRSA, and isoniazid and pyrazinamide against Mycobacterium tuberculosis. Based on this template, we generated a new library of peptides (>200) with enhanced

antimicrobial activities. For example, IFX-027, IFX-135, IFX-145, IFX-146, IFX-154, and IFX-301 showed MIC = 1.5-25 µg/mL against Gram+ve and Gram-ve bacteria. Several of the lead compounds demonstrated synergistic activity with

several other antibiotics with fractional inhibitory concentration (FIC) indices ranging from 0.3-0.5. Our lead peptides (IFX- 031, IFX-031-1, and IFX-111) also reduced biofilm formation by MRSA and P. aeruginosa. IFX-301 was found to be nontoxic at a dose level of 50 mg/kg in mice, and all peptides were not toxic against human red blood cells (hRBC)

(HC50>500 μg/mL). In Aim 1, we will establish a structure-activity relationship (SAR) based on the six lead peptides to

obtain insights into the structural determinants responsible for the molecules’ selectivity towards bacterial pathogens. The

most potent compounds will be further evaluated for their stability, cytotoxicity, and development over time to antimicrobial

resistance. The proposed milestones for Aim 1 are to identify five lead peptide analogs with MIC ≤5 µg/mL and MIC ≤10 µg/mL respectively against Gram+ve and Gram-ve bacteria, and hRBC hemolysis of ≤5% at a concentration of 20 times

the MIC value. In Aim 2, we will evaluate the in vivo efficacy and toxicity, preliminary pharmacokinetics (e.g., Cmax, tmax,

t1/2), and efficacy of the 2-3 lead antimicrobial peptide analogs identified in Aim 1 on a murine infection model against four

pathogenic bacteria. At the successful completion of Phase I, the most potent compound with a large therapeutic index will be advanced to Phase II studies and be the focus for an IND application.