AMPs in general do not have specific targets (unlike traditional antibiotics) as their mode of action involves nonspecific interactions with the cytoplasmic membrane of bacteria, essentially “punching holes” in the membrane and killing the organism. Development of resistance is not expected since this would require substantial changes in the lipid composition of bacterial membranes. The issue has been that naturally occurring, AMPs also disrupt eukaryotic membranes, hence their toxicity. Researchers have generally attempted to manipulate the structure of naturally-occurring AMPs to maximize antimicrobial activity and minimize toxicity, generally assessed by hemolysis of human red blood cells; this clearly has not been a successful approach to date towards agents for systemic administration. In addition, researchers have also attempted to maintain the broad-spectrum antimicrobial activity of AMPs, i.e., a one-size fits all approach, which, although theoretically useful, has likely inhibited development of therapeutic AMPs. In contrast to other researchers, our approach has been one of synthetic peptide de novo design1-11, utilizing a structural framework, or template, encompassing factors known to be important for antimicrobial activity: the presence of preformed or inducible secondary structure (α-helix, in our case), with hydrophobic amino acid residues making up the non-polar face of the helix and basic (positively charged) residues making up the polar face. This 26-residue template would then be able to accept amino acid substitutions with minimal effects on peptide properties and stability other than the ones under investigation. We also intended, not only to manipulate this template to eliminate toxicity, but also to select for anti-Gram-negative organism activity, such organisms representing the most serious antibiotic resistance challenges. We hypothesized that such a de novo synthetic peptide approach to examining incremental changes in hydrophilicity/hydrophobicity, amphipathicity and helicity of AMPs would enable rapid progress in the rational design of novel peptide therapeutics.5
Panel A represents four major milestones for our de novo design approach: (1) our discovery of “specificity determinants, the substitution of two positively charged Lys residues into the center of the non-polar face of the synthetic AMP prevents transmembrane penetration and toxicity to normal cells, thereby dramatically decreasing hemolytic activity, selecting for Gram-negative organisms and removing high affinity binding to serum proteins4-7; and (2) the use of D-amino acids, as opposed to naturally-occurring L-isomers, also makes the peptides resistance to enzymatic degradation, a key concern when considering drug half-life in a patient; (3) substitution of unusual positively charged amino acids diaminobutyric acid (Dab) and diaminopropionic acid (Dap) on the polar face, in conjunction with our specificity determinants on the non-polar face, eliminates toxicity as exemplified by hemolysis under very stringent conditions, whilst maintaining excellent antimicrobial activity against antibiotic-resistant organisms2. These results are unprecedented in AMP research; (4) as shown in Figure 1, Panel B nature’s AMPs have only access to the positively charged residues, Arg and Lys. It is clear that shortening the side-chain of Lys on the polar face (4 carbon atoms in its sidechain to two or one carbon atoms in Dab and Dap residues, respectively) dramatically lowers lysis of human red blood cells (never before reported in AMP research). Further, of profound importance to the future of peptide-based therapeutic agents, we have demonstrated that modification of native AMPs Piscidin 1 and Dermaseptin S4 with specificity determinants produced similar results of improved antimicrobial activity and dramatically decreased hemolytic activity5.
We have also determined the effect of changing locations of positively charged residues on the polar face of our AMPs as well as eliminated a single positively charged residue from the C-terminal which allows future development of Pegylated AMPs on a C-terminal cysteine residue if prolonged half-life is necessary1. When the positively charged residues are at positions 3, 7, 11, 18, 22, and 26 on the polar face we have denoted this orientation as -1 at the end of the peptide name. When the positively charged residues are at positions 3, 7, 14, 15, 22, and 26 on the polar face we have denoted this orientation as -2 at the end of the peptide name. These results are shown in Figure 2. Interestingly, the location of positively charged residues on the polar face had a major effect on hemolysis and the best location was dependent on whether Dab or Dap residues were used, i.e., side-chain length, number of carbon atoms and residue location all appear to affect hemolysis. The two best peptides are D89(6Dab-2) and D105(6Dap-1). We are now poised to continue this promising approach further with a view of identifying promising lead compounds for eventual clinical studies, and formulation development. We strongly believe our work to be a key player in the challenge of overcoming global bacterial resistance. In addition, we have no competition at the present time.
Our results strongly suggest that our novel compounds will replace existing antibiotics and have the potential to solve the world-wide crisis. We have filed two critical patent applications on our technology. The international PCT patent application was filed in April 2018 with a follow-up US provisional patent application in January 18, 2019 with the PCT application due January 17, 2020. Though we already have potential lead compounds, the follow-up research is critical to ensure the broadest patent coverage surrounding our existing lead compounds. Milestone 1 – synthesis, purification and analysis on some 31 peptides. These peptides will be tested for antimicrobial activity against 7 Acinetobacter baumannii strains resistant to antibiotics of last resort (colistin and polymyxin B) and hemolytic activity against human red blood cells. Milestone 2 – to address the question of whether pegylation of our lead AMPs, using a C-terminal cysteine residue will maintain antimicrobial activity and negligible hemolytic activity. This methodology would be a major asset to extending half-life of our AMPs. Milestone 3 – to screen our six best compounds for cytotoxicity against a series of 4 primary human cell lines (primary human hepatocytes, primary cardiomyocytes, primary renal proximal tubule cells and human endothelial cells). Milestone 4 – formulation and stability studies. Milestone 5 – non-GLP screening toxicity and pharmacokinetic/toxicokinetic (PK/TK) studies in rats of 4 potential compounds for selecting a lead compound. Milestone 6 – to screen our best 4 potential lead compounds for in vivo efficacy in a rat model of infection with Acinetobacter baumannii blood strain 649 (IP infection with bacteria and IV injection of our peptides). This protocol was developed in our laboratory. We already have estimates for the GMP manufacture of one of our lead peptides at 50g quantity from 3 leading GMP peptide manufacturers in the U.S. We intend to advance the commercialization process towards completing a Phase I safety trial in humans with GMP-manufactured material.