Antibiotic resistance is increasing at an alarming rate, especially for the drug resistant form of Mycobacterium tuberculosis which killed an estimated 170,000 people in 2012 according to the U.S. Centers for Disease Control and Prevention. Alternatives to traditional antibiotics are urgently needed to combat these resistant bacteria. Disrupting bacterial, but not mammalian, outer membrane integrity with peptides is one such strategy to destroy pathogenic bacteria in a highly selective manner (Zasloff, Nature, 415:389-395 (2002)). Design strategies to develop potent, stable antimicrobial peptides (“AMPs”) are urgently needed.
AMPs are typically short, cationic peptides that usually adopt an alpha helical conformation. Upon discovery of naturally-occurring AMPs, many were tested for activity against M. tuberculosis including human and rabbit defensins and porcine protegrins. The most potent of these displayed >90% killing of M. tuberculosis at 50 μg/mL and acted by a mechanism which produced visible lesions on the mycobacterial outer membrane (Miyakawa, Ratnakar, et al., Infect. Immun., 64(3):926-932 (1996)). Subsequently, several of the broadly active natural peptides were modified and tested against M. tuberculosis with minimum inhibitory concentrations (“MICs”) as low as 10 μM (Linde, Hoffner, et al., J. Antimicrob. Chemother., 47(5):575-580 (2001); Sonawane, Santos, et al., Cell. Microbiol., 13(10):1601-1617 (2011)). Large, entirely synthetic libraries were also tested against M. tuberculosis with MICs reported as low as 1 μM (Ramon-Garcia, Mikut, et al., Antimicrob. Agents Chemother., 57(5):2295-2303 (2013)). In addition, peptoids, which are more resistant to degradation than peptides, were developed with MIC values as low as 6 μM (Kapoor, Eimerman, et al., Antimicrob. Agents Chemother., 55(6):3058-3062 (2011)).
Despite clear evidence of their efficacy, the mechanism of action of AMPs remains debated, though it is believed that the majority of AMPs act through disruption of microbial membranes. Recently, many insights have been gained into the motifs that govern the effectiveness of short alpha helical AMPs. The three main parameters that guide effectiveness are peptide hydrophobicity, peptide charge and the distribution of charged and hydrophobic residues. Activity is dependent on a mixture of hydrophobic and cationic residues, arranged to form an amphipathic peptide (Zelezetsky and Tossi, Biochim. Biophys. Acta, 1758(9):1436-1449 (2006)). It has been proposed that the cationic portion targets the peptide to the negatively charged bacterial membrane, while the hydrophobic portion allows for intercalation into the membrane and subsequent disruption of the membrane via a number of proposed mechanisms (Yeaman and Yount, Pharmacol. Rev., 55(1):27-55 (2003); Wimley, ACS Chem. Biol., 5(10):905-917 (2010)). This amphipathic character lends itself to design due to the periodicity of the alpha helical arrangement. Peptides can be visualized in two dimensions using helical wheel diagrams (Tossi, Sandri, et al., Biopolymers, 55(1):4-30 (2000)) and sequences bearing separate cationic and hydrophobic faces can be designed (Tossi, Tarantino, et al., Eur. J. Biochem., 250(2):549-558 (1997)).
The majority of prior studies have focused on either optimizing naturally-occurring peptides or screening large random synthetic libraries to develop potential drug candidates against a specific microbial target or investigating the general mechanism of action. We have developed a novel method for designing a novel peptide that uses bioinformatics and rational design informed by known mechanistic rules, to develop a set of more potent initial peptides than those found in nature while avoiding the need to screen large randomly constructed libraries. Specifically, we have combined a de novo design approach called Database Filtering with protein engineering, rational design, and three dimensional (“3-D”) modeling to design potent AMPs against a selected microbial target. Database Filtering uses a library of peptides with reported activity against the bacterium of choice to determine a characteristic peptide length, overall charge and hydrophobicity, and commonly occurring residues, resulting in a set of amino acids (WO 2013/078217; Mishra and Wang, J. Am. Chem. Soc., 134(30):12426-12429 (2012)). Our method then employs rational design including the use of helical wheel diagrams to arrange the set of amino acids in a way that maximizes the amphipathic nature of the peptide. 3-D modeling is then employed to verify an alpha-helical conformation and proper distribution of amino acids to generate the amphipathic surface.
We've successfully used our novel method to design novel AMPs which demonstrated high potency against M. tuberculosis and other microbes, such as streptococcus bacteria.