Introduction to the field

Interest in amphiphilic molecules.

A major problem facing medical science is the increasing occurrence of resistance to antimicrobial and anticancer agents, which has led to the need for new lead compounds in strategies of drug design.  Such agents need to be able to target specific cells and disrupt their function hence efficacy can be reduced by either poor selectivity or poor activity at the target site.

A key feature of interest is the membrane surrounding the cell since this maybe the target site for any new agent or if not it will need to be traversed for the agent to reach the cell interior. These membranes form a phospholipid barrier around the cell and exhibit a hydrophobic interior and hydrophilic exterior. The function of many biologically active molecules is therefore dependent upon their  own amphiphilic structure either because the membrane is the site of action or because it is their physicochemical characteristics that will determine their translocation across the bilayer and their subsequent compartmentalization within the cell. Currently, the main focus for our group’s interest is therefore to investigate the structure function relationships underpinning the membrane interactions of known biologically active molecules and the role of amphiphilicity in the activity of such biomolecules.

Work is currently focused in two areas. The first is investigation into the properties of membrane interactive peptides such as antimicrobial peptides – a number of which have also been seen to exhibit anticancer properties. The second involves the creation of synthetic amphiphilic molecules which may have potential in the targeting and destruction of bacterial cells or tumour cells. Investigations of this kind can not only give insight into the functionality of the molecules themselves but as we gain understanding of how they cause lysis we can apply this knowledge to aid our understanding of a wide range of key biological processes that require ongoing membrane fusion and lysis events.

1. Antimicrobial peptides

Antimicrobial peptides (AMPs) were first reported in the late 1980s and are effectors of innate immunity that generally exert antimicrobial activity through permeabilising the membranes of target organisms, including those with multiple drug resistance. Moreover, there is currently little evidence of microbial resistance to α-AMPs possibly as a consequence of the relatively non-specific mechanisms used in their antimicrobial action.  For all known α-AMPs, these mechanisms of action are believed to involve membrane invasion although in some cases this appears to lead to membrane translocation and attack upon intracellular targets. In general, the ability of α-AMPs to invade microbial membranes primarily depends upon their positive charge, which allows them to target and bind anionic components of these membranes, and their amphiphilicity, which facilitates partitioning into these membranes although an increasing number of negatively charged AMPs are being identified.

bio2

Fig. 1. The final configuration of aurein 2.3 showing insertion of the peptide into the DMPC bilayer is due to phenylalanine.

Many amphiphilic α-AMPs are surface active and interact with the bilayer such that their orientation is approximately parallel to the membrane surface albeit, sometimes, as an initial step leading to further membrane interactions or cell internalisation.  This orientation enables the polar face of the helices to interact with the bilayer head group region whilst their polar face penetrates the hydrophobic membrane core [1].  It has been shown that many AMPs adopt an oblique orientated α-helical structure [2-4], where the peptide penetrates the membrane at an angle between 30o and 60o.  Furthermore, this oblique orientated α-helical structure facilitates destabilisation of lipid packing for example, by causing hydrophobic mismatch where the hydrophobic stretches of a peptide/protein do not match the thickness of the hydrophobic core of a bilayer.  Most recently, biophysical studies have shown that the amphibian α-helical antimicrobial peptides (α-AMPs) aurein 2.3 destabilise membranes via angled bilayer penetration [5].  Although the lysine residues of aurein 2.3 also help to the stabilize helical they are also able to snorkel allowing deeper penetration into the membrane hydrophobic core.  Molecular dynamics has shown that other amio acid residues such as phenylalanine plays a crucial role in the membrane perturbation as shown by Figure 1, where the phenylalanine penetrates into the membrane as expected with hydrophobic mismatch.

Furthermore, Dennison et al., [6] showed that individually mutating the phenylalanine residues of aurein 2.5 to leucine had no major effect on the levels of phosphatidylglycerol and phosphatidylethanolamine interactions, suggesting that these residues are not essential to the membrane interactions of the peptide, contrasting to other aureins where corresponding phenylalanine residues are required for efficient membrane interaction and antibacterial activity. This difference in the requirement is suggested to relate to the surface architecture as proposed by the concept of the molecular perturbation potential (Figure 2).  Hence the peptide’s overall architecture is key to its ability to disrupt bilayers [7]. It has previously been shown that [6] the initial stages of membrane association is driven by amphiphilicity and is dependent on hydrophobic grooves on the helix surface.

peptides

Fig. 2. Potential surfaces of peptide aurein 1.2 (A), aurein 2.1 (B), aurein 2.2 (C) and Aurein 2.5 (D). Hydrophobic grooves are boarded by the positively charged amino acid residues. The juxtaposition of the cationic residues are thought to be MPP Blue indicates basic residues, red, acidic residues and white hydrophobic and neutral residues.

Some naturally occurring AMPs carry a C-terminal amide moiety which has been shown to be required for antimicrobial activity.  These peptides are attractive for development as novel antimicrobial agents and, in this capacity; attempts to optimize their lytic activity and understand their mechanism of interaction are of great importance. In order to gain an insight into the effects of amidation on the mechanism of membrane interaction, Dennison and Phoenix [8, 9] investigated the role of amidation on the synthetic AMP, Modelin-5.  Although both peptides are able to initially bind to bilayer structures amidation was key to helix stabilisation and hence overcome a key rate limiting step generating higher local concentrations of peptide at the bilayer interface, which in turn would be predicted to increase efficacy.

2. Foldemers and Synthetic compounds

The group has been investigating a range of membrane interactive molecules which are expected to adopt structures at a lipid interface that could be disruptive and hence which might provide lead compounds for the design of antimicrobial agents [10].  For example trans-benzanilide 1 and cis-benzanilide were shown to be membrane interactive although the cis-benzanilide had greater potential for development ofnew lead compounds.

Since a significant number of AMPs use α-helical structures in their mechanism of membrane interactions synthetic oligourea molecules have been used to create a helical scaffold in investigate the helical relevance in the mechanism of AMP action.  Based on research surrounding the antimicrobial activity of naturally occurring helical oligopeptides, [11-14], new synthetic oligoureas such as m-phenylenediamine are being developed in our laboratory to investigate whether they are able mimic the membrane interaction and antimicrobial activity of naturally occurring helical oligopeptides in order to seek new lead compounds.

 

References

 

1.         Papo, N. and Y. Shai, Host defense peptides as new weapons in cancer treatment. Cell Mol Life Sci, 2005. 62(7-8): p. 784-90.

2.         Dennison, S.R., F. Harris, and D.A. Phoenix, Are oblique orientated alpha-helices used by antimicrobial peptides for membrane invasion? Protein Pept Lett, 2005. 12(1): p. 27-9.

3.         Dennison, S.R., F. Harris, and D.A. Phoenix, The interactions of aurein 1.2 with cancer cell membranes. Biophys Chem, 2007. 127(1-2): p. 78-83.

4.         Marcotte, I., et al., Interaction of antimicrobial peptides from Australian amphibians with lipid membranes. Chem Phys Lipids, 2003. 122(1-2): p. 107-20.

5.         Mura, M., et al., Aurein 2.3 functionality is supported by oblique orientated alpha-helical formation. Biochim Biophys Acta, 2012.

6.         Dennison, S.R., F. Harris, and D.A. Phoenix, A study on the importance of phenylalanine for aurein functionality. Protein Pept Lett, 2009. 16(12): p. 1455-8.

7.         Wang, G., Y. Li, and X. Li, Correlation of three-dimensional structures with the antibacterial activity of a group of peptides designed based on a nontoxic bacterial membrane anchor. J Biol Chem, 2005. 280(7): p. 5803-11.

8.         Dennison, S.R. and D.A. Phoenix, Effect of cholesterol on the membrane interaction of Modelin-5 isoforms. Biochemistry, 2011. 50(50): p. 10898-909.

9.         Dennison, S.R. and D.A. Phoenix, Influence of C-terminal amidation on the efficacy of modelin-5. Biochemistry, 2011. 50(9): p. 1514-23.

10.       Dennison, S.R., T.J. Snape, and D.A. Phoenix, Thermodynamic interactions of a cis and trans benzanilide with Escherichia coli bacterial membranes. Eur Biophys J, 2012. 41(8): p. 687-93.

11.       Dennison, S.R., et al., Amphiphilic alpha-helical antimicrobial peptides and their structure/function relationships. Protein and Peptide Letters, 2005. 12(1): p. 31-39.

12.       Harris, F., S.R. Dennison, and D.A. Phoenix, Anionic Antimicrobial Peptides from Eukaryotic Organisms. Current Protein & Peptide Science, 2009. 10(6): p. 585-606.

13.       Huang, Y., J. Huang, and Y. Chen, Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein & cell, 2010. 1(2): p. 143-52.

14.       Conlon, J.M., J. Kolodziejek, and N. Nowotny, Antimicrobial peptides from the skins of North American frogs. Biochimica Et Biophysica Acta-Biomembranes, 2009. 1788(8): p. 1556-1563.

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