Abstract
Context: Learning from biology, the focus for this work is the design of versatile and novel antibiotics, based around natural antimicrobial-active peptides, with significant potential for intelligent design and delivery - this will address a major health-care problem, even in developed countries, of fighting infection. Some bacterial infections are newly discovered with limited means available to control them (MRSA, C. diff, S. bureau), especially in the aged and in those with compromised immune systems, and other more established bacterial infections have developed resistance due to over (self in some countries) prescription of known antibiotics. Some highly effective natural antimicrobial peptides (AMPs) are known, notably from amphibian epidermis (frog skin), and understanding the mechanism of their action can help significantly in the design of new AMPs. Added to this, natural proteins (notably from viruses) are capable of penetrating the outer membrane of cells, effectively delivering their cargo into a new host. Building on these highly developed systems through evolution, we will use a bottom-up approach to design new AMPs using both natural (21 are available) and unnatural (unlimited diversity) amino acids.Solid state NMR (Oxford) will be used to give high resolution (sub-Å) distance constraints and help define peptide secondary structure (helices, beta-sheets), information about folding and stability, details of molecularly specific interactions of peptides with lipids, and membrane perturbation. Molecular dynamics (Edinburgh) will aid in initial peptide design, and then rationalization of input experimental data which will also come FTIR and CD studies (NPL) and sample morphology coming from TEM. Sample optimization for various experimental methods will be between Oxford and NPL, using significant cumulative experience from both labs. This proposal therefore brings together three well-established research teams with highly complementary expertise to focus on a major health-care problem at the fundamental and molecular sciences level.Aims and Objectives: The systems of choice, initially, are known AMPs with essential elements of membrane association and disruption, namely peptides in the maganin family. Sequence information will be used to design new homologues of maganins, but with rationally inserted or changed amino acids to change function. Coupled to this will be studies of a small protein, gp41, derived from the HIV-1 virus with membrane active properties, namely membrane perturbing and hence potential for cell penetration and/or uptake.The final goal is to gain a fundamental understanding of the design principles required for new potential antibiotics which can be followed through to clinical trials and market.Potential applications and benefits: This NPL/EPSRC application has two key components, firstly bringing to NPL access to new state-of-the-art high resolution (sub-Å) distance measurement methodology, with one of the world's highest field and specialized solid state NMR instruments (at Oxford), and secondly joining a new NPL lead international consortium on "Length-scale Bridging Measurements in Biophysical Systems", with strong future business opportunities and cutting-edge research. The potential applications are through the production of newly designed AMPs which could fine use in combating bacterial resistance, and give principles on which resistance can be addressed and overcome, either through flexible design or through generalized properties which avoid resistance. The benefits of the research are clearly varied, from academic interest of membrane-protein interactions in all its multitude of situations, through to therapeutic use for the patient. Clearly new avenues and intellectual input is required if we are to understand the mechanisms and devise new ways to combat microbial resistance, and the approaches suggested here offer such opportunities, with potential obvious benefit.
Planned Impact
Beneficiaries from this research include academe, potentially business and/or industry, and in the longer term, the public.Curiosity-driven research into the mechanistic details of molecular recognition by membranes of surface active peptides has a very long history. Biophysical and spectroscopic studies reveal that lipid recognition sites in peptides are now recognised as important. This binding is tightly regulated, often involving phosphatidyl inositides, and examples include pleckstrin homology and C2 domains, which are among the largest domain families in the proteome (Lemon, 2008, Nat. Rev. Mol. Cell Bio., 9, 99-111). Direct structural data and subcellular localisation studies have given new insights into control of binding, and resultant trafficking in regulated in cells.As a potential route to HIV-1 therapy, it has been reported that peptides, hydrophobic drugs and specific monoclonal antibodies (2F5, 4E10) recognise gp41 ectodomain pre-transmembrane sequences at the membrane interface. These in vitro studies suggested two different mechanisms - membrane association prevents epitope immersion and membrane association allows recognition of the epitope.Indeed, efficacy of potential HIV therapy directed to gp41, has been ascribed to the interaction of defined aromatic (tryptophan) residues (Scherer, et al., 2010, PNAS, 107, 1529-1534), which are well recognised as having specific interactions with acyl-C=O moieties of bilayer lipids, from others and our own work (de Planque, et al., 2003, Biochemistry, 42, 5341-5348).The nature of the lipid component is additionally a vital aspect in surface recognition. Here lipid asymmetry and "domain" composition can influence dramatically the behaviour of a surface active peptide (van Meer, et al., 2008, Nat. Rev. Mol. Cell Biol., 9, 112-124).Impact is thus demonstrated in potential therapy with both academic interest and potential follow-through to industrial/ pharmaceutical design. How beneficiaries might benefit from this research:Not only is 'life-or-death' for a cell delineated by a ~3-4nm plasma membrane, it is also the site of initial interaction for cell destroying strategies. Destroying unwanted or pathogenic cells, be they viruses or bacteria, is so common-place in anti-viral and anti-microbials, that resistance is becoming a common obstacle to further development. Simply in economic impact, the cost of treating infections is ~$4-5 billion in the US, with the (uncontrolled) way in which antimicrobial agents are prescribed being a major risk determinant in eliciting resistance.In terms of patient numbers, 1.7 million illnesses and 100,000 deaths in the US alone a year occur through infections - that is 7 times deaths from AIDS. Virtually all bacteria have become less responsive to classic antimicrobials, many of which act specifically on the bacterial cell wall.Thus, the economic benefit of designing new antimicrobial peptides, of specific targeting capacity, is overwhelming.Technologically, manufacturing and producing such AMPs for toxicity trials for eventual human use is a well-proven process, and any new technology will need testing, especially if bioavailability or administration is a challenge.
