Finding Novel Antimicrobial Peptides with AMPLY

Rise of the superbugs
The rise of antibiotic resistant strains of microbes (bacteria, parasites, viruses and fungi) is probably the leading threat facing humankind. Increasingly desperate warnings about the real-world implications of the increasing resistance are now front page news. The problem is a multifaceted one. Antimicrobial resistance is not just about the loss of human life, but inextricably intertwined with increased patient morbidity and massive economic consequences for global healthcare systems. There are two possible solutions, either a socio-political/behavioural change or a technical/scientific response. Humankind has shown itself remarkably intransigent when faced with doom laden prophecies that require behavioural modification to circumvent (see also Climate Change), therefore it is probably prudent to assume that a managed technical response may be our best hope. But new antibiotics are unlikely to arise spontaneously. Mokyr highlights one of the issues with relying on the existing pharmaceutical industry to address the problem: “…few economies have ever left [decisions like these] entirely to the decentralized decision-making processes of competitive firms. The market test by itself is not always enough” (Mokyr, 1998).

Discovery of AMPs

Figure 1: A cationic, helical AMP (Taliecin-1)

The discovery of AMPs dates back to 1939, when Dubos extracted an antimicrobial agent from a soil Bacillus strain. The designation of AMPs has been extended to encompass a general view of them as a group of anionic antimicrobial proteins/peptides; host defence peptides; cationic amphipathic peptides and cationic AMPs. In contrast to acquired immune mechanisms these endogenous peptides provide a fast and effective means of defence against pathogens as part of the innate immune response. Antimicrobial peptides are evolutionary ancient weapons and their ubiquity throughout the animal and plant kingdoms supports the hypothesis that they have played a key role in the successful evolution of complex multi-cellular organisms. Such is their diversity they can be found in locations as disparate as the skin secretions of a frog to the defensive arsenal of a protozoa.

Dolby Bioinformatics

Figure 2: The Dolby certification logo (dolby.com)

One specific feature of AMPs that makes them difficult to find is that they’re small (often less than 20 amino acids in length – which is comparatively tiny compared to typical proteins). In a typical ‘omic dataset containing, potentially millions and millions of datapoints, isolating interesting AMPs for synthesis and testing is a challenging test. For inspiration we can look to the music industry. In the mid-20th century recordings were made on magnetic tape and engineers wrestled with an ever present low level of hissing noise in the background that threatened to drown out the music. Various ingenious solutions were deigned to mitigate the persistent hiss from forms of “low-noise” tape which recorded more signal; running the tape at a higher speed, or using dynamic pre-emphasis during recording and a form of dynamic de-emphasis during playback. This latter approach became the backbone of the Dolby noise reduction system, which became all pervasive in home audio equipment from the late 60s onwards. The audio engineer’s struggle to maximise signal-to-noise is the same core problem that faces computational biologists and the ongoing analysis of ‘omic “big data” in the search for tiny novel AMPs. There is music there, but at the moment the hiss is tremendous.

The detection of AMPs in metagenomic data is a tantalising low-hanging fruit for computational biologists, however. Post-computational wet-lab work is relatively cheap with spot synthesis of peptides up to around 25aas long possible from a wide array of third party companies with prices from as low as £2.50 per amino acid. A well organised screening program can screen in excess of 100 peptides a day, per person, against a model bacterial organism to test for activity. As a potential workflow the rapid assessment of multiple ‘omic datasets; identification of homologues of pattern matched AMPs; rapid synthesis and screening and a rush to publication would appear to provide a grant-friendly drug-discovery goldmine! But to tap this rich vein, improving the hit rate of putative AMPs from ‘omic data needs to be streamlined and improved.

The AMPLY Pipeline
Finding small sequences (you’re interested in) that often look a lot like other small sequences (you’re not interested in) in datafiles that can contain potentially gigabytes of data is a trickier task than it first appears. Annotation in metagenomics is an art and the determination of what’s real and what’s not often relies purely on defining mutually agreed thresholds. However, as the length of the aligned data being identified starts to shorten, a lot of the assumptions on PercentageID, BitScore and E-Value thresholds begins to fall away. It’s here we return to the Dolby signal-to-noise analogy – the “music” of the AMPs in metagenomic datasets are often drowned out by the sheer volume of background noise and to find them we need to adopt a novel strategy of aggressive emphasis.

Designed by Ben Thomas at Aberystwyth University in the CreeveyLab (http://www.creeveylab.org/) and funded by Life Science Wales (https://www.lifescienceshubwales.com/), AMPLY (http://amply.info) is a pipeline designed to plug this gap between the ‘omic data and lab work. AMPLY is designed to provide a basis to sift-out AMPs suitable as synthesis candidates and provide potential regions for crude synthesis by adopting a hyper-wide “balance of evidence” approach. AMPLY passes over data with a series of detection methods, then wrapping the summative results of both them and presenting the final results into a final tableau (known as the “bitpad”) where each potential AMP can be evaluated on the strength of a series of hundreds of datapoints, rather than just a couple of numeric values.


Figure 3: The AMPLY workflow

To date, AMPLY has been used to find, characterise and synthesise over 800 potentially novel AMPs which have been lab screened in partnership with Tika Diagnostics (http://tikadiagnostics.com/) at St. George’s Hospital in London. Among the AMPs discovered by AMPLY many are highly active against MRSA (a key superbug) and offer encouraging potential treatment avenues for future development. While there is still much work to be done, results so far have been extremely promising: AMPLY has been used to find bioactive AMPs in datasets as diverse as the skin of Peruvian poison dart frogs to the testicles of a Salamander so the only limitation in the AMPLY pipeline is the diversity of the stream of ‘omic data provided to it….

So, if you’re reading this blog and have interesting data and would like to be part of the drive to find new antimicrobials then get in touch for potential collaborations. We are always interested.

Contact Ben Thomas at bet16@aber.ac.uk, or via Twitter @flwrs4algrnon



References
Mokyr, Joel. “The political economy of technological change.” Technological revolutions in Europe (1998): 39-64.

Invasive Weed Species as a Source of Antimicrobials – Making the Best of a Bad Situation

Invasive Weed Species as a Source of Antimicrobials – Making the Best of a Bad Situation

Humans have always been dependant on nature to cater for their basic needs such a food and shelter but also for medicines. Initially medicines were in the form of crude treatments such as tinctures, teas, poultices, powders and other herbal formulations. The specific plants and methods of applications were originally passed down through oral history untill the information were recorded in herbals. In more recent history the use of natural products as medicines involves the isolation of active compounds [1]. The first active compound to be isolated in this way was morphine from opium by Friedrich Setürner in 1804 [2]. Drug discovery from plants also led to the isolation of many early drugs such as cocaine, codeine, digitoxin and quinine; some of which are still in use today. Due to the vast diversity of natural products ranging from teraestrial plants to marine organisms also incuding microorganisms and their infinite possible applications the isolation and characterisation for medicinal purposes continues today.
Plants have been the single most productive source of leads for the development of drugs, particularly as anti-cancer agents and anti-infectives [3]. Eventhough natural products have been a plentyful and continuous stream of useful drugs their use has dimished in the past two decade due to the major pharaceutical companies deminishing their interest in natural products. Due to slow nature of natural product discovery and its incompatiblity with high throughput screening (HTS) directed at moleculat targets [4]. Many large screening collections have been dissapointing in practice (these libraries containing a range of compounds from many different sources) natural products are the most diverse class of compounds with a significantly higher hit rate compared to fully synthetic and combinatorial libraries [5]. Furthermore, it has been shown that 83% of core ring scaffolds that are present in natural products are not present in commercially available screening libraries leading to fewer drug leads [6]. It is unsurprisingly that even with the introduction of new methods and technologies natural products have contributed massively to the drugs which have been approved in recent years (see Fig.1).

image1
Figure 1: Contributuion of Natural Products to Approved Drugs between 1981-2010; n=1355. (Adapted from Newman and Cragg 2012 [7])

My PhD funded by the Life Sciences Research Network Wales (http://www.lsrnw.ac.uk/). The project is based on the discovery of antimicrobal compounds form invasive weed species. Invasive non-native weed species are a significant global concern. These are resposible for a loss of biodiversity, altering ecological processes, impacting ecosystem services resulting in a cost of $35 billion annually in the USA [8-10]. If antimicrobial or any bioactive compounds could be sources from these problematic plants then we could at least draw one positive from their unwanted presence within our environment. This project includes the traditional extraction, isolation and characterisation of active compounds form plants followed by biological assays to test a range of biological activites of the compounds extracted. These techinques are also combined with the genomic and bioinformational approaches to aid and improve drug discovery. A wide range of plants were selected for this study and a range of compounds have been extracted from each with a range of interesting biological activites; especially antimicrobial activity. The most active plants tested were Japanese knotweed and Himalayan balsam.

image 2
Resveratrol was found to be the most active antimicrobial compounds present in Japanese Knotweed. This compounds is also found in spermatophytes, such as grapevines and has been linked to a wide variety of biological activites. It has been reported to have antioxidant, anticancer, anti-inflammatory, prevent post-menopausal bone loss, and a range of positive metabolic effects. Resveratrol has also been suggested as the causal link between increased red wine consuption and decreased risk of heart disease [11].
A key compound has been found in Himalayan balsam which is by far the most potant antimicrobial compound in all the plants studied. It has a minimum inhibitor concentration of between 3-15 µg/mL agaisnt a range of Staphylococcal species. This compound has also been found to be non-toxic against mammlian cells. Similar compounds have also been show to have anti-cancer and anti-fungal activity.
The mode of action of these compounds are currently being elucidated using genomic, metabolomic and proteominc approaches combined with novel assays and cytometric techniques. In addition to this I aim to improve the activity of these compounds using computer aided drug design (CADD) through the Life Sciences Reseach Network Wales CADD Platform (http://www.lsrnw.ac.uk/platform-technologies/welsh-computer-aided-drug-design-cadd-platform/).
Natural products have been a source of drugs which have revolutionalised treatment of disease. It is clear that natural sources will contiune to play a significant role in the fight against disease and should be combined with new inovative methods which are currently being developed to form a multidisciplinary approach to treat disease.

References
1. Balunas, M.J. and A.D. Kinghorn, Drug discovery from medicinal plants. Life sciences, 2005. 78(5): p. 431-441.
2. Schmitz, R., Friedrich Wilhelm Sertürner and the discovery of morphine. Pharmacy in history, 1985. 27(2): p. 61-74.
3. Harvey, A.L., Natural products in drug discovery. Drug discovery today, 2008. 13(19): p. 894-901.
4. Harvey, A.L., R. Edrada-Ebel, and R.J. Quinn, The re-emergence of natural products for drug discovery in the genomics era. Nature Reviews Drug Discovery, 2015. 14(2): p. 111-129.
5. Sukuru, S.C.K., et al., Plate-based diversity selection based on empirical HTS data to enhance the number of hits and their chemical diversity. Journal of biomolecular screening, 2009. 14(6): p. 690-699.
6. Hert, J., et al., Quantifying biogenic bias in screening libraries. Nature chemical biology, 2009. 5(7): p. 479-483.
7. Newman, D.J. and G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010. Journal of natural products, 2012. 75(3): p. 311-335.
8. Simberloff, D., et al., Impacts of biological invasions: what’s what and the way forward. Trends in ecology & evolution, 2013. 28(1): p. 58-66.
9. Hulme, P.E., et al., Bias and error in understanding plant invasion impacts. Trends in ecology & evolution, 2013. 28(4): p. 212-218.
10. Pimentel, D., R. Zuniga, and D. Morrison, Update on the environmental and economic costs associated with alien- invasive species in the United States. Ecol. Econ., 2005. 52(3): p. 273-288.
11. King, R.E., J.A. Bomser, and D.B. Min, Bioactivity of resveratrol. Comprehensive Reviews in Food Science and Food Safety, 2006. 5(3): p. 65-70.

Post by Dai Fazakerley.
Dai is a PhD student with Prof. Luis Mur and is one of our Biochemistry BSc graduates.