High quality reagents for innate immunity research, vaccine development and drug discovery

		A screen of our Puretitre library reveals new ways to reverse antibiotic resistance The emergence of bacteria which are resistant to multiple antibiotics is an increasing concern for global healthcare systems. Historically, this issue has been countered by the discovery and development of new classes of antibiotics. However, as resistance to these new antibiotics frequently emerges within several years of their introduction [1], attention has focussed more recently on the identification of agents that block the mechanisms used by bacteria to resist the effects of the antibiotic. So-called “resistance reversing” drugs have been successfully deployed in the clinic to treat infections where the chief mechanism of resistance is via overexpression of enzymes that break down antibiotics of the beta-lactam family. For example, the combination of amoxicillin with the natural product clavulanic acid (together called Augmentin) works much more effectively than amoxicillin alone when treating infections with bacteria expressing lactamase enzymes as their chief means of resistance. However, there are many other mechanisms by which bacteria resist antibiotics, and lactamase inhibitors are not effective against these other forms of resistance. In a recent screen of our Puretitre library of 200 natural compounds, 4 hits were discovered to significantly inhibit the resistance of a clinically relevant strain of Escherichia coli to the antibiotic tetracycline [2]. The strain examined is a clinical isolate that commonly causes urinary tract infection in man, and is resistant to many different classes of antibiotic. Further study of these hits showed that they were also effective at reversing resistance to antibiotics of several other classes, including chloramphenicol, trimethoprim and tobramycin. One of the hits, cepharanthine, was found to potently inhibit molecular efflux from the resistant cells - a commonly used method of resistance in such microbes. Notably, very few inhibitors of Gram-negative efflux pumps have been discovered previously. Two of the other hits, propyl gallate and ellagic acid, significantly inhibited the uptake of nutrients into the cell, and slowed the rate of growth of the resistant bacteria. Preliminary studies of the potential toxicity of the compounds revealed that three of the top four hits (propyl gallate, ellagic acid and cinchonidine), showed negligible toxicity in an in vitro mammalian cell culture model, at doses higher than those required to block resistance. This is consistent with the use of propyl gallate and ellagic acid as approved food additives in some territories, and the rationale for the library of focussing on plants with a history of safe oral use in man. By screening collections of molecules with low toxicity from the outset, the aim is to accelerate progression of hits from such screens to animal studies or nutraceutical trials. The relatively high hit rate of this screen (~4%) also suggests that traditional medicines may be richer in molecules with antibiotic potentiating activity than previously thought. Therefore, although there are many other potential mechanisms of resistance waiting to be explored, it is likely that natural product screening could provide useful structural leads for the development of new drugs to target resistance to multiple different classes of antibiotic. References [1] WHO Global Action Plan on Antimicrobial Resistance. http://www.who.int/antimicrobial-resistance/en/ [2] Jenic et al, Reversal of tetracycline resistance by cepharanthine, cinchonidine, ellagic acid and propyl gallate in a multi-drug resistant Escherichia coli. Natural Products and Bioprospecting (2020) http://link.springer.com/article/10.1007/s13659-020-00280-y Nobel prize awarded for natural product-based drug discovery The 2015 Nobel prize in Physiology or Medicine has been awarded to three researchers for their contributions to drug discovery from natural product screening programmes [1]. Professors Satoshi Omura and William C. Campbell were awarded the prize for their discovery of Avermectin, from which the widely used anti-parasite agent Ivermectin is derived, and Professor Tu Youyou was rewarded for her contribution to the discovery of Artemisinin - the latest front-line medicine in the fight against malaria. Avermectin’s remarkable activity was discovered by screening the conditioned media of thousands of bacteria cultured from soil samples collected by Omura’s team from around Japan. Those with promising potential were sent to Campbell’s laboratory at Merck in the US for further analysis. Campbell’s team then screened the samples for activity against parasitic worms in a mouse model. The products of one microbe in particular, (Streptomyces avermitilis, cultured from soil taken near a golf course in Ito city), demonstrated remarkable worm-killing activity, yet very low toxicity. Activity-guided separation led to the discovery of the novel compound, avermectin, with a highly unusual new scaffold. Simple modifications of this scaffold led to the development of two compounds (termed Ivermectin) which together demonstrated even lower toxicity and greater anti-parasite activity. The impact of Ivermectin on human and animal health is difficult to overstate. The drug displays not only broad-spectrum activity against a wide array of clinically relevant parasites, but also low toxicity in animals and man. Millions of West Africans have been spared the blight of river blindness (caused by the parasitic worm Onchocerca volvulus), or lymphatic filariasis (also caused by parasitic worms), largely due to the impact of Ivermectin. The drug is now included on the World Health Organization's List of Essential Medicines - those which are considered to be the safest and most effective in modern healthcare. A more focussed natural product screening approach was taken to the discovery of Artemisinin. Following an outbreak of malaria strains resistant to the anti-malarial drug chloroquine, the Chinese government initiated a state-wide effort to identify new anti-malarials. In the early years of the initiative, ~40,000 synthetic compounds were screened, but without success. Mindful of this, Youyou’s team aimed to maximise the chances of success by focussing their screens on phytochemicals, together with evidence from a collation and reappraisal of ancient texts relating to Chinese traditional medicines. Those plants reportedly prescribed for fever or other symptoms of malaria were of particular interest, and were selected for biological assay. These ethnopharmacologically focussed screens led to the discovery that cold water extracts of Artemisia annua L. (sweet wormwood), displayed remarkable activity against malaria parasites. Activity guided separation enabled the identification of a highly complex molecule with activity against several Plasmodium species, but low toxicity in mice. The unusual endoperoxide ring of the compound was found to be essential for antiparasite activity, but unlike anything a chemist could synthesise, or likely had conceived of before. Artemisinin-based therapies are now the recommended first-line therapy for P. falciparum malaria globally, and are estimated to have saved millions of lives to date [2]. These two wonderful examples of the successful application of natural product screening programmes lend further proof to the immense potential of natural compounds, particularly when coupled with the rich evidence base from traditional medicine, for the development of new drugs. As our medical needs transition away from infectious disease towards chronic non-communicable diseases over the coming decades, returning to these rich resources is likely to be critical in our efforts to develop the next generation of therapeutic compounds. [1] Shen B. A New Golden Age of Natural Products Drug Discovery. Cell 163:1297-1300 (2015) http://www.cell.com/cell/fulltext/S0092-8674(15)01550-0 [2] Su X, Miller LH. Artemisinin: discovery from the Chinese herbal garden. Cell 146:855-858 (2011) http://www.cell.com/cell/fulltext/S0092-8674(11)00950-0 Discovery of new class of antibiotic through innovative natural product library screen A novel approach to natural product screening has enabled the discovery of an entirely new type of antibiotic [1]. To date, the majority of antibiotics have been discovered by screening the secreted products of soil microbes. However, 99% of soil micro-organisms do not grow when cultivated in the lab under standard conditions. Now, researchers led by Dr Kim Lewis in the Department of Biology, at Northeastern University, Massachusetts, have turned to a novel approach to cultivate a greater fraction of these so-called “uncultivable” bacteria. Their approach is based on the recent finding that most soil microbes depend on metabolites secreted by their neighbours for growth [2]. To enable this, the group cultured individual soil microbes within many thousands of tiny microwells, separated from their surroundings by a permeable membrane which allowed small molecules but not bacteria to cross. This cassette was then buried in the same type of soil from which the microbes were isolated, in order to perfuse the chambers with the unique mixture of compounds derived from their soil microbe neighbours necessary for their growth. After several weeks of culture in this conducive environment, pure colonies of thousands of previously uncultivable organisms were obtained. A screen of crude extracts of the secreted products of these new colonies revealed that many displayed antimicrobial activity against Staphylococcus aureus - a clinically relevant microbe which is commonly associated with serious hospital acquired infections and resistance to multiple antibiotics. Activity-guided separation, using C18 high performance liquid chromatography (HPLC), was then used to isolate an active compound of 1,242 Daltons which was not reported in natural product databases. Nuclear magnetic resonance (NMR) was then used to establish the structure of the compound, revealing it to be a unusual depsipeptide containing enduracididine, methylphenylalanine, and four D-amino acids. The new compound, called Teixobactin, displayed antimicrobial activity against a wide range of clinically relevant bacteria, and the mode of action was found to involve the inhibition of cell wall synthesis by binding to highly conserved motifs of two critical components of the Gram-positive bacterial cell wall: peptidoglycan and teichoic acid. Remarkably, cultivation of Teixobactin in a model of bacterial evolution revealed no emergence of antibiotic resistance over multiple generations, suggesting that the targets are relatively resistant to the emergence of escape mutants [1]. The discovery is particularly timely, as the incidence of antibiotic resistant infections in hospitals around the world is increasing rapidly, and some strains are resistant to all front-line antibiotics. The WHO has called for greatly expanded research efforts on the discovery of new antibiotics, in order to avert large increases in deaths due to untreatable infections [3]. The implications of the study for natural product screening are also profound. Cultivating the 99% of previously uncultivated microbes in soil alone has the potential to markedly increase the diversity of the known natural product chemical space. The new biological space has potential to reduce rates of re-discovery of compounds of the same or similar scaffolds to existing compounds, and increases potential for identification of compounds with activity against many diverse targets, including those of non-bacterial origin. [1] Ling LL, et al. A new antibiotic kills pathogens without detectable resistance. Nature 517:455-459 (2015) http://www.nature.com/nature/journal/v517/n7535/full/nature14098.html [2] Nichols D, et al. Use of ichip for high-throughput in situ cultivation of ‘uncultivable’ microbial species. Appl Environ Microbiol 76:2445-2450 (2010) http://aem.asm.org/content/76/8/2445.full [3] WHO Global Action Plan on Antimicrobial Resistance. http://www.who.int/antimicrobial-resistance/en/ No decrease in rate of discovery of new natural product scaffolds between 1990 and 2015 It has been claimed that the rate of discovery of natural products with truly novel structures has been in decline since the ‘golden era’ of discovery between the 1950s and 1980s [1]. However, a new study published in PNAS reveals that the rate of discovery of novel scaffolds from natural sources has remained roughly at the same level since the peak was reached ~30 years ago [2]. Roger Linington at Simon Fraser University, Canada, and his co-workers applied rigorous definitions of compound similarity and used computational chemoinformatics to formally address this question [1]. By combining structures from published databases and searches of the more recent published literature, the team assembled a database of 52,395 unique marine and micro-organism-derived compounds discovered between 1941 and 2015. The researchers then calculated Tanimoto similarity scores between all molecule pairs in their database. The rate of discovery of those with a Tanimoto similarity score below 0.4 - i.e. a new scaffold - peaked at around 200 per year in the late 1980s. However, the rate of new scaffold discovery has remained at pretty much the same level until at least 2015. The authors comment that this unabated rate of discovery is surprising, given the decline of interest in natural products from industry in the early 1990s. However, greater interest among workers in academia, together with improvements in compound identification and the mining of novel taxa from ever more remote niches, has maintained the healthy rate of new scaffold discovery. The researchers also point out that when accounting for dereplication, ~1,500 novel natural product structures were also reported per year during the past 30 years. This indicates that although the majority of reported structures are variants of existing scaffolds, ~13% are compounds with no structurally related equivalent. Thus, even after 70 years of exploration, new scaffolds continue to be discovered. Crucially, the authors point out that while much progress has been made in understanding of the structural diversity of natural products in recent years, very little information is available with respect to the potential bioactivities of these molecules. Indeed, of the tens of thousands of mammalian targets with potential for drug development, only a small fraction has been screened in any campaign, and only ~400 have been drugged successfully to date. Together, these findings support the view that the recent slow-down in approval of natural product derived drugs is not due to a reduced rate of scaffold discovery. Instead, it is more likely a symptom of the refocusing of large pharmaceutical firms to synthetic library screening. A re-inclusion of the wealth inherent in Nature’s chemical space in modern screening programmes clearly retains great potential for the discovery of new compounds with therapeutic potential. [1] Kong DX, Guo MY, Xiao ZH, Chen LL, Zhang HY. Historical variation of structural novelty in a natural product library. Chem Biodivers 8:1968-77 (2011) http://onlinelibrary.wiley.com/doi/10.1002/cbdv.201100156/abstract [2] Pye CR, Bertin MJ, Lokey RS, Gerwick WH, Linington RG. Retrospective analysis of natural products provides insights for future discovery trends. Proc Natl Acad Sci USA 114:5601-5606 (2017) http://www.pnas.org/content/114/22/5601.abstract

 

Resources General FAQ Phytotitre FAQ Free data Natural product news Publications

Company About us Contact us

Ordering How to place orders Custom products and services

 

Caithness Biotechnologies Ltd., 72 Boston Road, Leicester, UK, LE4 1HB. Telephone: +44 (0) 116 326 3802 | email: contact@caithnessbiotechnologies.com