Successful Drug Discovery Informed by Actinobacterial Systematics Verrucosispora HPLC-DAD analysis of culture filtrate Structures of Abyssomicins Biological activity T DAD1, 7.382 (196 mAU,Up2) of 002-0101.D V. maris AB-18-032 mAU CH3 CH3 T extract H3C H3C Antibacterial activity (MIC): S. leeuwenhoekii C34 maris AB-18-032 175 mAU DAD1 A, Sig=210,10 150 C DAD1 B, Sig=230,10 O O DAD1 C, Sig=260,20 125 7 7 500 Rt 7.4 min DAD1 D, Sig=280,20 O O O O Growth inhibition of Gram-positive bacteria DAD1 , Sig=310,20 100 Abyssomicins DAD1 F, Sig=360,40 C 75 DAD1 G, Sig=435,40 Staphylococcus aureus (MRSA) 4 µg/ml DAD1 H, Sig=500,40 50 400 O O 25 O O Staphylococcus aureus (iVRSA) 13 µg/ml 0 CH CH3 300 400 500 nm 3 DAD1, 7.446 (300 mAU,Dn1) of 002-0101.D 300 mAU Mode of action: C HO atrop-C HO 250 atrop-C CH3 CH3 CH3 CH3 200 H C H C H C inhibitior of pABA biosynthesis 200 Rt 7.5 min H3C 3 3 3 Proximicin A Proximicin 150 HO O HO O O O O O O O O O A 100 O covalent binding to Cys263 of PabB 100 N 50 O O HO O O Sea of Japan B O O N O O (4-amino-4-deoxychorismate synthase) by 0 CH CH3 CH3 CH3 3 300 400 500 nm HO HO HO HO Michael addition -289 m 0 B D G H 2 4 6 8 10 12 14 16 min Newcastle Michael Goodfellow, School of Biology, University Newcastle University, Newcastle upon Tyne Atacama Desert In This Talk I will Consider: • Actinobacteria as a key group in the search for new therapeutic drugs. • Taxonomic approaches to the selection of actinobacteria from extreme habitats. • Atacama desert soil as a source of creative actinobacteria. • Selection of novel actinobacteria for chemical screening. • Conclusions and some new directions of travel. Actinobacteria from Atacama Desert Need for a New Generation of Antibiotics 2008-2012 New antibacterial agents approved by the Food and Drug Administration (FDA) in the United States between 1983-2009. Adapted from Bassetti et al. (2011). 3 Twin Track Approach Novel New Drug Target Screens Sample extracts Biological Material Dereplication Filters H I T Compound Evaluation & Characterization L E A D Goodfellow & Fiedler (2010). Antonie van Leeuwenhoek 98:119-142. Actinobacteria What are they? • A phylogenetically defined, metabolically active group of slow-growing, gram-positive bacteria which have DNA rich in guanine plus cytosine (> 55% mol. GC), unique 16S and 23S rRNA signatures and conserved indels in some proteins. Why prioritize them for drug discovery? • Unravelled capacity to synthesize natural products with a wide spectrum of bioactivity. • Account for almost half of all microbial bioactive compounds; nearly 80% produced by Streptomyces strains. • Culture-independent studies show that <1% of actinobacterial taxa in natural habitats have been cultivated hence scope for the discovery of new chemical entities from cultivable novel taxa is enormous. • Diverse populations of novel actinobacteria present in extreme habitats are able to produce new specialised metabolites. Whole Genome Sequence of Streptomyces coelicolor Strain A3 (2) Contains 27 natural product biosynthetic gene clusters Hopwood et al., 2002. Nature 417:141-147. Specialised Metabolites produced by Streptomyces coelicolor A3(2) Actinorhodin Actinorhodin 1955 / Actinorhodin 1980 / Undecylprodigiosin ∆act ∆red Prodiginine (cultivated in R2 for 5 days) Prodiginine 1976 / Methylenomycin 1978 / Calcium Dependent Antibiotic (SCP1 encoded) We do a poor job in interpreting a cell’s potential from its genomic sequence. However, we are getting better. Prokaryotic Systematics is not a Luxury Prokaryotic systematics is a core discipline practiced by few. However, the implementation of taxonomic concepts and practices underpins taxonomic approaches to drug discovery: • Well delineated natural classification. • Provision of a stable, universally accepted nomenclature. • Establishing cultivable actinobacterial diversity in natural habitats. • Improved procedures for selective isolation of novel and neglected taxa. • Rational choice of strains for bioprospecting and biotechnology. • Inferring the course of prokaryotic evolution. • Legislation (e.g. Hazard groups, patents of living biological material, Convention on Biological Diversity). 8 Polyphasic Taxonomy 1960-2014 Integrated use of genotypic and phenotypic data: Chemotaxonomy Sound Classification Genomics Polyphasic Taxonomy = Stable Nomenclature Molecular Systematics Reliable Identification Numerical Taxonomy Outcomes: Sound infrastructure for pure and applied microbiology and for designing and implementing scientific policies. 9 Hierarchic Classification of Actinobacteria based on Phylogeny Phylum Actinobacteria 5 Classes 19 Orders Bergey’s Manual Trust is The next edition of a nonprofit organisation Bergey’s Manual will be that is supported by 50 Families an electronic one that will royalty income. be frequently updated. 220 Genera 3,000 Species Goodfellow et al. 2012. Bergey’s Manual of Systematic Bacteriology, 2nd Edition, Volume 5, Springer, New York. Our Strategy Extreme/Neglected habitats Selective isolation Selection of isolates Dereplication technologies Novel actinobacteria • Recent approaches to drug discovery (eg. combinatorial chemistry and fragment based drug design) have yet to make a significant Screening impact. Dereplication of active compounds • Need to screen novel actinobacteria to avoid costly rediscovery of known bioactive New natural products compounds. Current bottleneck Commercial success Culture-dependent bioprospecting strategy Why Focus on Actinobacterial Diversity from Extreme Habitats? • Isolation of actinobacteria from extreme biomes rests on the premise that harsh environmental conditions give rise to unique taxa (species, genera) with novel chemistry. • Evidence of a coupling between taxonomic and chemical diversity means that taxonomic diversity can be used as a surrogate for chemical diversity. • Screening common actinobacteria from well studied habitats leads to the costly rediscovery of known bioactive compounds Decrease in the number of new drugs coming onto the market http://smellslikescience.com/a-need-for-new-antibiotics/ Actinobacteria for Low Throughput Screens Objectives: Focus on rare and previously unknown taxa, including novel species of Streptomyces. Key taxonomic methods designed to meet these aims: • Choice of selective isolation procedures. • Selection of cultivation media. • Recognition of novel taxa by comparative sequencing of conserved genes / proteins, notably 16S rRNA genes. Screening strategies: • Cell based targets- plug assays using panel of pathogenic strains. • Reporter Bacillus subtilis strains to detect cellular targets. • Choice of production media. • Chemical analysis of extracts for novel specialised metabolites. Discovery dependent on dereplication at all stages of the procedure. Atacama Desert The Atacama Desert located in northern Chile is the oldest and driest desert on the planet. ALMA Lomas Byas Most Atacama Desert soils and regoliths are either hyper- arid (ratio of mean annual rainfall to mean annual evaporation is < 0.05) , or extreme hyper-arid (corresponding ratio < 0.002). Yungay Low concentrations of organic matter, high salinity, the presence of inorganic oxidants and high UV radiation Salar de conditions. Atacama The underlining premise is that these extreme conditions will have given rise to a unique actinobacterial diversity and consequential chemical diversity. Sampling Sites Salar de Atacama Yungay Hyper-arid area Extreme hyper-arid area ALMA Lomas Bayas High altitudes, Very high UV radiation Most extreme hyper arid area Selective Isolation of Actinobacteria Media Selective agents Target organism (s) References Glucose-yeast extract Rifampicin (20µg ml-1) Actinomadura spp. Athalye et al. (1981) agar Gause’s No.1 agar Nalidixic acid (10µg ml-1) Rare or uncommon Zakharova et al. (2003) actinomycetes Humic acid-vitamin agar Humic acid (1g/L) Streptosporangiaceae Hayakawa & Nonomura spp. (1984) Oligotrophic agar Low carbon and nitrogen Rare actinomycetes Busarakam (2013) sources Raffinose-histidine agar Common Streptomyces spp. do Rare or uncommon Vickers et al. (1984) not grow well in the presence streptomycetes of these nutrients Starch-casein agar High carbon to nitrogen ratio Streptomyces spp. Küster and Williams (1964) SM1and SM2* agars Neomycin (4 µg ml-1), D(+) Amycolatopsis spp. Tan et al. (2006) sorbitol (1%, w/v)* Cycloheximide and nystatin (each at 25 µg ml-1) used to control fungi. 16 Selective Isolation Range of selective isolation media Sprinkle plates Dilution plates Isolation of Streptomyces Strains Isolation of Amycolatopsis Strains Oligotrophic agar Humic-acid-vitamin-agar SM 1 agar Starch-casein vitamin agar Isolation of Modestobacter Strains Arginine-vitamin agar Humic-acid-vitamin agar R2A agar Oligotrophic agar Starch-casein-vitamin agar Actinobacteria (cfu/g dry weight soil) growing on Selective Media inoculated with Suspensions of Atacama Desert Soils and Incubated at 28oC for 3 Weeks Media Soil Gause’s No.1 Humic acid Oligotrophic Starch-casein SM1 agar agar agar agar Salar de 1.1x104 1.3x104 0.3x102 0.1x102 0.3x102 Atacama Yungay 3x102 5x103 3.3x102 2.3x102 4.7x102 Actinobacteria were not isolated on glucose-yeast extract or raffinose-histidine agars. Dereplication Term used for differentiating phenotypically similar isolates (and metabolites) to select representatives to facilitate efficient screening and to minimize costs and time in maintaining large culture collections. • Choice of methods: Chemical: fatty acid analyses, pyrolysis and MALDI-TOF mass spectrometry. Molecular: rep-PCR,