COMMENTARY
COMMENTARY
Atlas for drug discovery Pierre Stallfortha and Jon Clardyb,1 a Junior Research Group ‘Chemistry of Microbial Communication,’ Leibniz Institute for Natural Product Reserach and Infection Biology, Hans-Knöll-Institute, 07745 Jena, Germany; and b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
Reports of increasing antibiotic resistance and sagging drug discovery rates are appearing with increasing frequency, and many warn that we are living on the wrong side of an antibiotic peak—a period in which everfewer new antibiotics are being discovered at ever-increasing costs (1). In response, efforts to discover new antibiotics and other drugs have taken many forms, including looking at formerly productive sources that were thought exhausted, much as rising oil prices led to new extraction techniques for abandoned oil fields. For several decades, small molecules produced by soil bacteria were our most important source of new drugs, as represented by the antibiotics erythromycin and vancomycin, the anticancer agents bleomycin and mitomycin, and the immunomodulators cyclosporin and rapamycin (2). In the last two decades, almost all pharmaceutical companies have abandoned bacterially based drug discovery because it seemingly fits poorly with the high-throughput screening and medicinal chemistry approaches that define the industry’s favored discovery paradigm, and because its high rate of rediscovering previously known compounds indicated that it was unlikely to yield new drugs (3). More recently, genomic revelations have dramatically altered our view of soil bacteria. The revelations were of two sorts: (i) most bacteria (∼99%) cannot be cultured under typical laboratory conditions, and (ii) even those that can be cultured produce only a fraction (∼10%) of the small molecules encoded in their genomes (4–6). These dual shortfalls in culturing and expression pointed to a bonanza of potentially useful small molecules remaining to be discovered and led to innovative technical approaches to finding them. In PNAS, Charlop-Powers et al. (7) use cultureindependent DNA sequencing on a broad environmental scale to systematize where different types of bacterially produced small molecules are likely to be found. Their “chemicalbiogeographic survey” provides insights into microbial ecology along with a practical guide for microbial genome prospectors. The potential scale of such a survey is staggering. Bacteria are the most diverse organisms www.pnas.org/cgi/doi/10.1073/pnas.1400516111
on the planet, and a single gram of soil can contain between a thousand and a million distinct species according to a pioneering survey (8). Species definition in bacteria has its whimsical aspects, but it usually means that a characteristic DNA sequence from one strain has, or does not have, a specified similarity to the sequence from another strain. If the similarity is high enough, say 98%, then the two strains are said to belong to the
Charlop-Powers et al. use culture-independent DNA sequencing on a broad environmental scale to systematize where different types of bacterially produced small molecules are likely to be found.
pathways: the polyketide (PK) pathway and the nonribosomal peptide (NRP) pathway, which assemble either carbon chains or amino acid chains from smaller subunits. These two pathways, either singly or in combination, make all of the examples mentioned earlier. The specific genes that were selected are necessary elements in both pathways, namely the genes that encode the proteins that select the smaller subunits. This necessary restriction has consequences, and antibiotics such as fosfomycin—whose biosyn-thesis follows a pathway different from PK or NRP pathways—would be overlooked. However, these limitations are obvious and could easily be addressed in future studies. To ensure a faithful representation of geographically resolved biosynthetic richness, the authors collect environmental DNA samples from more than 90 different locations. These samples served as templates for the preparation of ketosynthase (KS) and adenylation (AD) amplicons. Although these conditions introduced a modest amplification-dependent bias, the resulting amplicons closely mirror the variety of AD and KS domains found in the metagenome of the soil-dwelling bacteria. Subsequent 454 pyrosequencing of these amplicons allowed a classification system based on biosynthetic OTUs to be constructed. The OTU-based classification of environments was linked to environmental soil data including pH, moisture, and mineral composition among others gathered at the collection site, and the two data sets—biosynthetic OTUs and environmental parameters—could be cross-analyzed. A principal component analysis revealed three principal types of soils with different biosynthetic potential. For example, arid soil environments showed the largest biosynthetic potential.
same operational taxonomic unit (OTU). Researchers typically use the 16S ribosomal RNA gene sequence, a highly conserved sequence whose utility was established decades ago (9). Researchers isolate DNA from an environment—from desert soil to the human gut—and use PCR to amplify selected sequences from the sample for sequencing. These selected and amplified fragments of DNA are called amplicons. Amplicons of 16S rRNA have been widely used to create biogeographic surveys of soil bacteria—a catalog Biosynthetic Repertoires of Soil Types of what bacteria are found in different envi- The rich data set provided by this chemicalronments (9, 10). biogeographic survey provides two interesting insights. The first is that bacteria Biosynthesis-Based Genotyping of Soil in similar environments produce, or at least Microbiomes Charlop-Powers et al. used a similar ap- have amplicons that suggest they would proach to answer a different question: where produce, similar small molecules. A famous can we find genes that are likely to encode the production of useful molecules? Answering this question requires deciding on what genes, really what conserved DNA sequences on the genes, to look for. The authors select two important and well-studied biosynthetic
Author contributions: P.S. and J.C. wrote the paper. The authors declare no conflict of interest. See companion article on page 3757. 1
To whom correspondence should be addressed. E-mail: jon_clardy@ hms.harvard.edu.
PNAS | March 11, 2014 | vol. 111 | no. 10 | 3655–3656
generalization of microbial diversity notes that “Everything is everywhere, the environment selects.” (11) Soil variables, especially pH, largely control the environmental selection (10). These soil variables presumably select for bacteria with primary metabolisms that match a given environment. The present study demonstrates clearly that a plausible, but previously unproven, consequence of the environmental determinism of microbial biogeographic diversity is reflected in the secondary metabolism—the similar bacteria found in similar soils will produce similar compounds. This extreme environmental determinism in which soil variables control both biological and chemical diversity has no correlate in the biogeography of macroorganisms. Arid soils in geographically separated regions will, for example, host distinctly different plants that will produce different small molecules. Bacteria do not produce small molecules so that we can mine them for drugs; the molecules must confer some survival benefit. However, the ecological roles of these bacterially produced small molecules are poorly known, and their discovery represents a challenge for microbial ecology.
3656 | www.pnas.org/cgi/doi/10.1073/pnas.1400516111
A second conclusion, that arid soils have greater biosynthetic potential than pine forest soils or brackish sediments, might seem puzzling at first. Selman Waksman largely initiated the Golden Age of Antibiotics and won the 1952 Nobel Prize in Physiology or Medicine for his discovery of antibiotics, especially streptomycin, from swampy soils in New Jersey. The stream of antibiotics from Waksman’s laboratory, which all came from actinomycetes found in these soils, reflects his pioneering spirit that allowed him to succeed even with sampling suboptimal environments. Finally, Charlop-Powers et al. (7) provide a treasure map for drug prospec-
tors, and it will be interesting to see how finer-grained geographic and molecular analyses expand on this work. The chemical-biogeographic biodiversity approach could also be useful in exploring complex environments such as the human microbiome. We have increasingly good maps of what bacteria occupy different regions (and metabolic environments) of the human body. Will there be a similar correlation of chemistry and environment? This work also highlights the need for new techniques to convert genomic data into molecules, which is perhaps the greatest challenge facing genome-based drug discovery efforts.
1 Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Drugs for bad bugs: Confronting the challenges of antibacterial discovery. Nat Rev Drug Discov 6(1):29–40. 2 Nett M, Ikeda H, Moore BS (2009) Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat Prod Rep 26(11):1362–1384. 3 Baltz RH (2008) Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 8(5):557–563. 4 Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296(5570):1127–1129. 5 Winter JM, Behnken S, Hertweck C (2011) Genomics-inspired discovery of natural products. Curr Opin Chem Biol 15(1):22–31. 6 Challis GL (2008) Genome mining for novel natural product discovery. J Med Chem 51(9):2618–2628.
7 Charlop-Powers Z, Owen JG, Reddy BVB, Ternei MA, Brady SF (2014) Chemical-biogeographic survey of secondary metabolism in soil. Proc Natl Acad Sci USA 111:3757–3762. 8 Torsvik V, Øvreås L, Thingstad TF (2002) Prokaryotic diversity— magnitude, dynamics, and controlling factors. Science 296(5570): 1064–1066. 9 Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276(5313):734–740. 10 Fierer N, Jackson RB (2006) The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci USA 103(3): 626–631. 11 de Wit R, Bouvier T (2006) ‘Everything is everywhere, but, the environment selects’: What did Baas Becking and Beijerinck really say? Environ Microbiol 8(4):755–758.
Stallforth and Clardy
COMMENTARY
Atlas for drug discovery Pierre Stallfortha and Jon Clardyb,1 a Junior Research Group ‘Chemistry of Microbial Communication,’ Leibniz Institute for Natural Product Reserach and Infection Biology, Hans-Knöll-Institute, 07745 Jena, Germany; and b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
Reports of increasing antibiotic resistance and sagging drug discovery rates are appearing with increasing frequency, and many warn that we are living on the wrong side of an antibiotic peak—a period in which everfewer new antibiotics are being discovered at ever-increasing costs (1). In response, efforts to discover new antibiotics and other drugs have taken many forms, including looking at formerly productive sources that were thought exhausted, much as rising oil prices led to new extraction techniques for abandoned oil fields. For several decades, small molecules produced by soil bacteria were our most important source of new drugs, as represented by the antibiotics erythromycin and vancomycin, the anticancer agents bleomycin and mitomycin, and the immunomodulators cyclosporin and rapamycin (2). In the last two decades, almost all pharmaceutical companies have abandoned bacterially based drug discovery because it seemingly fits poorly with the high-throughput screening and medicinal chemistry approaches that define the industry’s favored discovery paradigm, and because its high rate of rediscovering previously known compounds indicated that it was unlikely to yield new drugs (3). More recently, genomic revelations have dramatically altered our view of soil bacteria. The revelations were of two sorts: (i) most bacteria (∼99%) cannot be cultured under typical laboratory conditions, and (ii) even those that can be cultured produce only a fraction (∼10%) of the small molecules encoded in their genomes (4–6). These dual shortfalls in culturing and expression pointed to a bonanza of potentially useful small molecules remaining to be discovered and led to innovative technical approaches to finding them. In PNAS, Charlop-Powers et al. (7) use cultureindependent DNA sequencing on a broad environmental scale to systematize where different types of bacterially produced small molecules are likely to be found. Their “chemicalbiogeographic survey” provides insights into microbial ecology along with a practical guide for microbial genome prospectors. The potential scale of such a survey is staggering. Bacteria are the most diverse organisms www.pnas.org/cgi/doi/10.1073/pnas.1400516111
on the planet, and a single gram of soil can contain between a thousand and a million distinct species according to a pioneering survey (8). Species definition in bacteria has its whimsical aspects, but it usually means that a characteristic DNA sequence from one strain has, or does not have, a specified similarity to the sequence from another strain. If the similarity is high enough, say 98%, then the two strains are said to belong to the
Charlop-Powers et al. use culture-independent DNA sequencing on a broad environmental scale to systematize where different types of bacterially produced small molecules are likely to be found.
pathways: the polyketide (PK) pathway and the nonribosomal peptide (NRP) pathway, which assemble either carbon chains or amino acid chains from smaller subunits. These two pathways, either singly or in combination, make all of the examples mentioned earlier. The specific genes that were selected are necessary elements in both pathways, namely the genes that encode the proteins that select the smaller subunits. This necessary restriction has consequences, and antibiotics such as fosfomycin—whose biosyn-thesis follows a pathway different from PK or NRP pathways—would be overlooked. However, these limitations are obvious and could easily be addressed in future studies. To ensure a faithful representation of geographically resolved biosynthetic richness, the authors collect environmental DNA samples from more than 90 different locations. These samples served as templates for the preparation of ketosynthase (KS) and adenylation (AD) amplicons. Although these conditions introduced a modest amplification-dependent bias, the resulting amplicons closely mirror the variety of AD and KS domains found in the metagenome of the soil-dwelling bacteria. Subsequent 454 pyrosequencing of these amplicons allowed a classification system based on biosynthetic OTUs to be constructed. The OTU-based classification of environments was linked to environmental soil data including pH, moisture, and mineral composition among others gathered at the collection site, and the two data sets—biosynthetic OTUs and environmental parameters—could be cross-analyzed. A principal component analysis revealed three principal types of soils with different biosynthetic potential. For example, arid soil environments showed the largest biosynthetic potential.
same operational taxonomic unit (OTU). Researchers typically use the 16S ribosomal RNA gene sequence, a highly conserved sequence whose utility was established decades ago (9). Researchers isolate DNA from an environment—from desert soil to the human gut—and use PCR to amplify selected sequences from the sample for sequencing. These selected and amplified fragments of DNA are called amplicons. Amplicons of 16S rRNA have been widely used to create biogeographic surveys of soil bacteria—a catalog Biosynthetic Repertoires of Soil Types of what bacteria are found in different envi- The rich data set provided by this chemicalronments (9, 10). biogeographic survey provides two interesting insights. The first is that bacteria Biosynthesis-Based Genotyping of Soil in similar environments produce, or at least Microbiomes Charlop-Powers et al. used a similar ap- have amplicons that suggest they would proach to answer a different question: where produce, similar small molecules. A famous can we find genes that are likely to encode the production of useful molecules? Answering this question requires deciding on what genes, really what conserved DNA sequences on the genes, to look for. The authors select two important and well-studied biosynthetic
Author contributions: P.S. and J.C. wrote the paper. The authors declare no conflict of interest. See companion article on page 3757. 1
To whom correspondence should be addressed. E-mail: jon_clardy@ hms.harvard.edu.
PNAS | March 11, 2014 | vol. 111 | no. 10 | 3655–3656
generalization of microbial diversity notes that “Everything is everywhere, the environment selects.” (11) Soil variables, especially pH, largely control the environmental selection (10). These soil variables presumably select for bacteria with primary metabolisms that match a given environment. The present study demonstrates clearly that a plausible, but previously unproven, consequence of the environmental determinism of microbial biogeographic diversity is reflected in the secondary metabolism—the similar bacteria found in similar soils will produce similar compounds. This extreme environmental determinism in which soil variables control both biological and chemical diversity has no correlate in the biogeography of macroorganisms. Arid soils in geographically separated regions will, for example, host distinctly different plants that will produce different small molecules. Bacteria do not produce small molecules so that we can mine them for drugs; the molecules must confer some survival benefit. However, the ecological roles of these bacterially produced small molecules are poorly known, and their discovery represents a challenge for microbial ecology.
3656 | www.pnas.org/cgi/doi/10.1073/pnas.1400516111
A second conclusion, that arid soils have greater biosynthetic potential than pine forest soils or brackish sediments, might seem puzzling at first. Selman Waksman largely initiated the Golden Age of Antibiotics and won the 1952 Nobel Prize in Physiology or Medicine for his discovery of antibiotics, especially streptomycin, from swampy soils in New Jersey. The stream of antibiotics from Waksman’s laboratory, which all came from actinomycetes found in these soils, reflects his pioneering spirit that allowed him to succeed even with sampling suboptimal environments. Finally, Charlop-Powers et al. (7) provide a treasure map for drug prospec-
tors, and it will be interesting to see how finer-grained geographic and molecular analyses expand on this work. The chemical-biogeographic biodiversity approach could also be useful in exploring complex environments such as the human microbiome. We have increasingly good maps of what bacteria occupy different regions (and metabolic environments) of the human body. Will there be a similar correlation of chemistry and environment? This work also highlights the need for new techniques to convert genomic data into molecules, which is perhaps the greatest challenge facing genome-based drug discovery efforts.
1 Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Drugs for bad bugs: Confronting the challenges of antibacterial discovery. Nat Rev Drug Discov 6(1):29–40. 2 Nett M, Ikeda H, Moore BS (2009) Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat Prod Rep 26(11):1362–1384. 3 Baltz RH (2008) Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 8(5):557–563. 4 Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296(5570):1127–1129. 5 Winter JM, Behnken S, Hertweck C (2011) Genomics-inspired discovery of natural products. Curr Opin Chem Biol 15(1):22–31. 6 Challis GL (2008) Genome mining for novel natural product discovery. J Med Chem 51(9):2618–2628.
7 Charlop-Powers Z, Owen JG, Reddy BVB, Ternei MA, Brady SF (2014) Chemical-biogeographic survey of secondary metabolism in soil. Proc Natl Acad Sci USA 111:3757–3762. 8 Torsvik V, Øvreås L, Thingstad TF (2002) Prokaryotic diversity— magnitude, dynamics, and controlling factors. Science 296(5570): 1064–1066. 9 Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276(5313):734–740. 10 Fierer N, Jackson RB (2006) The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci USA 103(3): 626–631. 11 de Wit R, Bouvier T (2006) ‘Everything is everywhere, but, the environment selects’: What did Baas Becking and Beijerinck really say? Environ Microbiol 8(4):755–758.
Stallforth and Clardy
