Natural products in drug discovery: advances and opportunities - Nature
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Reviews
Natural products in drug discovery:
advances and opportunities
Atanas G. Atanasov 1,2,3,4 ✉, Sergey B. Zotchev2, Verena M. Dirsch 2
, the International
Natural Product Sciences Taskforce* and Claudiu T. Supuran 5 ✉
Abstract | Natural products and their structural analogues have historically made a major
contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless,
natural products also present challenges for drug discovery, such as technical barriers to screening,
isolation, characterization and optimization, which contributed to a decline in their pursuit by
the pharmaceutical industry from the 1990s onwards. In recent years, several technological and
scientific developments — including improved analytical tools, genome mining and engineering
strategies, and microbial culturing advances — are addressing such challenges and opening up
new opportunities. Consequently, interest in natural products as drug leads is being revitalized,
particularly for tackling antimicrobial resistance. Here, we summarize recent technological
developments that are enabling natural product-based drug discovery, highlight selected
applications and discuss key opportunities.
sp3 carbon atoms Historically, natural products (NPs) have played a key ‘bioactive’ compounds covering a wider area of chemical
Tetravalent carbon atoms role in drug discovery, especially for cancer and infec- space compared with typical synthetic small-molecule
forming single covalent bonds tious diseases1,2, but also in other therapeutic areas, libraries13.
with other atoms within the including cardiovascular diseases (for example, statins) Despite these advantages and multiple successful
molecular structure. A higher
fraction of sp3 carbons within
and multiple sclerosis (for example, fingolimod)3–5. drug discovery examples, several drawbacks of NPs have
molecules is a descriptor that NPs offer special features in comparison with con- led pharmaceutical companies to reduce NP-based drug
indicates more complex ventional synthetic molecules, which confer both advan- discovery programmes. NP screens typically involve a
3D structures. tages and challenges for the drug discovery process. library of extracts from natural sources (Fig. 1), which
1
Institute of Genetics and NPs are characterized by enormous scaffold diversity may not be compatible with traditional target-based
Animal Biotechnology of the and structural complexity. They typically have a higher assays14. Identifying the bioactive compounds of inter-
Polish Academy of Sciences, molecular mass, a larger number of sp3 carbon atoms and est can be challenging, and dereplication tools have to
Jastrzebiec, Poland.
oxygen atoms but fewer nitrogen and halogen atoms, be applied to avoid rediscovery of known compounds.
2
Department of
higher numbers of H-bond acceptors and donors, lower Accessing sufficient biological material to isolate and
Pharmacognosy, University
of Vienna, Vienna, Austria. calculated octanol–water partition coefficients (cLogP characterize a bioactive NP may also be challenging15.
3
Institute of Neurobiology, values, indicating higher hydrophilicity) and greater Furthermore, gaining intellectual property (IP) rights
Bulgarian Academy of molecular rigidity compared with synthetic compound for (unmodified) NPs exhibiting relevant bioactivities
Sciences, Sofia, Bulgaria. libraries1,6–9. These differences can be advantageous; for can be a hurdle, since naturally occurring compounds
4
Ludwig Boltzmann Institute example, the higher rigidity of NPs can be valuable in in their original form may not always be patented (legal
for Digital Health and Patient drug discovery tackling protein–protein interactions10. frameworks vary between countries and are evolving)16,
Safety, Medical University
Indeed, NPs are a major source of oral drugs ‘beyond although simple derivatives can be patent-protected
of Vienna, Vienna, Austria.
Lipinski’s rule of five’11. The increasing significance of (Box 1). An additional layer of complexity relates to the
5
Università degli Studi di
Firenze, NEUROFARBA drugs not conforming to this rule is illustrated by the regulations defining the need for benefit sharing with
Dept, Sezione di Scienze increase in molecular mass of approved oral drugs over countries of origin of the biological material, framed
Farmaceutiche, Florence, Italy. the past 20 years12. NPs are structurally ‘optimized’ in the United Nations 1992 Convention on Biological
*A full list of members and by evolution to serve particular biological functions1, Diversity and the Nagoya Protocol, which entered into
affiliations is presented including the regulation of endogenous defence mech- force in 2014 (ref.17), as well as recent developments con-
at the end of the paper.
anisms and the interaction (often competition) with cerning benefit sharing linked to use of marine genetic
✉e-mail: a.atanasov.
other organisms, which explains their high relevance for resources18.
mailbox@gmail.com;
claudiu.supuran@unifi.it infectious diseases and cancer. Furthermore, their use Although the complexity of NP structures can be
https://doi.org/10.1038/ in traditional medicine may provide insights regarding advantageous, the generation of structural analogues to
s41573-020-00114-z efficacy and safety. Overall, the NP pool is enriched with explore structure–activity relationships and to optimize
200 | March 2021 | volume 20 www.nature.com/nrdReviews
Problem 1 Solutions for 1 and 2
Not possible to culture the • New methods for culturing
organism out of the natural • New methods for in situ
habitat analysis Problem 6 Solution for 6
Unknown molecular New methods
Problem 2 Solution for 2 targets for NPs for molecular
Relevant NPs are not produced New methods for NP synthesis identified with mode of action
when the organism is taken out induction and heterologous phenotypic assays elucidation
of the natural habitat expression of biosynthetic genes
Multiple
rounds of
fractionation
Crude Single
Organisms Fractions
extracts compounds
Problem 3 Solution for 3 Bioactivity
Presence of NPs that are known New methods for evaluation
dereplication
Problem 4
Presence of NPs that do not Solution for 4 and 5
have drug-like properties New methods for
extraction and
Problem 5 pre-fractionation
Bioactive NPs are present in of extracts
insufficient amounts
Lipinski’s rule of five
This guideline for the likelihood
of a compound having oral
bioavailability is based on Fig. 1 | Outline of traditional bioactivity-guided isolation steps in natural product drug discovery. Steps in the
several characteristics process are shown in purple boxes, with associated key limitations shown in red boxes and advances that are helping to
containing the number 5. address these limitations in modern natural product (NP)-based drug discovery shown in green boxes. The process begins
It predicts that a molecule is with extraction of NPs from organisms such as bacteria. The choice of extraction method determines which compound
likely to have poor absorption classes will be present in the extract (for example, the use of more polar solvents will result in a higher abundance of polar
or permeation if it has more compounds in the crude extract). To maximize the diversity of the extracted NPs, the biological material can be subjected
than one of the following to extraction with several solvents of different polarity. Following the identification of a crude extract with promising
characteristics: there are
pharmacological activity, the next step is its (often multiple) consecutive bioactivity-guided fractionation until the pure
>5 H-bond donors and
>10 H-bond acceptors; the
bioactive compounds are isolated. A key limitation for the potential of this approach to identify novel NPs is that many
molecular weight is >500; potential source organisms cannot be cultured or stop producing relevant NPs when taken out of their natural habitat.
or the partition coefficient These limitations are being addressed through development of new methods for culturing, for in situ analysis, for NP
LogP is >5. Notably, natural synthesis induction and for heterologous expression of biosynthetic genes. At the crude extract step, challenges include
products were identified as the presence in the extracts of NPs that are already known, NPs that do not have drug-like properties or insufficient
common exceptions at the amounts of NPs for characterization. These challenges can be addressed through the development of methods for
time of publication in 1997. dereplication, extraction and pre-fractionation of extracts. Finally, at the last stage, when bioactive compounds are
identified by phenotypic assays, significant time and effort are typically needed to identify the affected molecular targets.
Dereplication
This challenge can be addressed by the development of methods for accelerated elucidation of molecular modes of action,
Pharmacological screening of
natural product extracts yields
such as the nematic protein organization technique (NPOT), drug affinity responsive target stability (DARTS), stable isotope
hits potentially containing labelling with amino acids in cell culture and pulse proteolysis (SILAC-PP), the cellular thermal shift assay (CETSA) and an
multiple natural products that extension known as thermal proteome profiling (TPP), stability of proteins from rates of oxidation (SPROX), the similarity
need to be considered for ensemble approach (SEA) and bioinformatics-based analysis of connectivity (connectivity map, CMAP)23,189–192.
further study to identify
the bioactive compounds. NP leads can be challenging, particularly if synthetic areas: analytical techniques, genome mining and engi-
Dereplication is the process of
recognizing and excluding from
routes are difficult. Also, NP-b ased drug leads are neering, and cultivation systems. In the concluding sec-
further study such hit mixtures often identified by phenotypic assays, and deconvolu- tion, we highlight promising future directions for NP
that contain already known tion of their molecular mechanisms of action can be drug discovery.
bioactive compounds. time-consuming19. Fortunately, there have been sub-
stantial advances20 both in the development of screening Application of analytical techniques
Phenotypic assays
Assays that rely on the ability assays (for example, harnessing the potential of induced Classical NP-based drug research starts with biological
of tested compounds to exert pluripotent stem cells and gene editing technologies) screening of ‘crude’ extracts to identify a bioactive ‘hit’
desired phenotypic changes and in strategies to identify the modes of action of active extract, which is further fractionated to isolate the active
in cells, isolated tissues, organs compounds (reviewed previously21–23). NPs. Bioactivity-guided isolation is a laborious process
or animals. They offer a
complementary strategy
Here, we discuss recent technological and scien- with a number of limitations, but various strategies and
to target-based assays for tific advances that may help to overcome challenges in technologies can be used to address some of them (Fig. 2).
identifying new potential drugs. NP-based drug discovery, with an emphasis on three For example, to create libraries that are compatible
NaTure RevIewS | DRug DiScOvERy volume 20 | March 2021 | 201Reviews
Box 1 | Natural products that activate the KEAP1/NRF2 pathway with high-throughput screening, crude extracts can be
pre-fractionated into sub-fractions that are more suit-
An example of a pathway affected by diverse natural products (NPs) is the KEAP1/NRF2 able for automated liquid handling systems. In addi-
pathway. This pathway regulates the expression of networks of genes encoding proteins tion, fractionation methods can be adjusted so that
with versatile cytoprotective functions and has essential roles in the maintenance
sub-fractions preferentially contain compounds with
of redox and protein homeostasis, mitochondrial biogenesis and the resolution of
drug-like properties (typically moderate hydrophilicity).
inflammation196–199.
Activation of this pathway can protect against damage by most types of oxidants and Such approaches can increase the number of hits com-
pro-inflammatory agents, and it restores redox and protein homeostasis200. The pathway pared with using crude extracts, as well as enabling more
has therefore attracted attention for the development of drugs for the prevention efficient follow-up of promising hits24.
and treatment of complex diseases, including neurological conditions such as Metabolomics was developed as an approach to
relapsing–remitting multiple sclerosis201 and autism spectrum disorder202. simultaneously analyse multiple metabolites in biologi
Dimethyl fumarate (DMF), the methyl ester of the NP fumarate (a tricarboxylic acid cal samples. Enabled by technological developments in
(TCA) cycle intermediate that is found in both animals and plants), is one of the earliest chromatography and spectrometry, metabolomics was
discovered inducers of the KEAP1/NRF2 pathway203,204. The origins of the development of historically applied first in other research fields, such as
DMF as a drug date back to the use in traditional medicine of the plant Fumaria officinalis.
biomedical and agricultural sciences2. Advances in the
Initially, fumaric acid derivatives were used for the treatment of psoriasis as it was
analytical instrumentation used in NP research25,26, cou-
thought that psoriasis is caused by a metabolic deficiency in the TCA cycle that could
be compensated for by repletion of fumarate205. Despite this erroneous assumption, pled with computational approaches that can generate
DMF is effective in treating psoriasis, both topically and orally, and is the active principle plausible NP analogue structures and their respective
of Fumaderm, which has been used clinically for several decades in the treatment of plaque simulated spectra27, have also enabled application of
psoriasis in Germany. More recently, a DMF formulation developed by Biogen has been ‘omics’ approaches such as metabolomics in NP-based
tested in other immunological disorders, with successful phase III trials in multiple drug discovery. Metabolomics can provide accurate infor-
sclerosis206,207 leading to its approval by the FDA and EMA in 2013. mation on the metabolite composition in NP extracts,
The isothiocyanate sulforaphane, isolated from broccoli (Brassica oleracea)208, is among thus helping to prioritize NPs for isolation, to accelerate
the most potent naturally occurring inducers of the KEAP1/NRF2 pathway209 and has dereplication28,29 and to annotate unknown analogues
protective effects in animal models of Parkinson210, Huntington211 and Alzheimer212
and new NP scaffolds. Moreover, metabolomics can
diseases, traumatic brain injury213, spinal cord contusion injury214, stroke215, depression216
detect differences between metabolite compositions in
and multiple sclerosis217. Sulforaphane-rich broccoli extract preparations are being
developed as preventive interventions in areas of the world with unavoidable exposure various physiological states of producing organisms and
to environmental pollutants, such as China; the initial results of a randomized clinical trial enable the generation of hypotheses to explain them,
showed rapid and sustained, statistically significant increases in the levels of excretion and can also provide extensive metabolite profiles to
of the glutathione-derived conjugates of benzene and acrolein218, and a follow-up trial underpin phenotypic characterization at the molecular
(NCT02656420) also demonstrated dose–response-dependent benzene detoxification219. level30. Both options are very useful in understanding the
In a placebo-controlled, double-blind, randomized clinical trial in young individuals molecular mechanisms of action of NPs.
(age 13–27 years) with autism spectrum disorder, sulforaphane reversed many of the For metabolite profiling, NP extracts are analysed by
clinical abnormalities202; these encouraging findings led to a recently completed clinical NMR spectroscopy or high-resolution mass spectrom-
trial in children (age 3–12 years) (NCT02561481; results of the trial are not yet publicly
etry (HRMS), or respective combined methods involv-
available). An α-cyclodextrin complex of sulforaphane known as SFX-01 (developed by
ing upstream liquid chromatography (LC)31,32, such as
Evgen Pharma) is being clinically studied for its potential to reverse resistance to endocrine
therapies in patients with ER+HER2- metastatic breast cancer (phase II trial completed220) LC–HRMS, which can separate numerous isomers pres-
and in patients with subarachnoid haemorrhage (phase II trial NCT02614742 recently ent in NP extracts33. Moreover, such combined methods
completed; results not yet publicly available). Currently, a clinical trial of SFX-01 in patients might integrate HRMS and NMR, allowing the simulta-
hospitalized with COVID-19 is in its final stages of preparation. neous use of the advantages of both techiques34,35. NMR
Finally, the pentacyclic triterpenoids bardoxolone methyl (also known as RTA 402) and analysis of NP extracts is simple and reproducible, and
omaveloxolone (RTA 408), which are semi-synthetic derivatives of the NP oleanolic acid, provides direct quantitative information and detailed
are the most potent (active at nanomolar concentrations) activators of the KEAP1/NRF2 structural information, although it has relatively low sen-
pathway known to date221. These compounds have shown protective effects in numerous sitivity, meaning that it generally enables profiling only
animal models of chronic disease222, and are currently in clinical trials for a wide range
of major constituents33. The applications of NMR in NP
of indications, such as chronic kidney disease in type 2 diabetes, pulmonary arterial
research are versatile36 and the technique is used both
hypertension, melanoma, radiation dermatitis, ocular inflammation and Friedreich’s
ataxia200. Most recently, bardoxolone methyl has entered a clinical trial in patients directly for metabolomics of unfractionated NP extracts
hospitalized with confirmed COVID-19 (NCT04494646). and for structural characterization of compounds and
O fractions obtained with appropriate separation methods,
O
O S most often LC. HRMS is the gold standard for qualita-
O S C
N tive and quantitative metabolite profiling33 and is most
O
commonly applied in combination with LC. HRMS can
Dimethyl fumarate Sulforaphane also be used in the direct infusion mode (called DIMS)37,
whereby samples are directly profiled by MS without a
chromatography step, or in MS imaging (MSI)38, which
O
HH
O
HH O
enables determination of the spatial distribution of NPs
O
within living organisms. HRMS enables routine acqui-
N N N
H
sition of accurate molecular mass information, which
F
O F together with appropriate heuristic filtering can pro-
O O vide unambiguous assignment of molecular formulae
H H for hundreds to thousands of metabolites within a sin-
Bardoxolone methyl (RTA 402) Omaveloxolone (RTA 408) gle extract over a dynamic range that may exceed five
202 | March 2021 | volume 20 www.nature.com/nrdReviews
a
Bacterial strains isolated from
marine sediment from the Image-based phenotypic LC–HRMS-based metabolomics
coastal areas of Panama were bioactivity profiling of the data also recorded with the 234
used for the preparation of 234 NP extracts in HeLa cells NP extracts
234 NP extracts
O OH
R
Integration and clustering of the
Me N biological and chemical datasets
N N revealed 13 unique clusters
H
O
Quinocinnolinomycin A R =
Quinocinnolinomycin B R =
Quinocinnolinomycin C R = One of the clusters was
prioritized for further study, and
a combination of LC–MS and NMR
Quinocinnolinomycin D R = analysis led to the identification
of quinocinnolinomycins A–D
b
156 FACs generated containing Selected 56 FACs predicted
56 FACs transformed into the
unique BGCs from three to contain uncharacterized
heterologous expression host
species from the Aspergillus BGCs (i.e. BGCs with no
(A. nidulans) and NP extracts
genus known product or well-
prepared
characterized homologue)
15 new metabolites and their Metabolomics data analysed
BGCs were characterized through with FAC-Score algorithm
NP extracts subjected to
combination of gene deletions that filters out signals present
untargeted LC–HRMS
within the BGCs and additional in host extracts or in more
LC–MS and NMR analysis than one FAC strain
Fig. 2 | Applications of advanced analytical technologies empowering modern natural product-based drug discovery.
a | An illustrative example of the application of liquid chromatography–high-resolution mass spectrometry (LC–HRMS)
metabolomics in the screening of natural product (NP) extracts is the work of Kurita et al.58, in which 234 bacterial extracts
were subjected to image-based phenotypic bioactivity screening and LC–HRMS metabolomics. Clustering of the resulting
data allowed prioritization of promising extracts for further analysis, resulting in the discovery of the new NPs, quinocin-
nolinomycins A–D. b | Another illustrative example of LC–HRMS screening of NP extracts is the work of Clevenger et al.85,
who obtained novel NP extracts through heterologous expression of fungal artificial chromosomes (FACs) containing
uncharacterized biosynthetic gene clusters (BGCs) from diverse fungal species in Aspergillus nidulans. Analysis of the
LC–HRMS metabolomics data with a FAC-Score algorithm directed the simultaneous discovery of 15 new NPs and
the characterization of their BGCs.
orders of magnitude31,39. However, challenges remain in In this respect, the Global Natural Products Social
data mining and in the unambiguous identification of (GNPS) molecular networking platform developed
the metabolites using various workflows relying on open in the Dorrestein laboratory is an important addition
web-based tools40. to the toolbox41. Molecular networking organizes thou-
Dereplication of secondary metabolites in bioactive sands of sets of MS/MS data recorded from a given set
extracts includes the determination of molecular mass of extracts and visualizes the relationship of the ana-
and formula and cross-searching in the literature or lytes as clusters of structurally related molecules. This
structural NP databases with taxonomic information, improves the efficiency of dereplication by enabling
which greatly assists the identification process. Such annotation of isomers and analogues of a given metab-
metadata, which are difficult to query in the literature, olite in a cluster42. The recorded experimental spectra
are often compiled in proprietary databases, such as the can be searched against putative structures and their
Dictionary of Natural Products, which encompasses corresponding predicted MS/MS spectra generated
all NP structures reported with links to their biological by tools such as competitive fragmentation modelling
sources (see Related links). However, a comprehensive (CFM-ID)43. Based on such approaches, vast databases
experimental tandem mass spectrometry (MS/MS) data- of theoretical NP spectra have been created and applied
base of all NPs reported to date does not exist, and a in dereplication44. The GNPS molecular networking
search for experimental spectra across various platforms approach has limitations, however, such as better appli-
is hindered by the lack of standardized collision energy cability to some classes of NPs than others and the
conditions for fragmentation in LC–MS/MS25. uncertainty of structural assignment among possible
NaTure RevIewS | DRug DiScOvERy volume 20 | March 2021 | 203Reviews
predicted candidates. Efforts to address such issues are of biosynthetically prolific bacteria, which enabled the
ongoing45–47, including overlaying molecular networks of identification of new bioactive polyketides59.
large NP extract libraries with taxonomic information to Finally, advances in analytical technologies con-
improve the confidence of annotation48. Overall, molec- tinue to support the rigorous structure determination
ular networking mainly allows better prioritization of NPs of interest. The progressive development of
of the isolation of unknown compounds by strengthen- higher-field NMR instruments and probe technology60,61
ing the dereplication process and elucidating relation- has enabled NP structure determination from very small
ships between NP analogues, and rigorous structure quantities (below 10 µg)62,63, which is important, as the
elucidation for NPs of interest should not be neglected. available quantities of NPs are often limited. In addi-
Another useful platform for metabolite identifica- tion, microcrystal electron diffraction (MicroED) has
tion is METLIN49, which includes a high-resolution recently emerged as a cryo-electron microscopy-based
MS/MS database with a fragment similarity search technique for unambiguous structure determination
function that is useful for identification of unknown of small molecules64 and is already finding important
compounds. Other databases and in silico tools such applications in NP research65. The increased resolution
as Compound Structure Identification (CSI): FingerID and sensitivity of analytical equipment can also help
and Input Output Kernel Regression (IOKR) can be used address problems associated with ‘residual complex-
to search available fragment ion spectra, as well as to ity’ of isolated NPs; that is when biologically potent
generate predicted spectra of fragment ions not present but unidentified impurities in an isolated NP sample
in current databases50. A novel computational platform (which could include structurally related metabolites or
for predicting the structural identity of metabolites conformers) lead to an incorrect assignment of structure
derived from any identified compound has also been and/or activity66,67. To avoid futile downstream develop-
recently reported51, which should increase the searchable ment efforts, Pauli and colleagues recommended that
chemical space of NPs. lead NPs should undergo advanced purity analysis at an
To accelerate the identification of bioactive NPs in early stage using quantitative NMR and LC–MS67.
extracts, metabolomics data can be matched to the bio-
logical activities of these extracts52. Various chemometric Genome mining and engineering
methods such as multivariate data analysis can correlate Advances in knowledge on biosynthetic pathways for NPs
the measured activity with signals in the NMR and MS and in developing tools for analysing and manipulating
spectra, enabling the active compounds to be traced in genomes are further key drivers for modern NP-based
complex mixtures with no need for further bioassays53–55. drug discovery. Two key characteristics enable the iden-
Furthermore, several analytical modules involving dif- tification of biosynthetic genes in the genomes of the
ferent bioassays and detection technologies can be producing organisms. First, these genes are clustered in
linked to allow simultaneous bioactivity evaluation and the genomes of bacteria and filamentous fungi. Second,
identification of compounds present in small amounts many NPs are based on polyketide or peptide cores,
(analytical scale) in complex compound mixtures34,35. and their biosynthetic pathways involve enzymes —
Metabolomics data can be integrated with data polyketide synthases (PKSs) and nonribosomal peptide
obtained by other omics techniques such as transcrip- synthetases (NRPSs), respectively — that are encoded by
tomics and proteomics and/or with imaging-b ased large genes with highly conserved modules68.
screens. For example, Acharya et al. used this approach ‘Genome mining’ is based on searches for genes
to characterize NP-mediated interactions between a that are likely to govern biosynthesis of scaffold struc-
Micromonospora species and a Rhodococcus species56. tures, and can be used to identify NP biosynthetic gene
In another interesting example, Kurita et al. developed a clusters69–71. Prioritization of gene clusters for further
compound activity mapping platform for the prediction work is facilitated by advances in biosynthetic know
of identities and mechanisms of action of constituents ledge and predictive bioinformatics tools, which can pro-
from complex NP extract libraries by integrating cyto- vide hints about whether the metabolic products of the
logical profiling57 with untargeted metabolomics data clusters have chemical scaffolds that are new or known,
from a library of extracts58, and identified quinocinno- thereby supporting dereplication72,73. Such predictive
linomycins as a new family of NPs causing endoplasmic tools for gene cluster analysis can be applied in combi-
reticulum stress58 (Fig. 2a). nation with spectroscopic techniques to accelerate the
Analytical advances that enable the profiling of identification of NPs65 and determine the stereochem-
responses to bioactive molecules at the single-c ell istry of metabolic products66. Furthermore, to extend
level can also accelerate NP-based drug discovery. Irish, genome mining from a single genome to entire genera,
Bachmann, Earl and colleagues developed a high- microbiomes or strain collections, computational tools
Phylogenomic approach throughput platform for metabolomic profiling of bio- have been developed, such as BiG-SCAPE, which enables
The use of genomic data to
activity by integrating phospho-specific flow cytometry, sequence similarity analysis of biosynthetic gene clusters,
reveal evolutionary
relationships. In the context of single-cell chemical biology and cellular barcoding with and CORASON, which uses a phylogenomic approach
natural product drug discovery, metabolomic arrays (characterized chromatographic to elucidate evolutionary relationships between gene
the use of phylogenomics is microtitre arrays originating from biological extracts)59. clusters74.
based on the assumption that Using this platform, the authors studied the single-cell Phylogenetic studies of known groups of talented
organisms that have closer
evolutionary relationships are
responses of bone marrow biopsy samples from patients secondary metabolite producers can also empower
more likely to produce similar with acute myeloid leukaemia following exposure to discovery of novel NPs. Recently, a study comparing
natural products. microbial metabolomic arrays obtained from extracts secondary metabolite profiles and phylogenetic data in
204 | March 2021 | volume 20 www.nature.com/nrdReviews
Taxonomic distance myxobacteria demonstrated a correlation between the To transfer a biosynthetic gene cluster of such size, a plat-
The distance of compared taxa taxonomic distance and the production of distinct sec- form based on transformation-associated recombination
on a constructed phylogenetic ondary metabolite families75. In filamentous fungi, it was (TAR) cloning was developed. This platform enables
tree (also known as an likewise shown that secondary metabolite profiles are direct cloning and manipulation of large biosynthetic
evolutionary tree). Closer
distance of compared taxa
closely correlated with their phylogeny76. These organ- gene clusters in S. cerevisiae, maintenance and manipula-
indicates a closer evolutionary isms are rich in secondary metabolites, as demonstrated tion of the vector in E. coli, and heterologous expression
relationship. by LC–MS studies of their extracts under laboratory of the cloned gene clusters in Actinobacteria (such as
conditions77. Concurrent genomic and phylogenomic S. coelicolor) following chromosomal integration88, and
analyses implied that even the genomes of well-studied is an alternative to BACs for heterologous expression of
organism groups harbour many gene clusters for sec- large biosynthetic gene clusters.
ondary metabolite biosynthesis with as yet unknown Heterologous expression has limitations, such as
functions78. The phylogeny of biosynthetic gene clusters, the need to clone and manipulate very large genome
together with analysis of the absence of known resistance regions occupied by biosynthetic gene clusters and the
determinants, was recently used to prioritize members of difficulty of identifying a suitable host that provides all
the glycopeptide antibiotic family that could have novel conditions necessary for the production of the corre-
activities. This led to the identification of the known sponding NPs. These limitations can be circumvented
antibiotic complestatin and the newly discovered cor- by activating biosynthetic gene clusters directly in the
bomycin as compounds that act through a previously native microorganism through targeted genetic manip-
uncharacterized mechanism involving inhibition of ulations, generally involving the insertion of activating
peptidoglycan remodelling79. regulatory elements or deletion of inhibitory elements
Many microorganisms cannot be cultured, or tools such as repressors or their binding sites. For example,
for their genetic manipulation are not sufficiently a derepression strategy of deleting gbnR, a gene for a
developed, which makes it more challenging to access transcriptional repressor in Streptomyces venezuelae
their NP-producing potential. However, biosynthetic ATCC 10712 was used by Sidda et al. in the discovery of
gene clusters for NPs can be cloned and heterologously gaburedins, a family of γ-aminobutyrate-derived ureas89.
expressed in organisms that are well-characterized An example of the activator-based strategy is the consti-
and easier to culture and to genetically manipulate tutive expression of the samR0484 gene in Streptomyces
(such as Streptomyces coelicolor, Escherichia coli and ambofaciens ATCC 23877, which led to the discovery
Saccharomyces cerevisiae) 80. The aim is to achieve of stambomycins A–D, 51-membered cytotoxic glyco-
higher production titres in the heterologous hosts sylated macrolides72. Alternatively, silent biosynthetic
than in wild-type strains, improving the availability of gene clusters can be activated using repressor decoys90,
lead compounds80–82. Vectors that can carry large DNA which have the same DNA nucleotide sequence as the
inserts are needed for the cloning of complete NP bio- binding sites for the repressors that prevent the expres-
synthetic gene clusters. Cosmids (which can have inserts sion of the clusters. When these decoys are introduced
of 30–40 kb), fosmids (which can harbour 40–50 kb) and into the bacteria, they sequester the respective repres-
bacterial artificial chromosomes (BACs; which can have sors, and the ‘endogenous’ binding sites in the genome
inserts of 100 kb to >300 kb) have been developed83. For remain unoccupied, leading to derepression of the pre-
fungal gene clusters, self-replicating fungal artificial viously silent biosynthetic genes and production of the
chromosomes (FACs) have been developed, which can corresponding NPs. This approach has been applied to
have inserts of >100 kb (ref.84). FACs in combination activate eight silent biosynthetic gene clusters in multi-
with metabolomic scoring were used to develop a scal- ple streptomycetes and led to the characterization of a
able platform, FAC-MS, allowing the characterization novel NP, oxazolepoxidomycin A90. The repressor decoy
of fungal biosynthetic gene clusters and their respec- strategy is simpler, easier and faster to perform than the
tive NPs at unprecedented scale85. The application of deletion of genes encoding regulatory factors. However,
FAC-MS for the screening of 56 biosynthetic gene clus- it has the same limitation as other approaches that rely
ters from different fungal species yielded the discovery on the introduction of recombinant DNA molecules into
of 15 new metabolites, including a new macrolactone, cells: it is necessary to develop protocols for efficient
valactamide A85 (Fig. 2b). introduction of DNA into the targeted host strain, and
Even in culturable microorganisms, many biosyn- the decoy must be maintained on a high-copy plasmid
thetic gene clusters may not be expressed under con- to ensure efficient repressor sequestration.
ventional culture conditions, and these silent clusters Another approach focused on exchange of regu
could represent a large untapped source of NPs with latory elements is based on the CRISPR–Cas9 technol-
drug-like properties86. Several approaches can be pur- ogy. The promise of this technique is exemplified in a
sued to identify such NPs. One approach is sequencing, recent work by Zhang et al., which demonstrated that
bioinformatic analysis and heterologous expression CRISPR–Cas9-mediated targeted promoter introduction
of silent biosynthetic gene clusters, which has already can efficiently activate diverse biosynthetic gene clusters
led to the discovery of several new NP scaffolds from in multiple Streptomyces species, leading to the produc-
cultivable strains87. Direct cloning and heterologous tion of unique metabolites, including a novel polyketide
expression was also used to discover the new antibiotic in Streptomyces viridochromogenes91. The CRISPR–Cas9
taromycin A, which was identified upon the transfer technology was also used to knock out genes encoding
of a silent 67 kb NRPS biosynthetic gene cluster from two well-k nown and frequently rediscovered anti
Saccharomonospora sp. CNQ-490 into S. coelicolor88. biotics in several actinomycete strains, which led to the
NaTure RevIewS | DRug DiScOvERy volume 20 | March 2021 | 205Reviews
a
DNA isolation from
Sequencing Bioinformatics analysis NP chemical
specific microorganism/
(genome mining) synthesis
environment/microbiota
Heterologous Genetic manipulations
expression of the native host
NP isolation and
characterization
b
Analysed sequences for BGCs Selected a soil sample rich in BGCs
Sequenced that code for lipopeptides from a tree branch not associated
metagenomes of with calcium-binding motifs with the BGCs for known
2,000 soil samples and clustered to create a calcium-binding antibiotics and
phylogenetic tree cloned DNA in a cosmid library
O
HO O
H H
N N O
N O
H
O
N O HN OH OH
O OH Identified a predicted BGC
O
and transferred into
HN O NH O Streptomyces albus using
O O transformation-associated
H H recombination
N N
N N
H H
O OH O
O
R NH2
Malacidin A R = Me
Malacidin B R = Et
Malacidins isolated from cultures and
their structures elucidated using a Extracts from cultures of S. albus
combination of mass spectrometry and harbouring the BGC found to
NMR data, supported by bioinformatics show antibacterial activity
analysis of the BGC against Staphylococcus aureus
c
Analysed genomic sequence Chemically synthesized 25
data from human microbiome ‘synthetic–bioinformatic’
for gene clusters predicted Identified 57 unique
NP-like compounds predicted
to encode large (≥5 residues) NRPS gene clusters
to be encoded by the analysed
nonribosomal peptides gene clusters
OH
O O R1 O R2 O
H H H H
N N N N
N N N OH
H H H
OH O O O O
OH
OH OH
Humimycin A R1 = L-Phe, R2 = L-Val
Humimycin B R1 = L-Tyr, R2 = L-Ile
Tested the 25 NP-like
Identified humimycins as compounds for activity
new antibiotics active against a panel of common
against methicillin-resistant human commensal and
S. aureus pathogenic bacteria
206 | March 2021 | volume 20 www.nature.com/nrdReviews
◀ Fig. 3 | Strategies for genome mining-driven discovery of natural products and was derived from a set of ~100 genes through variable
natural product-like compounds. a | Genome mining-based approaches to explore peptide processing96.
the biosynthetic capacity of microorganisms rely on DNA extraction, sequencing and Some bioactive compounds initially isolated from
bioinformatics analysis. The vast majority of microbes from different environments marine organisms might be products of symbionts, and
and microbiota communities have not been cultured, and their capacity to produce
genome mining can facilitate the characterization of
natural products (NPs) was largely inaccessible until recently. In the case of unculturable
microorganisms, the bioinformatics analysis step can be followed by either targeted
such NPs. For example, it has been shown that bioactive
heterologous expression of biosynthetic gene clusters (BGCs) prioritized as being likely compounds from the sponge Theonella swinhoei are pro-
to yield relevant new NPs or direct chemical synthesis of ‘synthetic–bioinformatic’ duced by bacterial symbionts97, and characterization of
NP-like compounds. b,c | These two approaches are exemplified by the recent discoveries the symbiont ‘Candidatus Entotheonella serta’ using
of malacidins (panel b) and humimycins (panel c), respectively93,94. A major strength of the single-cell genomics led to the discovery of gene clus-
‘synthetic–bioinformatic’ approach is that it is entirely independent of microbial culture ters for misakinolide and theonellamide biosynthesis98.
and gene expression. Its limitations are the accuracy of computational chemical structure Another example of a marine NP produced by a bacte-
predictions and the feasibility of total chemical synthesis. NRPS, nonribosomal peptide rial symbiont is ET-743 (trabectedin), originally isolated
synthetase. from the tunicate Ecteinascidia turbinate. A meta-omics
approach developed by Rath et al. revealed that the
production of different rare and previously unknown producer of this clinically used anticancer agent is the
variants of antibiotics that were otherwise obscured, bacterial symbiont ‘Candidatus Endoecteinascidia
including amicetin, thiolactomycin, phenanthroviridin frumentensis’99.
and 5-chloro-3-formylindole92. Similarly, plant microbiomes also represent a large
Approaches that rely on sequencing, bioinformat- reservoir for the identification of novel bioactive NPs
ics and heterologous expression can also enable the (such as the antitumour agents maytansine, paclitaxel
identification of novel NPs from bacterial strains that and camptothecin, which were initially isolated from
have not yet been cultivated (Fig. 3a) . For example, plants and later shown to be produced by microbial
Hover et al. searched the metagenomes of 2,000 soil endophytes)100 that can be tapped by genome mining
samples for biosynthetic gene clusters for lipopeptides approaches. An illustrative example is a recent work
with calcium-binding motifs. This led to the discov- by Helfrich et al. that identified hundreds of novel bio
ery of malacidins, members of the calcium-dependent synthetic gene clusters by genome mining of 224 bac
antibiotic family, via heterologous expression of a 72 kb terial strains isolated from Arabidopsis thaliana leaves101.
biosynthetic gene cluster from a desert soil sample in a A combination of bioactivity screening and imaging mass
Streptomyces albus host strain93 (Fig. 3b). However, in spectrometry was used to select a single species for fur-
comparison with some of the other above-discussed ther genomic analysis and led to the isolation of a NP with
strategies 72,89,90, this metagenome-b ased discovery an unprecedented structure, the trans-acyltransferase
approach is more suited to finding new members of PKS-derived antibiotic macrobrevin101.
known NP classes rather than discovery of entirely Targeted genetic engineering of NP biosynthetic gene
new classes. In another study, Chu et al. developed a clusters can be of high value if the producing organism
human microbiome-based approach that identified is difficult to cultivate or the yield of a NP is too low
nonribosomal linear heptapeptides called humimycins to allow comprehensive NP characterization. Rational
as novel antibiotics active against methicillin-resistant genetic engineering and heterologous expression con-
Staphylococcus aureus (MRSA)94 (Fig. 3c). The structure tributed to increase the production of vioprolides, a
of the NPs was predicted via bioinformatics analysis of depsipeptide class of anticancer and antifungal NPs in
gene clusters found in human commensal bacteria, fol- the myxobacterium Cystobacter violaceus Cb vi35, by
lowed by their chemical synthesis. A major strength of several orders of magnitude. In addition, non-natural
this innovative approach is that it is entirely independent vioprolide analogues were generated by this approach102.
of microbial cultivation and heterologous gene expres- Similarly, promoter engineering and heterologous
sion. Nevertheless, there are limitations related to the expression of biosynthetic gene clusters was reported
accuracy of computational chemical structure predic- to result in a 7-fold increase in the production of the
tions and the feasibility of total chemical synthesis if cytotoxic NP disorazol103, and a 328-fold increase in
structures are complex. the production of spinosad, an insecticidal macrolide
The genomes of plants or animals can also be mined produced by the bacterium Saccharopolyspora spinosa104.
for novel NPs. For example, mining of 116 plant genomes Besides increasing NP yields, targeted gene manip-
enabled by identification of a precursor gene for the ulation can also be used to alter biosynthetic pathways
biosynthesis of lyciumins, a class of branched cyclic in a predictable manner to produce new NP analogues
ribosomal peptides with hypotensive action produced with improved pharmacological properties, such as
by Lycium barbarum (popularly known as goji), identi- higher specific activity, lower toxicity and better pharma
fied diverse novel lyciumin chemotypes in seven other cokinetics. Such biosynthetic engineering approaches
plants, including crops such as soybean, beet, quinoa and depend on a solid understanding of the biosynthetic
eggplant95. Genome mining in the animal kingdom is pathway leading to a specific NP, access to the genes
exemplified by the work of Dutertre et al., which used specifying this pathway and the ability to manipulate
an integrated transcriptomics and proteomics approach them in either the original or a heterologous host.
to discover thousands of novel venom peptides from Recent advances in biosynthetic engineering have
Conus marmoreus snails96. Proteomics analysis revea enabled faster and more efficient production of NP
led that the vast majority of the conopeptide diversity analogues, including the development of methods for
NaTure RevIewS | DRug DiScOvERy volume 20 | March 2021 | 207Reviews
accelerated engineering and recombination of modules identification of specific growth factors, allowing expan-
of PKS gene clusters105, NRPSs106,107 and NRPS–PKS sion of the number of species that can be successfully
assembly lines108, as well as elucidation of mechanisms cultured. This strategy was used by D’Onofrio et al. for
for polyketide chain release that are contributing to the identification of new acyl-desferrioxamine sidero-
NP structural diversification109,110. Examples of bio phores (iron-chelating compounds) as growth factors
synthetic engineering applied to several important NPs produced by helper strains promoting the growth of
include the generation of analogues of the immuno previously uncultured isolates from marine sedi-
suppressant rapamycin 111, the antitumour agents ment biofilm117,126. The siderophore-assisted growth is
mithramycin112 and bleomycin113, and the antifungal based on the property of these compounds to provide
agent nystatin114. iron for microbes unable to autonomously produce
It should be noted that biosynthetic engineering has siderophores themselves, and the application of this
limitations regarding the parts of the NP molecule that approach led to the isolation of previously uncultivated
can be targeted for modifications, and the chemical microorganisms126. The development of strategies to
groups that can be introduced or removed. Considering cultivate microbial symbionts that produce NPs only
the complexity of many NPs, however, total synthesis upon interaction with their hosts can promote access
may be prohibitively costly, and a combined approach to new NPs. Microbial symbionts interacting with
of biosynthetic engineering and chemical modification insects or other organisms are a highly promising reser-
can provide a viable alternative for identifying improved voir for the discovery of novel bioactive NPs produced
drug candidates. For example, biosynthetic engineering in a unique ecological context127–130. To stimulate NP
may create a ‘handle’ for addition of a beneficial chem- production, culturing strategies can be developed that
ical group by synthetic chemistry, as demonstrated for better mimic the native environment of microbial sym-
the biosynthetically engineered analogues of nystatin bionts of insects, including the use of media containing
mentioned above; further synthetic chemistry modifi- either lyophilized dead insects131 or l-proline, a major
cations resulted in compounds with improved in vivo constituent of insect haemolymph132.
pharmacotherapeutic characteristics compared with Strategies to mimic the natural environment even
amphotericin B115,116. more closely by harnessing in situ incubation in the
environment from which the microorganism is sam-
Advances in microbial culturing systems pled have been developed, dating back to more than
The complex regulation of NP biosynthesis in response 20 years ago with the biotech companies OneCell and
to the environment means that the conditions under Diversa. They developed platforms that allowed the
which producing organisms are cultivated can have a growth of some previously uncultivated microbes from
major impact on the chance of identifying novel NPs87. various environments based on diluting out and sus-
Several strategies have been developed to improve the pension in a single drop of medium120,133. More recently,
likelihood of identifying novel NPs compared with such strategies have been highlighted by the develop-
monoc ulture under standard laboratory conditions ment and application of a platform dubbed the iChip,
and to make ‘uncultured’ microorganisms grow in a in which diluted soil samples are seeded in multiple
simulated natural environment117 (Fig. 4). small chambers separated from the environment with
One well-established approach to promote the identi- a semipermeable membrane134. After seeding, the iChip
fication of novel NPs is the modulation of culture condi- is placed back into the soil from which the sample was
tions such as temperature, pH and nutrient sources. This taken for an in situ incubation period, allowing the cul-
strategy may lead to activation of silent gene clusters, tured microorganisms to be exposed to influences from
thereby promoting production of different NPs. The their native environment. The power of this culturing
term ‘One Strain Many Compounds’ (OSMAC) was approach was demonstrated by the discovery of a new
coined for this approach about 20 years ago118, but the antibiotic, teixobactin, produced by a previously uncul-
concept has a longer history119, with its use being routine tured soil bacterium135,136 (Fig. 4a). This platform may be
in industrial microbiology since the 1960s120. of great significance for NP drug discovery, given that
While OSMAC is still widely used for the identifi- it has been estimated that only 1% of soil organisms
cation of new bioactive compounds121,122, this approach have so far been successfully cultured using traditional
has limited capacity to mimic the complexities of natu- culturing techniques137.
ral habitats. It is difficult to predict the combination of The omics strategies discussed in previous sections
cues (which might also involve metabolites secreted by can complement efforts to explore NPs produced upon
other members of the microbial community) to which microbial interactions. The application of such a strategy
the microorganism has evolved to respond by switching is illustrated in the work of Derewacz et al., who analysed
metabolic programmes. To account for such kinds of the metabolome of a genome-sequenced Nocardiopsis
interactions, co-culturing using ‘helper’ strains can be bacterium upon co-culture with bacteria of the genera
applied123. This can enable the production and identifica- Escherichia, Bacillus, Tsukamurella and Rhodococcus138.
tion of new NPs, as illustrated by recent studies in which Around 14% of the metabolomic features found in
particular fungi were co-cultured with Streptomcyes co-cultures were undetectable in monocultures, with
species124,125. many of those being unique to specific co-culture gen-
Study of the molecular mechanisms underlying era, and the previously unreported polyketides ciro-
the ability of helper strains to increase the cultivabil- micin A and B, which possess an unusual pyrrolidinol
ity of previously uncultured microbes can lead to the substructure and displayed moderate and selective
208 | March 2021 | volume 20 www.nature.com/nrdReviews
a
Extracts from 10,000 iChip An extract from a new
iChip device with diluted soil
isolates screened for bacterial species, Eleftheria
samples incubated in soil to
antimicrobial activity terrae, showed good
simultaneously grow and
against Staphylococcus antimicrobial activity
isolate uncultured bacteria
aureus against S. aureus
O
O NH2 O
N
H Compound isolation from the
O E. terrae extract, and structure
O O O O HN
H H H H H NH elucidation by NMR and
N N N N N advanced Marfey’s analysis
N N N N O
H H H H N NH yielded teixobactin, a new
O O O O H antibiotic with activity against
OH OH
Gram-positive bacteria
Teixobactin
b
Microbial species selected Bioinformatics analysis Antigens representative of the
from human oral microbiome identified membrane proteins selected extracellular protein
for targeted isolation based with extracellular domains that domains designed, synthesized
on genomic sequence data could serve as antigens for and injected into rabbits for
antibody development antibody production
Three species of Saccharibacterium Oral microbiota samples stained
isolated along with their interacting with fluorescently labelled IgG purified from the rabbit
Actinobacterium hosts, as well as antibodies, followed by flow serum and fluorescently
SR1 bacteria that are members of cytometry and cell sorting to labelled
a candidate phylum with no isolate the targeted bacterial
previously cultured representatives species
Fig. 4 | Application of advanced microbial culturing approaches to identify new natural products. New strategies
for isolating previously uncultured microorganisms can enable access to new natural products (NPs) produced by them.
a | To recapitulate the effect of complex signals coming from the native environment, microorganisms can be cultivated
directly in the environment from which they were isolated. This concept is used with the iChip platform, in which diluted
environmental samples are seeded in multiple small chambers separated from the native environment with a semipermeable
membrane. The potential of this approach is illustrated by the recent discovery of teixobactin, a new antibiotic with
activity against Gram-positive bacteria134,135. b | Another important recent development involves obtaining information from
environmental samples using omics techniques such as metagenomics to identify and partially characterize microorganisms
present in a specific environment before culturing. An approach relying on such preliminary information was recently
used to engineer the capture of antibodies based on genetic information, which resulted in the successful cultivation of
previously uncultured bacteria from the human mouth145. This reverse genomics workflow was validated by the isolation
and cultivation of three species of Saccharibacteria (TM7) along with their interacting Actinobacteria hosts, as well as
SR1 bacteria that are members of a candidate phylum with no previously cultured representatives.
cytotoxicity, were identified138. Other examples include such ‘easy to culture’ microbes have already been charac-
a ‘culturomics’ approach that combines multiple culture terized, and conventional screening efforts tend to yield
conditions with MS profiling and 16S rRNA-based tax- disappointing returns associated with frequent rediscov-
onomy to identify prokaryotic species from the human ery of known NPs and their closely related congeners.
gut139, and an ultrahigh-throughput screening platform Therefore, culturing strategies aimed at previously unex-
based on microfluidic droplet single-cell encapsulation plored (or under-investigated) microbial groups, with
and cultivation followed by next-generation sequenc- the potential to produce NPs with entirely new scaffolds
ing and LC–MS, which allows investigation of pairwise and bioactivities (such as Burkholderia, Clostridium and
interactions between target microorganisms140. The lat- Xenorhabdus) are of high interest141,142. Closthioamide,
ter approach enabled identification of a slow-growing the first secondary metabolite from a strictly anaerobic
oral microbiota species that inhibits the growth of bacterium, was discovered from Clostridium cellulo-
S. aureus140. lyticum by this approach143. Targeted isolation of such
Historically early-a dopted microbial culturing species is important, and a genome-guided approach
approaches led to a bias reflected in the predominant to achieve this goal has recently been demonstrated
discovery of NPs from microorganisms that are easy to for Burkholderia strains in environmental samples144.
cultivate (such as streptomycetes and some common fil- Another highly innovative approach to the isolation
amentous fungi). As a result, a vast number of NPs from and cultivation of previously uncultured bacteria was
NaTure RevIewS | DRug DiScOvERy volume 20 | March 2021 | 209You can also read
