Natural products in drug discovery: advances and opportunities

Historically, natural products (NPs) have played a key role in drug discovery, especially for cancer and infectious diseases 1 , 2 , but also in other therapeutic areas, including cardiovascular diseases (for example, statins) and multiple sclerosis (for example, fingolimod) 3 – 5 . NPs offer special features in comparison with conventional synthetic molecules, which confer both advantages and challenges for the drug discovery process. NPs are characterized by enormous scaffold diversity and structural complexity. They typically have a higher molecular mass, a larger number of sp 3 carbon atoms and oxygen atoms but fewer nitrogen and halogen atoms, higher numbers of H-bond acceptors and donors, lower calculated octanol–water partition coefficients (cLogP values, indicating higher hydrophilicity) and greater molecular rigidity compared with synthetic compound libraries 1 , 6 – 9 . These differences can be advantageous; for example, the higher rigidity of NPs can be valuable in drug discovery tackling protein–protein interactions 10 . Indeed, NPs are a major source of oral drugs ‘beyond Lipinski’s rule of five ’ 11 . The increasing significance of drugs not conforming to this rule is illustrated by the increase in molecular mass of approved oral drugs over the past 20 years 12 . NPs are structurally ‘optimized’ by evolution to serve particular biological functions 1 , including the regulation of endogenous defence mechanisms and the interaction (often competition) with other organisms, which explains their high relevance for infectious diseases and cancer. Furthermore, their use in traditional medicine may provide insights regarding efficacy and safety. Overall, the NP pool is enriched with ‘bioactive’ compounds covering a wider area of chemical space compared with typical synthetic small-molecule libraries 13 . Despite these advantages and multiple successful drug discovery examples, several drawbacks of NPs have led pharmaceutical companies to reduce NP-based drug discovery programmes. NP screens typically involve a library of extracts from natural sources (Fig. 1 ), which may not be compatible with traditional target-based assays 14 . Identifying the bioactive compounds of interest can be challenging, and dereplication tools have to be applied to avoid rediscovery of known compounds. Accessing sufficient biological material to isolate and characterize a bioactive NP may also be challenging 15 . Furthermore, gaining intellectual property (IP) rights for (unmodified) NPs exhibiting relevant bioactivities can be a hurdle, since naturally occurring compounds in their original form may not always be patented (legal frameworks vary between countries and are evolving) 16 , although simple derivatives can be patent-protected (Box 1 ). An additional layer of complexity relates to the regulations defining the need for benefit sharing with countries of origin of the biological material, framed in the United Nations 1992 Convention on Biological Diversity and the Nagoya Protocol, which entered into force in 2014 (ref. 17 ), as well as recent developments concerning benefit sharing linked to use of marine genetic resources 18 . Fig. 1 Outline of traditional bioactivity-guided isolation steps in natural product drug discovery. Steps in the process are shown in purple boxes, with associated key limitations shown in red boxes and advances that are helping to address these limitations in modern natural product (NP)-based drug discovery shown in green boxes. The process begins with extraction of NPs from organisms such as bacteria. The choice of extraction method determines which compound classes will be present in the extract (for example, the use of more polar solvents will result in a higher abundance of polar compounds in the crude extract). To maximize the diversity of the extracted NPs, the biological material can be subjected to extraction with several solvents of different polarity. Following the identification of a crude extract with promising pharmacological activity, the next step is its (often multiple) consecutive bioactivity-guided fractionation until the pure bioactive compounds are isolated. A key limitation for the potential of this approach to identify novel NPs is that many potential source organisms cannot be cultured or stop producing relevant NPs when taken out of their natural habitat. These limitations are being addressed through development of new methods for culturing, for in situ analysis, for NP synthesis induction and for heterologous expression of biosynthetic genes. At the crude extract step, challenges include the presence in the extracts of NPs that are already known, NPs that do not have drug-like properties or insufficient amounts of NPs for characterization. These challenges can be addressed through the development of methods for dereplication, extraction and pre-fractionation of extracts. Finally, at the last stage, when bioactive compounds are identified by phenotypic assays, significan

Advances in knowledge on biosynthetic pathways for NPs and in developing tools for analysing and manipulating genomes are further key drivers for modern NP-based drug discovery. Two key characteristics enable the identification of biosynthetic genes in the genomes of the producing organisms. First, these genes are clustered in the genomes of bacteria and filamentous fungi. Second, many NPs are based on polyketide or peptide cores, and their biosynthetic pathways involve enzymes — polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), respectively — that are encoded by large genes with highly conserved modules 68 . ‘Genome mining’ is based on searches for genes that are likely to govern biosynthesis of scaffold structures, and can be used to identify NP biosynthetic gene clusters 69 – 71 . Prioritization of gene clusters for further work is facilitated by advances in biosynthetic knowledge and predictive bioinformatics tools, which can provide hints about whether the metabolic products of the clusters have chemical scaffolds that are new or known, thereby supporting dereplication 72 , 73 . Such predictive tools for gene cluster analysis can be applied in combination with spectroscopic techniques to accelerate the identification of NPs 65 and determine the stereochemistry of metabolic products 66 . Furthermore, to extend genome mining from a single genome to entire genera, microbiomes or strain collections, computational tools have been developed, such as BiG-SCAPE, which enables sequence similarity analysis of biosynthetic gene clusters, and CORASON, which uses a phylogenomic approach to elucidate evolutionary relationships between gene clusters 74 . Phylogenetic studies of known groups of talented secondary metabolite producers can also empower discovery of novel NPs. Recently, a study comparing secondary metabolite profiles and phylogenetic data in myxobacteria demonstrated a correlation between the taxonomic distance and the production of distinct secondary metabolite families 75 . In filamentous fungi, it was likewise shown that secondary metabolite profiles are closely correlated with their phylogeny 76 . These organisms are rich in secondary metabolites, as demonstrated by LC–MS studies of their extracts under laboratory conditions 77 . Concurrent genomic and phylogenomic analyses implied that even the genomes of well-studied organism groups harbour many gene clusters for secondary metabolite biosynthesis with as yet unknown functions 78 . The phylogeny of biosynthetic gene clusters, together with analysis of the absence of known resistance determinants, was recently used to prioritize members of the glycopeptide antibiotic family that could have novel activities. This led to the identification of the known antibiotic complestatin and the newly discovered corbomycin as compounds that act through a previously uncharacterized mechanism involving inhibition of peptidoglycan remodelling 79 . Many microorganisms cannot be cultured, or tools for their genetic manipulation are not sufficiently developed, which makes it more challenging to access their NP-producing potential. However, biosynthetic gene clusters for NPs can be cloned and heterologously expressed in organisms that are well-characterized and easier to culture and to genetically manipulate (such as Streptomyces coelicolor , Escherichia coli and Saccharomyces cerevisiae ) 80 . The aim is to achieve higher production titres in the heterologous hosts than in wild-type strains, improving the availability of lead compounds 80 – 82 . Vectors that can carry large DNA inserts are needed for the cloning of complete NP biosynthetic gene clusters. Cosmids (which can have inserts of 30–40 kb), fosmids (which can harbour 40–50 kb) and bacterial artificial chromosomes (BACs; which can have inserts of 100 kb to >300 kb) have been developed 83 . For fungal gene clusters, self-replicating fungal artificial chromosomes (FACs) have been developed, which can have inserts of >100 kb (ref. 84 ). FACs in combination with metabolomic scoring were used to develop a scalable platform, FAC-MS, allowing the characterization of fungal biosynthetic gene clusters and their respective NPs at unprecedented scale 85 . The application of FAC-MS for the screening of 56 biosynthetic gene clusters from different fungal species yielded the discovery of 15 new metabolites, including a new macrolactone, valactamide A 85 (Fig. 2b ). Even in culturable microorganisms, many biosynthetic gene clusters may not be expressed under conventional culture conditions, and these silent clusters could represent a large untapped source of NPs with drug-like properties 86 . Several approaches can be pursued to identify such NPs. One approach is sequencing, bioinformatic analysis and heterologous expression of silent biosynthetic gene clusters, which has already led to the discovery of several new NP scaffolds from cultivable strains 87 . Direct cloning and heterologous expressio

The technological advances discussed above have the potential to reinvigorate NP-based drug discovery in both established and emerging areas. NPs have long been the key source of new drugs against infectious diseases, especially antibiotics (reviewed elsewhere 151 , 152 ). Selected NPs with antimicrobial properties discovered by leveraging advances discussed in the sections above, including strategies to exploit the human microbiome for novel NPs 94 , 153 are highlighted in Figs 3 , 4 . Along with the search for new NPs with antimicrobial activities, researchers are continuing to develop and optimize already known NP classes, making use of advances in biosynthetic engineering 154 , total synthesis 155 or semi-synthetic strategies 156 , 157 . In addition, antivirulence strategies could represent an alternative approach to fighting infections 158 , for which NPs targeting bacterial quorum sensing could be of interest 159 . NPs also have a successful history as cancer therapeutics, which has been well covered in other reviews 160 – 163 . An important new opportunity in this field is the capacity of some NPs to trigger a selective yet potent host immune reaction against cancer cells, particularly given the intense interest at present in strategies that could improve response rates to immune checkpoint inhibitors by turning ‘cold’ tumours ‘hot’ 164 . For example, NPs such as cardiac glycosides 165 can increase the immunogenicity of stressed and dying cancer cells by triggering immunogenic cell death, characterized by the release of damage-associated molecular patterns (DAMPs), which could open new avenues for drug discovery or repurposing 166 – 168 . Botanical therapies containing complex mixtures of NPs have long attracted interest owing to the potential for synergistic therapeutic effects of components within the mixture 169 , 170 . However, the variability of the NP composition in the starting plant material owing to factors such as environmental variations in the location at which the plants were collected is a major challenge for the development of botanical drugs 1 . With the advances in technology for their characterization, such as metabolomics discussed above, as well as development of regulatory guidance for complex mixtures of NPs ( see Related links ), it is becoming more feasible to develop such mixtures as therapeutics, rather than to identify and purify a single active ingredient 171 . Since gut microbiota are considered to play a major role in health and disease 172 – 174 , and NPs are known to affect the gut microbiome composition 175 – 178 , this area is an emerging opportunity for NP-based drug discovery. However, drug discovery efforts in this area are still in their infancy, with many open questions remaining 179 . A future direction may be the characterization of single microbiota-derived species for particular therapeutic applications, and the advances in culturing strategies, genome mining and analytics discussed above will be of great importance in this respect. Many advances discussed above are supported by computational tools including databases (such as genomic, chemical or spectral analysis data; see ref. 180 for a recent review on NP databases) and tools that enable the analysis of genetic information, the prediction of chemical structures and pharmacological activities 181 , the integration of data sets with diverse information (such as tools for multi-omics analysis) 182 and machine learning applications 183 . Although this Review focuses on technologies that enable the discovery of novel NPs, it is important to acknowledge that unmodified NPs may possess suboptimal efficacy or absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. So, for development of NP hits into leads and ultimately into successful drugs, chemical modification may be required. In addition, bringing a compound into clinical development requires a sustainable and economically viable supply of sufficient quantities of the compound. Total chemical synthesis, semi-synthesis using a NP as a starting point for analogue generation and biosynthetic engineering modifying biosynthetic pathways of the producing organism will be of great importance in this context (Fig. 5 ). Recent advances in chemical synthesis and biosynthetic engineering technologies are strongly empowering NP-based drug discovery and development by enabling property optimization of complex NP scaffolds that were previously regarded as inaccessible. This allows the enrichment of screening libraries with NPs, NP hybrids, NP analogues and NP-inspired molecules, as well as superior structure functionalization approaches (including late-stage functionalization) for optimization of NP leads 94 , 105 – 108 , 184 – 188 . Fig. 5 Strategies to obtain natural product analogues with superior properties. Unmodified natural products (NPs) often possess suboptimal properties, and superior analogues need to be obtained in order to yield valu