Bioactive Peptide Natural Products as Lead Structures for Medicinal Use

Article pubs.acs.org/accounts

Bioactive Peptide Natural Products as Lead Structures for Medicinal Use Published as part of the Accounts of Chemical Research special issue “Chemical Biology of Peptides”. Tam Dang and Roderich D. Süssmuth* Technische Universität Berlin, Institut für Chemie, Fachgebiet Biologische Chemie, Strasse des 17. Juni 124, 10623 Berlin, Germany

CONSPECTUS: The need for new drugs for the treatment of various diseases is enormous. From the previous century until the present, numerous peptide and peptide-derived natural products have been isolated from bacteria and fungi. Hence, microorganisms play a pivotal role as sources for novel drugs with an emphasis on anti-infective agents. Various disciplines from biology, chemistry, and medicine are involved in early stages of the search for peptide natural products including taxonomy, microbiology, bioanalytics, bioinformatics, and medicinal chemistry. Under biochemical aspects, small peptide drugs are basically either ribosomally synthesized and post-translationally modified (RiPPs) or synthesized by multimodular nonribosomal peptide synthetases (NRPSs). Within the context of current developments on bioactive peptide natural products, this Account predominantly highlights recent discoveries, approaches, and research from our laboratory on RiPPs and NRPSs from bacteria and fungi. In our search for peptides showing bioactivities of interest, different approaches were applied: classical screening, in silico prediction, in vitro reconstitution, site-directed mutagenesis, chemoenzymatics, heterologous expression, and total synthesis including structure− activity relationship (SAR) studies in the research on the labyrinthopeptins, albicidin, and the cyclodepsipeptides (CDPs). The ribosomally synthesized labyrinthopeptins, class III lanthipeptides, which have been discovered in a classical screening campaign, display highly attractive antiallodynic (against neuropathic pain caused by dysfunction of the nervous system) and antiviral activities. Therefore, the biosynthetic assembly was investigated by extensive enzymatic studies of the modifying enzymes, and site-directed mutagenesis was performed for the generation of analogs. By genome mining, other class III lanthipeptides have been uncovered, while synthetic access proved to be an unmet challenge for the labyrinthopeptins. In contrast, for the gyrase inhibitor albicidin, the establishment of a chemical synthesis followed by medicinal chemistry studies was the only viable option to gain access to derivatives. Albicidin, which has been discovered investigating plant host−pathogen interactions, has a strong activity against Gram-negative bacteria, for example, Escherichia coli and Pseudomonas aeruginosa, and a future synthetic derivative may become a lead structure for development of an anti-Gram-negative drug. The compound class of the cyclodepsipeptides contributes already two marketed drugs, enniatin (fusafungine) and emodepside. Cyclodepsipeptides show general antibacterial and antifungal effects, whereas specific insecticidal and anthelmintic activities provide lead structures for drug development. Hence, exploiting the chances of reprogramming NRPSs, the generation of chimeric or otherwise designed synthetases could render a new untapped structural space and thus novel bioactivities. While current developments in the fields of genomics, bioinformatics, and molecular biology facilitate the search for new natural products and the design of new peptide structures, the next decade will show which compounds have been carried on further applications and whether current developments have led to an increase in drug candidates.

1. INTRODUCTION

the HIV fusion inhibitor enfuvirtide, and liraglutide, a glucagonlike peptide-1 receptor agonist for treatment of type II diabetes.

In recent years, peptides have received increased attention by the pharmaceutical industry. It is thought that they may fill the gap between small molecules and protein drugs combining advantages of both worlds. Examples are the hormone insulin, © 2017 American Chemical Society

Received: March 31, 2017 Published: June 26, 2017 1566

DOI: 10.1021/acs.accounts.7b00159 Acc. Chem. Res. 2017, 50, 1566−1576

Article

Accounts of Chemical Research

Figure 1. (a) General representation of RiPP biosynthesis. (b) Lanthipeptide classes and important PTMs. (c) RiPPs with unique properties for therapeutic use (unusual amino acids are highlighted in blue and orange color).

Bioinformatic tools aid in identifying biosynthetic gene clusters and predicting structures of secondary metabolites. According to their biosynthetic mechanism, peptide natural products are categorized into two fundamentally different types: ribosomally synthesized and post-translationally modified peptides (RiPPs) and nonribosomal peptides (NRPs).

These examples are isostructural or structural analogues of their endogenous physiological equivalents. In the bacterial and fungal world, there also exists a plethora of peptides that have antibacterial, antifungal, and cytotoxic effects and also have been widely used as drugs for decades, for example, vancomycin (antibacterial) or the echinocandins (antifungal). With a focus on bacteria and fungi, a number of key questions emerge in modern peptide natural product research. First is the discovery of new structural diversity and bioactivity, ideally against novel molecular targets. Second is the understanding of the biosynthesis on molecular biological and biochemical levels, followed by engineering of the biosynthetic machineries to design complex peptide architectures. In the search for new peptide structures, classical biological and chemical screening approaches are complemented by high-throughput sequencing technologies combined with bioinformatic tools such as genome mining. Genome mining, which comprises in silico1 or in vivo2 search of gene databases for individual genes or gene clusters, facilitates the handling of vast amounts of genomic data.

2. RIBOSOMALLY SYNTHESIZED AND POST-TRANSLATIONALLY MODIFIED PEPTIDES To date, there are >20 RiPP families (MW < 10 kDa) known,21 which are subdivided into members according to their characteristic structural and biosynthetic features. Some RiPPs have been considered as lead structures for drug use and a number of examples exist where RiPPs have entered clinical trials. Due to an increased understanding of biosynthetic principles combined with advances in strain and pathway engineering of the microbial producer, RiPPs have undergone a renaissance as potential drug leads. 1567


Article


Figure 2. (a) Principle of NRPS assembly. (b) Important NRPs with unique properties for therapeutic use, nonproteinogenic amino acids (green), and nonpeptide-derived substrates (blue).

Ser or Thr and Cys, respectively. A dehydration step rendering the 2,3-didehydroamino acid is followed by a Michael-type addition resulting in the formation of a thioether. Nisin is naturally produced by Lactococcus lactis and used as food preservative. Actagardine A is a fairly small tetracyclic lantibiotic, and the semisynthetic analogue NVB302 (aminoheptylamido-deoxyactagardine B) has entered clinical trials for the treatment of Clostridium dif ficile infections.24 Microbisporicin is one of the more recently discovered lantibiotics with activity against Grampositive bacteria. It resembles the N-terminal structure of nisin and the core structure of epidermin with some unusual amino acid modifications. While the subgroup of lantibiotics consists of a vast number of representatives, which mainly interfere with the bacterial cell wall biosynthesis of Gram-positive bacteria, a small number of lanthipeptides shows antiallodynic,9 antiviral,25 or morphogenetic activities.26 One representative is duramycin with an unusual lysinoalanine. It binds to phosphatidyl ethanolamine and was suggested for treatment of cystic fibrosis.27 The microviridins are a stand-alone family of RiPPs, which commonly occur as tricyclic depsipeptides. Their potential to inhibit serine proteases

A common biosynthetic feature of RiPPs is that the ribosomally synthesized precursor peptide (20−110 aa) undergoes post-translational processing. The precursor peptide commonly consists of two obligatory (leader and core) and two optional (signal and recognition) parts present in the amino acid sequences. The leader peptide facilitates recognition and processing by PTM enzymes. The modified peptide undergoes proteolytic cleavage of the leader peptide and the matured peptide is exported from the cell to exert its function (Figure 1). Among others, the lanthipeptides, the cyanobactins, the thiopeptides, the linear azol(in)e-containing peptides (LAPs), the lasso peptides, the microviridins, and the fungal amatoxins belong to the RiPP families. For many of these peptides, macrocyclization is an essential structural element for bioactivity and provides further benefits such as increased metabolic stability and improved cellular uptake.22 The well-known RiPP nisin belongs to the lanthipeptides and is subclassified as a lantibiotic due to its antibacterial activity.23 The characteristic structural feature is lanthionine (Lan) and β-methyllanthionine (MeLan), which are synthesized from 1568


Article

Accounts of Chemical Research Table 1. Selected RiPPs and NRPs with Proven or Potential Application As Drugs compound actagardine (gardimycin) α-amanitin albicidin duramycin (Moli1901, lancuvotide) griselimycin labyrinthopeptin lugdunin lysobactin (katanosin B) microbisporicin (NAI-107) microviridin nannocystin A nisin PF1022A teixobactin telomycin a

year of discovery

organisma

19763 19644 19855 19586

Actinoplanes garbadinensis (AB) Amanita phalloides (BM) Xanthomonas albilineans (PB) Streptomyces cinnamomeus forma azacoluta (AB) Streptomyces sp. (AB) Actinomadura namibiensis8 (AB) Staphylococcus lugdunensis11 (FC) Lysobacter sp. (PB) Microbispora corallina (AB) Microcystis viridis (CB) Nannocystis sp. (PB) Lactococcus lactis (FC) Rosellinia spp. (F) Elef theria terrae (PB) Streptomyces canus (AB)

19717 20109,10 201612 198813 200814 199015 201516 192817 199218 201519 195720

bioactivity antibacterial (Gram-positive) antitumor antibacterial (Gram-negative) antibacterial (Gram-positive), antiviral antituberculosis antiallodynic, antiviral antibacterial (Gram-positive) antibacterial (Gram-positive) antibacterial (Gram-positive) serine protease inhibitor antitumor antibacterial (Gram-positive) anthelmintic antibacterial (Gram-positive) antibacterial (Gram-positive)

RiPPs or NRPs

literature

RiPP RiPP NRP (+PKS) RiPP

3 4 5 6

NRP RiPP NRP NRP RiPP RiPP NRP (+PKS) RiPP NRP NRP NRP

7 8−10 11,12 13 14 15 16 17 18 19 20

Actinobacteria (AB), Cyanobacteria (CB), Firmicutes (FC), Proteobacteria (PB), Fungi (F), Basidiomycota (BM).

is a cyclodepsipeptide and active against Mycobacterium tuberculosis, which may become applicable for the treatment of tuberculosis.7 It targets the DnaN sliding clamp of bacterial DNA polymerase. Lysobactin, a cyclodepsipeptide with a high number of β-hydroxylated amino acids, shows strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).13 Teixobactin was recently discovered using the iChip technology developed for difficult-to-cultivate bacteria.19 Like lysobactin, teixobactin inhibits bacterial cell wall biosynthesis by binding to lipid II. Most recently, lugdunin was described from the commensal bacterium Staphylococcus lugdunensis living in the human nose.12 This cyclic peptide is particularly active against resistant pathogens such as VRE, MRSA, and glycopeptide-intermediate-resistant Staphylococcus aureus (GISA). Unlike the abovementioned NRPs, the cyclic peptide nannocystin A has a potent antiproliferative and antitumor activity and targets the eukaryotic translation factor 1.16,32

has gained recent attention, for example, for treatment of lung emphysema.28 Apart from RiPPs produced by bacteria, there are only a few RiPPs produced by fungi, for example, α-amanitin, synthesized by various Amanita species (“deathcap”). α-Amanitin is highly toxic due to its inhibition of the eukaryotic RNA polymerase II. For future drug use, it has been suggested as an antibody−drug conjugate in cancer treatment.29

3. NONRIBOSOMALLY SYNTHESIZED PEPTIDES NRPs are synthesized by multifunctional, modular enzyme complexes termed nonribosomal peptide synthetases (NRPSs).30 Compared to RiPPs, NRPs clearly have the bigger share among marketed peptide drugs. Biosynthetic investigations of the past decades have significantly leveraged the understanding of the modular NRP assembly: a common NRPS assembly line consists of initiation, elongation, and termination modules. A minimal module consists of three main domains: adenylation (A), thiolation or peptide carrier protein (T or PCP), and condensation (C). The A domain specifically recognizes and activates proteinogenic or nonproteinogenic amino acids, among other substrates, and directs covalent attachment to a 4′-phosphopantetheine (PPant) arm of the PCP domain (Figure 2). In the C domain, amide bonds are formed leading to the peptide elongation. The polypeptide termination by reaction with a nucleophile can occur in different ways, for example, by the thioesterase (TE) domain, a hydrolytic step rendering a linear peptide, by the N-terminus (head-to-tail cyclized peptide), or by the amino acid side chain. Additional domains exist for structural modification purposes: formylation (F), cyclization (Cy), oxidation (Ox), reduction (R, Red), epimerization (E), and methylation (M). Even if unusual substrates bring peculiar modifications along, the final polypeptide is often modified after termination of the NRPS assembly, for example, by glycosylations, acylations, or halogenations.30,31 Due to the vast number of NRPs known to date, only few peptides, which have more recently raised some interest, shall be mentioned. As the above selection of peptides (Table 1) shows, most of the NRPs are depsipeptides that display antibacterial activities. Telomycin is a cyclodepsipeptide with two unusual Trp modifications and shows strong activity against Gram-positive bacteria, particularly against Staphylococcus aureus.20 Griselimycin

4. THE SEARCH FOR NOVEL POTENTIAL DRUG LEADS An essential key element next to a promising bioactivity of a natural product peptide is certainly knowledge of the chemical structure. Nowadays, this is accompanied by genome sequencing of the producing organism, localization of the biosynthetic gene cluster, and assignment of biosynthetic functions. Since the structure provided by nature in many cases does not exhibit the required properties, an optimization by structure−activity relationship (SAR) studies is performed. In this context, it has to be assessed whether bioengineering approaches or chemical synthesis are appropriate avenues to follow. 4.1. Labyrinthopeptins, a Novel RiPP Class of Lanthipeptides

The labyrinthopeptins were found in a screening campaign from Actinomadura namibiensis DSM6313, isolated from a soil sample from Namibia’s desert.8 They belong to the class III lanthipeptides, and their particular structural feature is the triamino triacid labionin (Lab).9 Labyrinthopeptin A2 (LabA2) is an 18-mer peptide that contains two Lab moieties acting as a clamp for the formation of the A/A′- and B/B′-rings. The 20-mer peptide labyrinthopeptin A1 (LabA1) varies from LabA2 in the primary sequence and an extension of the B′-ring. 1569


Article


Figure 3. (a) Structure of labyrinthopeptins and other class III lanthipeptides. (b) Biosynthetic gene cluster and suggested PTMs due to processing of LabA2 by LabKC. The presentation of phosphorylation, elimination, and cyclization events is schematic and does not reflect the exact progression of the biosynthesis. (c) Structures of engineered variants of labyrinthopeptin. 1570


Article


Figure 4. (a) Proposed biosynthetic pathway of albicidin. Polyketide synthase (PKS)−nonribosomal peptide synthetase (NRPS) hybrids consist of several domains: acyl-CoA ligase (AL), acyl carrier protein (ACP), trans-acting acyltransferase (AT), ketosynthase (KS), dehydrogenase (DH), ketoreductase (KR), methyltransferase (MT), peptide carrier (T), condensation (C), adenylation (A), and thioesterase (TE). (b) Adenylation domain 1571


Article

Accounts of Chemical Research Figure 4. continued

model for activation of phenylalanine (GrsA) and p-aminobenzoic acids (albicidin). (c) Derivatization of albicidin at the N-terminus (building block A) and at the central L-Cya (building block C).

manipulation of A. namibiensis has been proven intractable and heterologous expression could not be achieved in Escherichia coli, unlike for the two-component lantibiotic lichenicidin.42 Heterologous expression in Streptomyces lividans enabled the design of a library of 39 mutants, which comprise amino acid exchanges, ring contractions and expansions, and rearrangements of the ring topology (Figure 3).43 This library of mutants generates analogues of labyrinthopeptin by genetic engineering, which will be employed for future studies on antiviral and antiallodynic properties.

The sequencing of the biosynthetic labyrinthopeptin (lab) gene cluster revealed a biosynthetic origin of Lab from a conserved Ser/Ser/Cys motif in the core peptide.10 The gene cluster only consists of five genes coding for the LabA1/LabA2 precursor peptides, the modifying enzyme LabKC, and the transporters LabT1 and LabT2. The in vitro reconstitution of the biosynthesis with the LabA2 precursor peptide and the enzyme LabKC consisting of a lyase, a Ser/Thr kinase, and a Lan cyclase domain revealed GTP as a cosubstrate. Bioactivity profiling rendered an unexpected antiallodynic activity of LabA2 in a mouse model for neuropathic pain (ED50 = 50 μg kg−1).10 While LabA1 was significantly less active, it showed a remarkable antiviral activity against the herpes simplex virus (HSV; EC50 = 0.29−2.8 μM) and the human immunodeficiency virus (HIV; EC50 = 0.7−3.3 μM).25 The remarkable bioactivities prompted the investigation on mechanistic details of the biosynthesis of class III lanthipeptides. Genome mining for related gene clusters combined with isolation and structural characterization revealed further class III lanthipeptides such as avermipeptin (Streptomyces avermitilis), erythreapeptin (Saccharopolyspora erythraea), griseopeptin (Streptomyces griseus), curvopeptin (Thermomonospora curvata), and stackepeptin (Stackebrandtia nassauensis DSM-44728).33−35 In stackepeptin, the lanthionine/labionin motif occurs 3-times instead of 2-times in all other class III lanthipeptides. For most of the class III lanthipeptides, a morphogenetic function in the cell cycle has been assumed.36 Interestingly, labionin and lanthionine structures have been found to coexist, and two biosynthetic modes have been suggested under the regime of the modifying enzyme LabKC.33 A great number of synthetic peptides of the LabA2 leader peptide have been investigated. Apart from a conserved ILELQ motif,37 N- or C-terminal truncations of the leader peptide mostly led to a severe impairment of the core peptide processing. With regard to cofactor demand, investigations have shown that some LanKC enzymes are specific, whereas others like CurKC accept various desoxyribonucleotides and ribonucleotides.34 The directionality of the three reactions, phosphorylation, dehydration, and cyclization, performed by LanKC is another important aspect for an understanding of the biosynthesis.38 Deuterium labeling of LabA peptides revealed a directionality from the C- to the N-terminus. The curvopeptin system39 revealed a more complex situation: while the first phosphorylation is installed at the N-terminus, further ones follow stepwise from the C-terminus toward the N-terminus. Interestingly, the proteolytic processing of class III lanthipeptides is still largely unclear due to a lack of a protease gene in the biosynthesis gene cluster. As an exception, the flavipeptin gene cluster (Kribella f lavida) revealed a prolyl oligopeptidase (POP)-type protease to obtain flavipeptin with an N-terminal overhang.40 However, the POP-type protease is only an initial step of proteolytic processing, and additional proteases are likely involved, since investigations of class III lanthipeptides showed sequential leader peptide removal in multiple steps. As a current hypothesis, we assume an unspecific processing by internal proteases encoded in the genome of actinomycetes. For sustainable production of new labyrinthopeptins, only a biotechnological process is a viable option, since chemoenzymatics or chemical synthesis41 pose unmet challenges. However, genetic

4.2. Albicidin, a NRP Consisting of Aminobenzoic Acids

Albicidin has been described as a pathogenic factor identified from the plant pathogen Xanthomonas albilineans,5 which infects sugar cane plants. The Gram-negative bacterium invades the xylem and causes chlorosis leading to leaf scald disease. Albicidin is a linear pentapeptide with an N-terminal methyl p-coumaric acid. Except for the unusual L-cyanoalanine (L-Cya), it consists of modified and unmodified p-aminobenzoic acids (pABAs).44 Genome mining of X. albilineans could pinpoint the albicidin (alb) gene cluster, which mainly remained cryptic without prior knowledge of the albicidin structure. The central part of the albicidin biosynthesis is a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (Figure 4). Hence, the N-terminal p-hydroxycoumaric acid (building block A) derived from the precursor p-hydroxybenzoic acid (pHBA) is assembled by the PKS, which is extended on an NRPS by pABA (building block B/D), pMBA (building block E/F), and L-Cya (building block C). L-Cya is the only amino acid with a stereocenter on the entire molecule. A remarkable feature of the albicidin megasynthetase is a novel type of A domain specificity, that is, the activation and coupling of pABAs as novel building blocks. An ATP-dependent O-carbamoyltransferase (Alb15) in the alb gene cluster optionally attaches a carbamoyl residue to the N-terminal phenolic group of the coumaric acid and thus contributes to microheterogeneity.45 In comparison to albicidin, carbamoyl-albicidin showed an approximately 6-fold higher inhibitory efficiency against bacterial gyrase.45 Albicidin is not only a phytotoxin blocking the chloroplast development but also a strong antibacterial against a wide range of Gram-positive and Gram-negative bacteria, particularly against E. coli. It inhibits the DNA gyrase (E. coli: MIC = 0.031−0.5 μg mL−1),46 which is an important bacterial target. Since it is not cytotoxic at 10 μM for mammalian cells,47 albicidin represents a promising and novel antimicrobial drug, particularly against Gram-negative bacteria. Due to low production yields of Xanthomonas strains and the limited options for structural diversification, we embarked on a total synthesis and library synthesis program employing an allyl protection group strategy and a triphosgene-mediated coupling strategy.46 One series of albicidins bears modifications at building block A,48 another series at building block C.49 The MIC was determined for several Gram-positive and Gram-negative bacteria, and it was found that lipophilic, large acyl moieties at the N-terminus increased antibacterial activities, while charged side chains replacing cyanoalanine decreased the antimicrobial activity.48,49 Future activities in our group are directed to an understanding of albicidin’s mode of action and an optimization of this lead structure. 1572


Article


Figure 5. Concept of NRPS engineering approaches on fungal depsipeptide synthetases based on module swapping.

to obtain modified PF1022-type compounds.54 A similar method including an in vivo approach by MBS uses a knockout strain of B. bassiana and an E. coli strain as heterologous producer for the new beauvericin55 and enniatin analogues. Combining the hydroxy acid-activating module of PF1022A synthetase with an amino acid-activating module of enniatin or beauvericin synthetase via CBS rendered chimeric synthetases to produce artificial CDP structures (Figure 5).57 Current work focuses on modulating the ring size of CDPs rendering cyclohexa- to cyclooctadepsipeptides. Crucial aspects for biotechnological processes are the choice of the host organism. Hence, the fungus Aspergillus niger is the organism of choice for heterologous expression of CDPs and CDP production up to multigram-scale per liter.58 It remains to be seen whether in the long-run chemical approaches facing racemization issues and difficult-to-couple N-methyl amino acids are competitive with the sustainable biotechnological production of CDPs.

4.3. Cyclodepsipeptides Synthesized by Iterative NRPSs

An interesting class of fungal peptides is the cyclodepsipeptides (CDPs), which consist of alternating units of N-methyl amino and α-hydroxy acids. These are the cyclohexadepsipeptides enniatin and beauvericin and the cyclooctadepsipeptides bassianolide and PF1022. They are of particular interest due to their broad antibacterial, antifungal, insecticidal, and anthelmintic activities. Two particular compounds from the group of CDPs are marketed drugs: fusafungine, a mixture of enniatins, is an antibacterial for the treatment of rhinosinusitis in nasal spray, and emodepside, a semisynthetic derivative of PF1022A, is used as anthelmintic against nematodes in veterinary medicine. Emodepside binds to a presynaptic latrophilin receptor and interacts with a calcium-activated potassium channel.50 Both modes of action cause paralysis and death of the nematode. All these CDPs are produced by highly homologous iterative bimodular NRPSs (PFSyn, ESyn, BeauvSyn, BassSyn).51 The first module activates α-hydroxy acids, while the second module activates amino acids. The second module contains an additional M domain responsible for methylation of the amino groups. Additional T and C domains are linked to the C-terminus of the second module to perform macrolactonization. Apart from chemical synthesis52 and chemoenzymatics (CHE),53−55 new CDPs have been generated by precursor-directed biosynthesis (PDB), mutational biosynthesis (MBS), and also combinatorial biosynthesis (CBS).56 An unusual substrate tolerance of the hydroxy acid-activating module of nearly all CDP synthetases facilitated the incorporation of various α-hydroxy acids by CHE

5. CONCLUSION AND OUTLOOK While classical screening approaches are still successful, in a modern search for new bioactive peptides from bacteria and fungi, bioinformatics has become essential and improved continuously. For instance, a new algorithm has been recently developed for RiPP prediction to deal with the complexity and diversity of RiPP structures.59 Additionally, the antiSMASH database has been recently advanced to improve the search and prediction for RiPP molecules.60 A recent innovation is, for example, mass spectrometry-based molecular networking61 to 1573



■

ACKNOWLEDGMENTS The authors acknowledge support by Cluster of Excellence Unifying Concepts in Catalysis (UniCat) and DFG (SU 239/24-1). We thank Dr. Rashed Al Toma, Dr. Andi Mainz, Simon Boecker, and Charlotte Steiniger for discussions on the manuscript.

ease the identification of secondary metabolites. Broad screening campaigns and more selective rational approaches mostly focus on particular bacteria, for example, Actinobacteria or fungi, for example, Ascomycota. The work on model organisms or pathogens commonly begins with genome sequencing followed by the identification and biosynthetic assignment of each metabolite encoded in the genome, for example, shown for Bacillus amyloliquefaciens or Paenibacillus larvae.62−64 Based on all these rapidly progressing approaches, it remains to be seen how big the structural space and the biosynthetic toolbox provided by nature still is. Hence, the increasing understanding of RiPP and NRP biosyntheses gives way to more adventurous but rationally designed bioengineering approaches: chemoenzymatics, mutasynthesis, site-directed mutagenesis, and combinatorial biosynthesis. Their application affords genetically manageable production hosts or heterologous producers. Since the number of biosynthesis systems and possibilities for engineering is nearly infinite, it is wise to restrict to systems that are relatively simple but meaningful with regard to structure and bioactivities. In this context, lanthipeptides (RiPPs) and the CDPs (NRPs) represent excellent model cases. Yet, there is still much to learn and still some way to go from proof-of-concept studies to general, easy-to-manipulate, and robust (cellular) production systems. Interestingly, total synthesis of many, even highly complex, peptides has been accomplished and industrial peptide synthesis as an improving technology, which moves away from simple linear peptides to more complex and challenging molecule architectures. Therefore, the next years will show whether production of peptide structures will experience a switch to sustainable biotechnological approaches or whether they can be provided by enhanced chemical synthesis methodologies.

■

Article

■

REFERENCES

(1) Challis, G. L.; Ravel, J. Coelichelin, a New Peptide Siderophore Encoded by the Streptomyces coelicolor Genome: Structure Prediction from the Sequence of Its Non-Ribosomal Peptide Synthetase. FEMS Microbiol. Lett. 2000, 187, 111−114. (2) Hornung, A.; Bertazzo, M.; Dziarnowski, A.; Schneider, K.; Welzel, K.; Wohlert, S.-E.; Holzenkämpfer, M.; Nicholson, G. J.; Bechthold, A.; Süssmuth, R. D.; Vente, A.; Pelzer, S. A Genomic Screening Approach to the Structure-Guided Identification of Drug Candidates from Natural Sources. ChemBioChem 2007, 8, 757−766. (3) Parenti, F.; Pagani, H.; Beretta, G. Gardimycin, a New Antibiotic from Actinoplanes. I. Description of the Producer Strain and Fermentation Studies. J. Antibiot. 1976, 29, 501−506. (4) Wieland, T. Peptides of Amanita phalloides. Pure Appl. Chem. 1964, 9, 145−158. (5) Birch, R. G.; Patil, S. S. Preliminary Characterization of an Antibiotic Produced by Xanthomonas albilineans Which Inhibits DNA Synthesis in Escherichia coli. Microbiology 1985, 131, 1069−1075. (6) Shotwell, O. L.; Stodola, F. H.; Michael, W. R.; Lindenfelser, L. A.; Dworschack, R. G.; Pridham, T. G. Antibiotics Against Plant Disease. III. Duramycin, A New Antibiotic from Streptomyces cinnamomeus forma azacoluta. J. Am. Chem. Soc. 1958, 80, 3912−3915. (7) Terlain, B.; Thomas, J. P. Structure of Griselimycin, Polypeptide Antibiotic Extracted from Streptomyces Cultures. 3. Products Related to Griselimycin. Bull. Soc. Chim. Fr. 1971, 6, 2363−2365. (8) Wink, J.; Kroppenstedt, R. M.; Seibert, G.; Stackebrandt, E. Actinomadura namibiensis sp. nov. Int. J. Syst. Evol. Microbiol. 2003, 53, 721−724. (9) Meindl, K.; Schmiederer, T.; Schneider, K.; Reicke, A.; Butz, D.; Keller, S.; Gühring, H.; Vértesy, L.; Wink, J.; Hoffmann, H.; Brönstrup, M.; Sheldrick, G. M.; Süssmuth, R. D. Labyrinthopeptins: A New Class of Carbacyclic Lantibiotics. Angew. Chem., Int. Ed. 2010, 49, 1151−1154. (10) Müller, W. M.; Schmiederer, T.; Ensle, P.; Süssmuth, R. D. In Vitro Biosynthesis of the Prepeptide of Type-III Lantibiotic Labyrinthopeptin A2 Including Formation of a C-C Bond as a PostTranslational Modification. Angew. Chem., Int. Ed. 2010, 49, 2436− 2440. (11) Freney, J.; Brun, Y.; Bes, M.; Meugnier, H.; Grimont, F.; Grimont, P. A. D.; Nervi, C.; Fleurette, J. Staphylococcus lugdunensis Sp. Nov. and Staphylococcus schleiferi sp. nov., Two Species from Human Clinical Specimens. Int. J. Syst. Bacteriol. 1988, 38, 168−172. (12) Zipperer, A.; Konnerth, M. C.; Laux, C.; Berscheid, A.; Janek, D.; Weidenmaier, C.; Burian, M.; Schilling, N. A.; Slavetinsky, C.; Marschal, M.; Willmann, M.; Kalbacher, H.; Schittek, B.; Brötz-Oesterhelt, H.; Grond, S.; Peschel, A.; Krismer, B. Human Commensals Producing a Novel Antibiotic Impair Pathogen Colonization. Nature 2016, 535, 511−516. (13) O’Sullivan, J.; McCullough, J. E.; Tymiak, A. A.; Kirsch, D. R.; Trejo, W. H.; Principe, P. A. Lysobactin, a Novel Antibacterial Agent Produced by Lysobacter sp. I. Taxonomy, Isolation and Partial Characterization. J. Antibiot. 1988, 41, 1740−1744. (14) Castiglione, F.; Lazzarini, A.; Carrano, L.; Corti, E.; Ciciliato, I.; Gastaldo, L.; Candiani, P.; Losi, D.; Marinelli, F.; Selva, E.; Parenti, F. Determining the Structure and Mode of Action of Microbisporicin, a Potent Lantibiotic Active Against Multiresistant Pathogens. Chem. Biol. 2008, 15, 22−31. (15) Ishitsuka, M. O.; Kusumi, T.; Kakisawa, H.; Kaya, K.; Watanabe, M. M. Microviridin. A Novel Tricyclic Depsipeptide from the Toxic Cyanobacterium Microcystis viridis. J. Am. Chem. Soc. 1990, 112, 8180− 8182. (16) Hoffmann, H.; Kogler, H.; Heyse, W.; Matter, H.; Caspers, M.; Schummer, D.; Klemke-Jahn, C.; Bauer, A.; Penarier, G.; Debussche, L.;

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Roderich D. Süssmuth: 0000-0001-7027-2069 Notes

The authors declare no competing financial interest. Biographies Tam Dang received his M.Sc. in Chemistry at Technische Universität Berlin (TU Berlin) in 2016. For his master thesis, he stayed at Pieter Dorrestein’s laboratory at University of California, San Diego, to learn about the application of mass spectrometry imaging on bacterial interactions and the data visualization guided with LC-MS on plants in a spatial manner. He is currently working on the investigation of mechanistic aspects of the nonribosomal peptide synthesis as a research assistant under the guidance of Roderich Sü ssmuth at TU Berlin. Roderich D. Süssmuth is the Rudolf-Wiechert-Professor of Biological Chemistry at the Department of Chemistry of the Technische Universität Berlin. Prior to his appointment, he was Assistant Professor at the Eberhard-Karls-Universität Tübingen. In 1999, Prof. Süssmuth received his doctoral degree in Organic Chemistry from Eberhard-KarlsUniversität Tübingen under the guidance of Günther Jung. His main research interests comprise the discovery of novel peptide antibiotics and their biosynthesis and chemical synthesis, as well as combinatorial synthesis and synthetic biology of bioactive peptides. 1574


Article

Accounts of Chemical Research Brö nstrup, M. Discovery, Structure Elucidation, and Biological Characterization of Nannocystin A, a Macrocyclic Myxobacterial Metabolite with Potent Antiproliferative Properties. Angew. Chem., Int. Ed. 2015, 54, 10145−10148. (17) Rogers, L. A.; Whittier, E. O. Limiting Factors in the Lactic Fermentation. J. Bacteriol. 1928, 16, 211−229. (18) Sasaki, T.; Takagi, M.; Yaguchi, T.; Miyadoh, S.; Okada, T.; Koyama, M. A New Anthelmintic Cyclodepsipeptide, PF1022A. J. Antibiot. 1992, 45, 692−697. (19) Ling, L. L.; Schneider, T.; Peoples, A. J.; Spoering, A. L.; Engels, I.; Conlon, B. P.; Mueller, A.; Schäberle, T. F.; Hughes, D. E.; Epstein, S.; Jones, M.; Lazarides, L.; Steadman, V. A.; Cohen, D. R.; Felix, C. R.; Fetterman, K. A.; Millett, W. P.; Nitti, A. G.; Zullo, A. M.; Chen, C.; Lewis, K. A New Antibiotic Kills Pathogens without Detectable Resistance. Nature 2015, 517, 455−459. (20) Misiek, M.; Fardig, O. B.; Gourevitch, A.; Johnson, D. L.; Hooper, I. R.; Lein, J. Telomycin, a New Antibiotic. Antibiot. Annu. 1957, 5, 852− 855. (21) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni, T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis, G. L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.; Donadio, S.; Dorrestein, P. C.; Entian, K.-D.; Fischbach, M. A.; Garavelli, J. S.; Göransson, U.; Gruber, C. W.; Haft, D. H.; Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars, M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.; Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Müller, R.; Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.; Patchett, M. L.; Piel, J.; Reaney, M. J. T.; Rebuffat, S.; Ross, R. P.; Sahl, H.-G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.; Sivonen, K.; Smith, L.; Stein, T.; Süssmuth, R. D.; Tagg, J. R.; Tang, G.-L.; Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.; Wenzel, S. C.; Willey, J. M.; van der Donk, W. A. Ribosomally Synthesized and Post-Translationally Modified Peptide Natural Products: Overview and Recommendations for a Universal Nomenclature. Nat. Prod. Rep. 2013, 30, 108−160. (22) Yu, Y.; Zhang, Q.; van der Donk, W. A. Insights into the Evolution of Lanthipeptide Biosynthesis. Protein Sci. 2013, 22, 1478−1489. (23) Schnell, N.; Entian, K. D.; Schneider, U.; Götz, F.; Zähner, H.; Kellner, R.; Jung, G. Prepeptide Sequence of Epidermin, a Ribosomally Synthesized Antibiotic with Four Sulphide-Rings. Nature 1988, 333, 276−278. (24) Boakes, S.; Dawson, M. J. Discovery and Development of NVB302, a Semisynthetic Antibiotic for Treatment of Clostridium dif f icile Infection. In Natural Products: Discourse, Diversity, and Design; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2014; pp 455−468. (25) Férir, G.; Petrova, M. I.; Andrei, G.; Huskens, D.; Hoorelbeke, B.; Snoeck, R.; Vanderleyden, J.; Balzarini, J.; Bartoschek, S.; Brönstrup, M.; Süssmuth, R. D.; Schols, D. The Lantibiotic Peptide Labyrinthopeptin A1 Demonstrates Broad Anti-HIV and Anti-HSV Activity with Potential for Microbicidal Applications. PLoS One 2013, 8, e64010. (26) Willey, J. M.; Gaskell, A. A. Morphogenetic Signaling Molecules of the Streptomycetes. Chem. Rev. 2011, 111, 174−187. (27) Zeitlin, P. L. Novel Pharmacologic Therapies for Cystic Fibrosis. J. Clin. Invest. 1999, 103, 447−452. (28) Ziemert, N.; Ishida, K.; Liaimer, A.; Hertweck, C.; Dittmann, E. Ribosomal Synthesis of Tricyclic Depsipeptides in Bloom-Forming Cyanobacteria. Angew. Chem., Int. Ed. 2008, 47, 7756−7759. (29) Moldenhauer, G.; Salnikov, A. V.; Lüttgau, S.; Herr, I.; Anderl, J.; Faulstich, H. Therapeutic Potential of Amanitin-Conjugated AntiEpithelial Cell Adhesion Molecule Monoclonal Antibody against Pancreatic Carcinoma. J. Natl. Cancer Inst. 2012, 104, 622−634. (30) Süssmuth, R. D.; Mainz, A. Nonribosomal Peptide SynthesisPrinciples and Prospects. Angew. Chem., Int. Ed. 2017, 56, 3770−3821. (31) Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D. Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis. Chem. Rev. 1997, 97, 2651−2673. (32) Krastel, P.; Roggo, S.; Schirle, M.; Ross, N. T.; Perruccio, F.; Aspesi, P.; Aust, T.; Buntin, K.; Estoppey, D.; Liechty, B.; Mapa, F.; Memmert, K.; Miller, H.; Pan, X.; Riedl, R.; Thibaut, C.; Thomas, J.; Wagner, T.; Weber, E.; Xie, X.; Schmitt, E. K.; Hoepfner, D.

Nannocystin A: An Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties. Angew. Chem., Int. Ed. 2015, 54, 10149−10154. (33) Völler, G. H.; Krawczyk, J. M.; Pesic, A.; Krawczyk, B.; Nachtigall, J.; Süssmuth, R. D. Characterization of New Class III LantibioticsErythreapeptin, Avermipeptin and Griseopeptin from Saccharopolyspora erythraea, Streptomyces avermitilis and Streptomyces griseus Demonstrates Stepwise N-Terminal Leader Processing. ChemBioChem 2012, 13, 1174−1183. (34) Krawczyk, B.; Völler, G. H.; Völler, J.; Ensle, P.; Süssmuth, R. D. Curvopeptin: A New Lanthionine-Containing Class III Lantibiotic and Its Co-Substrate Promiscuous Synthetase. ChemBioChem 2012, 13, 2065−2071. (35) Jungmann, N. A.; van Herwerden, E. F.; Hügelland, M.; Süssmuth, R. D. The Supersized Class III Lanthipeptide Stackepeptin Displays Motif Multiplication in the Core Peptide. ACS Chem. Biol. 2016, 11, 69−76. (36) Kodani, S.; Hudson, M. E.; Durrant, M. C.; Buttner, M. J.; Nodwell, J. R.; Willey, J. M. The SapB Morphogen Is a Lantibiotic-like Peptide Derived from the Product of the Developmental Gene ramS in Streptomyces coelicolor. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11448− 11453. (37) Müller, W. M.; Ensle, P.; Krawczyk, B.; Süssmuth, R. D. Leader Peptide-Directed Processing of Labyrinthopeptin A2 Precursor Peptide by the Modifying Enzyme LabKC. Biochemistry 2011, 50, 8362−8373. (38) Krawczyk, B.; Ensle, P.; Müller, W. M.; Süssmuth, R. D. Deuterium Labeled Peptides Give Insights into the Directionality of Class III Lantibiotic Synthetase LabKC. J. Am. Chem. Soc. 2012, 134, 9922−9925. (39) Jungmann, N. A.; Krawczyk, B.; Tietzmann, M.; Ensle, P.; Süssmuth, R. D. Dissecting Reactions of Nonlinear Precursor Peptide Processing of the Class III Lanthipeptide Curvopeptin. J. Am. Chem. Soc. 2014, 136, 15222−15228. (40) Völler, G. H.; Krawczyk, B.; Ensle, P.; Süssmuth, R. D. Involvement and Unusual Substrate Specificity of a Prolyl Oligopeptidase in Class III Lanthipeptide Maturation. J. Am. Chem. Soc. 2013, 135, 7426−7429. (41) Sambeth, G. M.; Süssmuth, R. D. Synthetic Studies toward Labionin, a New α,α-Disubstituted Amino Acid from Type III Lantibiotic Labyrinthopeptin A2. J. Pept. Sci. 2011, 17, 581−584. (42) Kuthning, A.; Mösker, E.; Süssmuth, R. D. Engineering the Heterologous Expression of Lanthipeptides in Escherichia coli by Multigene Assembly. Appl. Microbiol. Biotechnol. 2015, 99 (15), 6351−6361. (43) Krawczyk, J. M.; Völler, G. H.; Krawczyk, B.; Kretz, J.; Brönstrup, M.; Süssmuth, R. D. Heterologous Expression and Engineering Studies of Labyrinthopeptins, Class III Lantibiotics from Actinomadura namibiensis. Chem. Biol. 2013, 20, 111−122. (44) Cociancich, S.; Pesic, A.; Petras, D.; Uhlmann, S.; Kretz, J.; Schubert, V.; Vieweg, L.; Duplan, S.; Marguerettaz, M.; Noëll, J.; Pieretti, I.; Hügelland, M.; Kemper, S.; Mainz, A.; Rott, P.; Royer, M.; Süssmuth, R. D. The Gyrase Inhibitor Albicidin Consists of pAminobenzoic Acids and Cyanoalanine. Nat. Chem. Biol. 2015, 11, 195−197. (45) Petras, D.; Kerwat, D.; Pesic, A.; Hempel, B.-F.; von Eckardstein, L.; Semsary, S.; Arasté, J.; Marguerettaz, M.; Royer, M.; Cociancich, S.; Süssmuth, R. D. The O-Carbamoyl-Transferase Alb15 Is Responsible for the Modification of Albicidin. ACS Chem. Biol. 2016, 11, 1198−1204. (46) Kretz, J.; Kerwat, D.; Schubert, V.; Grätz, S.; Pesic, A.; Semsary, S.; Cociancich, S.; Royer, M.; Süssmuth, R. D. Total Synthesis of Albicidin: A Lead Structure from Xanthomonas albilineans for Potent Antibacterial Gyrase Inhibitors. Angew. Chem., Int. Ed. 2015, 54, 1969−1973. (47) Hashimi, S. M.; Wall, M. K.; Smith, A. B.; Maxwell, A.; Birch, R. G. The Phytotoxin Albicidin Is a Novel Inhibitor of DNA Gyrase. Antimicrob. Agents Chemother. 2007, 51, 181−187. (48) Kerwat, D.; Grätz, S.; Kretz, J.; Seidel, M.; Kunert, M.; Weston, J. B.; Süssmuth, R. D. Synthesis of Albicidin Derivatives: Assessing the Role of N-Terminal Acylation on the Antibacterial Activity. ChemMedChem 2016, 11, 1899−1903. 1575


Article

Accounts of Chemical Research (49) Grätz, S.; Kerwat, D.; Kretz, J.; von Eckardstein, L.; Semsary, S.; Seidel, M.; Kunert, M.; Weston, J. B.; Süssmuth, R. D. Synthesis and Antimicrobial Activity of Albicidin Derivatives with Variations of the Central Cyanoalanine Building Block. ChemMedChem 2016, 11, 1499− 1502. (50) Harder, A.; Holden-Dye, L.; Walker, R.; Wunderlich, F. Mechanisms of Action of Emodepside. Parasitol. Res. 2005, 97, S1−S10. (51) Weckwerth, W.; Miyamoto, K.; Iinuma, K.; Krause, M.; Glinski, M.; Storm, T.; Bonse, G.; Kleinkauf, H.; Zocher, R. Biosynthesis of PF1022A and Related Cyclooctadepsipeptides. J. Biol. Chem. 2000, 275, 17909−17915. (52) Scherkenbeck, J.; Lüttenberg, S.; Ludwig, M.; Brücher, K.; Kotthaus, A. Segment Solid-Phase Total Synthesis of the Anthelmintic Cyclooctadepsipeptides PF1022A and Emodepside. Eur. J. Org. Chem. 2012, 2012, 1546−1553. (53) Feifel, S. C.; Schmiederer, T.; Hornbogen, T.; Berg, H.; Süssmuth, R. D.; Zocher, R. In Vitro Synthesis of New Enniatins: Probing the α-DHydroxy Carboxylic Acid Binding Pocket of the Multienzyme Enniatin Synthetase. ChemBioChem 2007, 8, 1767−1770. (54) Müller, J.; Feifel, S. C.; Schmiederer, T.; Zocher, R.; Süssmuth, R. D. In Vitro Synthesis of New Cyclodepsipeptides of the PF1022-Type: Probing the α-D-Hydroxy Acid Tolerance of PF1022 Synthetase. ChemBioChem 2009, 10, 323−328. (55) Matthes, D.; Richter, L.; Müller, J.; Denisiuk, A.; Feifel, S. C.; Xu, Y.; Espinosa-Artiles, P.; Süssmuth, R. D.; Molnár, I. In Vitro Chemoenzymatic and in Vivo Biocatalytic Syntheses of New Beauvericin Analogues. Chem. Commun. 2012, 48, 5674−5676. (56) Boecker, S.; Zobel, S.; Meyer, V.; Süssmuth, R. D. Rational Biosynthetic Approaches for the Production of New-to-Nature Compounds in Fungi. Fungal Genet. Biol. 2016, 89, 89−101. (57) Zobel, S.; Boecker, S.; Kulke, D.; Heimbach, D.; Meyer, V.; Süssmuth, R. D. Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins. ChemBioChem 2016, 17, 283−287. (58) Richter, L.; Wanka, F.; Boecker, S.; Storm, D.; Kurt, T.; Vural, Ö .; Süßmuth, R.; Meyer, V. Engineering of Aspergillus niger for the Production of Secondary Metabolites. Fungal Biol. Biotechnol. 2014, 1, 4. (59) Skinnider, M. A.; Johnston, C. W.; Edgar, R. E.; Dejong, C. A.; Merwin, N. J.; Rees, P. N.; Magarvey, N. A. Genomic Charting of Ribosomally Synthesized Natural Product Chemical Space Facilitates Targeted Mining. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E6343− E6351. (60) Blin, K.; Medema, M. H.; Kottmann, R.; Lee, S. Y.; Weber, T. The antiSMASH Database, a Comprehensive Database of Microbial Secondary Metabolite Biosynthetic Gene Clusters. Nucleic Acids Res. 2017, 45, D555−D559. (61) Quinn, R. A.; Nothias, L.-F.; Vining, O.; Meehan, M.; Esquenazi, E.; Dorrestein, P. C. Molecular Networking As a Drug Discovery, Drug Metabolism, and Precision Medicine Strategy. Trends Pharmacol. Sci. 2017, 38, 143−154. (62) Müller, S.; Garcia-Gonzalez, E.; Genersch, E.; Süssmuth, R. D. Involvement of Secondary Metabolites in the Pathogenesis of the American Foulbrood of Honey Bees Caused by Paenibacillus larvae. Nat. Prod. Rep. 2015, 32, 765−778. (63) Kalyon, B.; Helaly, S. E.; Scholz, R.; Nachtigall, J.; Vater, J.; Borriss, R.; Süssmuth, R. D. Plantazolicin A and B: Structure Elucidation of Ribosomally Synthesized Thiazole/Oxazole Peptides from Bacillus amyloliquefaciens FZB42. Org. Lett. 2011, 13, 2996−2999. (64) Chen, X. H.; Koumoutsi, A.; Scholz, R.; Eisenreich, A.; Schneider, K.; Heinemeyer, I.; Morgenstern, B.; Voss, B.; Hess, W. R.; Reva, O.; Junge, H.; Voigt, B.; Jungblut, P. R.; Vater, J.; Süssmuth, R.; Liesegang, H.; Strittmatter, A.; Gottschalk, G.; Borriss, R. Comparative Analysis of the Complete Genome Sequence of the Plant Growth−promoting Bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol. 2007, 25, 1007−1014.

1576


Bioactive Peptide Natural Products as Lead Structures for Medicinal Use

Recommend Documents