Recent advances in natural products exploitation in Streptomyces via synthetic biology

Natural products of microbial origin have proven to be the wellspring of clinically useful compounds for human therapeutics. Streptomyces species are predominant sources of bioactive compounds, most of which serve as potential drug candidates. While the exploitation of natural products has been severely reduced over the past two decades, the growing crisis of evolution and dissemination of drug resistant pathogens have again attracted great interest in this field. The emerging synthetic biology has been heralded as a new bioengineering platform to discover novel bioactive compounds and expand bioactive natural products diversity and production. Herein, we review recent advances in the natural products exploitation of Streptomyces with the applications of synthetic biology from three major aspects, including recently developed synthetic biology tools, natural products biosynthetic pathway engineering strategies as well as chassis host modifications.     

Radioprotectors – the Evergreen Topic

To protect organisms from ionizing radiation (IR), and to reduce morbidity or mortality, various agents, called radioprotectors, have been utilized. Because radiation‐induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical‐scavenging properties are particularly promising as radioprotectors. Early development of such agents focused on thiol synthetic compounds, known as WR protectors, but only amifostine (WR‐2721) has been used in clinical trials as an officially approved radioprotector. Besides thiol compounds, various compounds with different chemical structure were investigated, but an ideal radioprotector has not been found yet. Plants and natural products have been evaluated as promising sources of radioprotectors because of their low toxicity, although they exhibit an inferior protection level compared to synthetic thiol compounds. Active plant constituents seem to exert the radioprotection through antioxidant and free radical‐scavenging activities. Our research established that plants containing polyphenolic compounds (raspberry, blueberry, strawberry, grape, etc.) exhibit antioxidative activities and protect genetic material from IR.     

Natural products for infectious microbes and diseases: an overview of sources,compounds, and chemical diversities

As coronavirus disease 2019 (COVID-19) threatens human health globally, infectious disorders have become one of the most challenging problem for the medical community. Natural products (NP) have been a prolific source of antimicrobial agents with widely divergent structures and a range vast biological activities. A dataset comprising 618 articles, including 646 NP-based compounds from 672 species of natural sources with biological activities against 21 infectious pathogens from five categories, was assembled through manual selection of published articles. These data were used to identify 268 NP-based compounds classified into ten groups, which were used for network pharmacology analysis to capture the most promising lead-compounds such as agelasine D, dicumarol, dihydroartemisinin and pyridomycin. The distribution of maximum Tanimoto scores indicated that compounds which inhibited parasites exhibited low diversity, whereas the chemistries inhibiting bacteria, fungi, and viruses showed more structural diversity. A total of 331 species of medicinal plants with compounds exhibiting antimicrobial activities were selected to classify the family sources. The family Asteraceae possesses various compounds against C. neoformans, the family Anacardiaceae has compounds against Salmonella typhi, the family Cucurbitacea against the human immunodeficiency virus (HIV), and the family Ancistrocladaceae against Plasmodium. This review summarizes currently available data on NP-based antimicrobials against refractory infections to provide information for further discovery of drugs and synthetic strategies for anti-infectious agents.

     

Ring E analogs of methyllycaconitine (MLA) as novel nicotinic antagonists.

We have prepared ring E analogs of the diterpenoid alkaloid methyllycaconitine. These compounds have been assayed for nicotinic activity and were found to act as functional antagonists on adrenal nicotinic receptors.     

Bacillus-based bionematicides: development,modes of action and commercialisation

Agricultural crops are severely damaged by root-knot nematodes causing extensive financial losses globally. Historically, agrochemicals have been the preferred method to combat these pests; however, threats to humans and the environment posed by these agrochemicals led to the need for developing new biocontrol agents. Importantly, the latter should adhere to biosafety regulations while being highly effective. Root-knot nematodes live in soil and thus the use of rhizobacteria such as Bacillus for biocontrol development have shown potential. Although various Bacillus species have been tested in this capacity, little is known about their secondary metabolites and the mechanisms of action responsible for their nematicidal activity. If these secondary metabolites can be qualitatively and quantitatively characterised, metabolic features could be synthetically engineered and used to combat root-knot nematodes. Although there is great potential for bionematicides, the commercialisation and development of such products can be difficult. This review summarises the importance of Bacillus species as natural antagonists of root-knot nematodes through the production of secondary metabolites. It provides an overview of the significance of root-knot nematodes in agriculture and the advances of chemical nematicides in recent years. The potential of Bacillus species as biocontrol agents, the known mechanisms of action responsible for the nematicidal activity demonstrated by Bacillus species, non-target effects of biocontrol agents and the commercialisation of Bacillus-based bionematicides are discussed.     

Activity of alpha7-selective agonists at nicotinic and serotonin 5HT3 receptors expressed in Xenopus oocytes

Nicotinic receptors containing alpha7 subunits are widely distributed in the central nervous system and are thought to be involved in a number of functions. However, it has been difficult to study alpha7-containing receptors in vivo because of a paucity of selective agonists. A new spirooxazolidinone compound, AR-R17779, was recently described as potent agonist at alpha7 receptors, but electrophysiological studies at other types of nicotinic receptors have not been carried out. We characterized the activity of AR-R17779 at alpha7, alpha4beta2, alpha3beta4, alpha3beta2, alpha3beta2alpha5 receptors expressed in Xenopus oocytes. In addition, since there is significant homology between nicotinic alpha7 and serotonin 5HT(3) receptors, the activity of AR-R17779 at expressed 5HT(3a) receptors was also examined. Finally, actions of tropisetron and ondansetron, two 5HT(3) antagonists, were explored. AR-R17779 was found to activate alpha7 receptors, but had no activity at other types of nicotinic receptors, and also had no activity at 5HT(3a) receptors. Tropisetron activated, while ondansetron acted as an antagonist, at alpha7 nicotinic receptors. The two 5HT(3) antagonists also acted as antagonists at alpha4beta2 and alpha3beta4 nicotinic receptors. Thus, AR-R17779 was confirmed to be a selective nicotinic alpha7 receptor agonist and to be without activity at 5HT(3) receptors. In contrast, the actions of tropisetron and ondansetron on nicotinic receptors were complex.     

Siderophore-Mediated microbial iron(III) uptake: An exercise in chiral recognition

Molecular recognition by microbial receptors for siderophores [natural iron(III) carriers] is examined with synthetic iron(III) carriers as structural probes. The iron(III) carriers have been designed to reproduce the two essential features of the natural siderophores: the capability to form octahedral iron(III) binding cavities and to fit specific membrane receptors. Specifically, analogs of tripodal ferrichrome and linear ferrioxamines have been prepared and examined. The ferrichrome analogs rely on C₃-symmetric binders that are assembled from triscarboxylates as anchors, amino acids as bridges, and terminal hydroxamate groups as binding sites. The ferrioxamine analogs are based on linear assemblies of three identical monomers, each derived from a chiral amino acid. The deliberate use of animo acid residues as variable building blocks enables us to systematically modify the molecules' envelopes and the preferred absolute configuration of the iron(III) complexes until optimal performance is reached. Examination of the synthetic analogs in Pseudomonas putida demonstrates that the domains around the iron(III) center and their chiral sense dictate the extent of recognition by the membrane receptors. It is also shown that the synthetic siderophore analogs may be designed to either exert a broader, or a more narrow range of microbial activity than the natural siderophores. The implications of these findings are discussed in relation to the possible design of species-specific antimicrobial agents. © 1993 Wiley-Liss, Inc.     

The purines: potent and versatile small molecule inhibitors and modulators of key biological targets

The goal of this review is to highlight the wide range of biological activities displayed by purines, with particular emphasis on new purine-based agents which find potential application as chemical-biology tools and/or therapeutic agents. The expanding interest in the biological properties of polyfunctionalized purine derivatives issues, in large part, from the development of rapid high-throughput screening essays for new protein targets, and the corresponding development of efficient synthetic methodology adapted to the construction of highly diverse purine libraries. Purine-based compounds have found new applications as inducers of interferon and lineage-committed cell dedifferentiation, agonists and antagonists of adenosine receptors, ligands of corticotropin-releasing hormone receptors, and as inhibitors of HSP90, Src kinase, p38alpha MAP kinase, sulfotransferases, phosphodiesterases, and Cdks. The scope of application of purines in biology is most certainly far from being exhausted. Testing purine derivatives against the multitude of biological targets for which small molecule probes have not yet been found should thus be a natural reflex.     

Integrating the discovery pipeline for novel compounds targeting ion channels

Many ion channels are attractive therapeutic targets for the treatment of neurological or cardiovascular diseases; there is a continuous need for selective channel antagonists and/or agonists. Recently, several technologies have been developed that make exploration of the enormous diversity of venom-derived peptidic toxins more feasible. Integration of exogenomics with synthetic methods such as diselenide or fluorous bridges, backbone spacers, and N-to-C cyclization provides an emerging technology that promises to accelerate discovery and development of natural products based compounds. These drug-discovery efforts are complemented by novel approaches to modulate the activities of ion channels and receptors. Taken together, these technologies will advance our knowledge and understanding of ion channels and will accelerate their expansion as targets for first-in-class therapeutics.     

Metal and metalloid containing natural products and a brief overview of their applications in biology,biotechnology and biomedicine

Bioinorganic natural product chemistry is a relatively unexplored but rapidly developing field with enormous potential for applications in biology, biotechnology (especially in regards to nanomaterial development, synthesis and environmental cleanup) and biomedicine. In this review the occurrence of metals and metalloids in natural products and their synthetic derivatives are reviewed. A broad overview of the area is provided followed by a discussion on the more common metals and metalloids found in natural sources, and an overview of the requirements for future research. Special attention is given to metal hyperaccumulating plants and their use in chemical synthesis and bioremediation, as well as the potential uses of metals and metalloids as therapeutic agents. The potential future applications and development in the field are also discussed.     

A role for acetylcholine receptors in the fusion of chick myoblasts

The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha-bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha-bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion-inducing agent that activates the nicotinic ACh receptor.     

Bioinformatic analysis of the origin, sequence and diversification of mu opioid receptors in vertebrates

Opioid receptors are a class of G protein-coupled receptors that mediate the effects of the different families of endogenous opioid peptides and natural alkaloid drugs such as morphine and its synthetic derivatives. In particular, the μ opioid receptor (MOR) represents the principal molecular target for morphine and it plays key roles in opioid analgesia and addiction. In this work, new putative MORs from different vertebrate species were identified in silico and their gene organization and predicted protein products are compared with the previously characterized MORs. Also, for the first time a new genomic organization in euteleleostei teleosts has been identified. Moreover, we suggest that MORs may be specific to craniate lineage. The analysis of functional mapping of MORs we present is an important contribution to the identification of their evolutionarily conserved regions.     

Synthetic biology of avermectin for production improvement and structure diversification

Natural products are still key sources of current clinical drugs and innovative therapeutic agents. Since wild‐type microorganisms only produce natural products in very small quantities, yields of production strains need to be improved by breaking down the precise genetic and biochemical circuitry. Herein, we use avermectins as an example of production improvement and chemical structure diversification by synthetic biology. Avermectins are macrocyclic lactones produced by Streptomyces avermitilis and are well known and widely used for antiparasitic therapy. Given the importance of this molecule and its derivatives, many efforts and strategies were employed to improve avermectin production and generate new active analogues. This review describes the current status of synthetic strategies successfully applied for developing natural‐product‐producing strains and discusses future prospects for the application of enhanced avermectin production.     

Synthesis of desformylflustrabromine and its evaluation as an alpha4beta2 and alpha7 nACh receptor modulator

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.     

Insect natural products and processes: new treatments for human disease

In this overview, some of the more significant recent developments in bioengineering natural products from insects with use or potential use in modern medicine are described, as well as in utilisation of insects as models for studying essential mammalian processes such as immune responses to pathogens. To date, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey and silk, for at least 4-7000 years, and have featured in folklore medicine for thousands of years. Particular examples of Insect Folk Medicines will briefly be described which have subsequently led through the application of molecular and bioengineering techniques to the development of bioactive compounds with great potential as pharmaceuticals in modern medicine. Insect products reviewed have been derived from honey, venom, silk, cantharidin, whole insect extracts, maggots, and blood-sucking arthropods. Drug activities detected include powerful antimicrobials against antibiotic-resistant bacteria and HIV, as well as anti-cancer, anti-angiogenesis and anti-coagulant factors and wound healing agents. Finally, the many problems in developing these insect products as human therapeutic drugs are considered and the possible solutions emerging to these problems are described.     

Insight into carrageenases: major review of sources,category, property,purification method,structure, and applications

Carrageenan, a kind of linear sulfated polysaccharides consisting of D-galactose with alternating α-1,3 and β-1,4 linkages, has been widely applied in the food and cosmetic industries as thickening and gelling agents due to excellent properties, such as gel-forming ability and chemical stability. It can be degraded by carrageenases to produce a series of even-numbered carrageenan oligosaccharides, which exhibit various fascinating functions, such as anti-inflammation, anti-coagulation, anti-tumor, and anti-thrombosis effects. Numerous carrageenases have been isolated and identified from various sources. The enzymes are grouped into three categories, namely κ-carrageenase, ι-carrageenase, and λ-carrageenase based on their substrate specificities and primary sequences, respectively. Elucidating the paradigm of the enzyme at every aspect would definitely enhance our understanding of the marine carbon cycling and natural evolution of glycoside hydrolases (GHs). The structural features of these enzymes have been fully illustrated, which will improve our knowledge of its catalytic mechanisms. In this review, we have summarized the recent progresses of major sources, category, and the enzyme’s biochemical characteristics. Additionally, structural characteristics and catalytic mechanisms have been introduced in detail. We conclude with a brief discussion of the potential of the carrageenases in possible future applications in preparing functional oligosaccharides with versatile activities. This comprehensive information should be helpful regarding the application of carrageenases.     

Chemopreventive agent-induced modulation of death receptors

The goals of chemoprevention of cancer are to inhibit the initiation or suppress the promotion and progression of preneoplastic lesions to invasive cancer through the use specific natural or synthetic agents. Therefore, a more desirable and aggressive approach is to eliminate aberrant clones by inducing apoptosis rather than merely slowing down their proliferation. The increased understanding of apoptosis pathways has directed attention to components of these pathways as potential targets not only for chemotherapeutic but also for chemopreventive agents. Activation of death receptors triggers an extrinsic apoptotic pathway, which plays a critical role in tumor immunosurveillance. An increasing number of previously identified chemopreventive agents were found to induce apoptosis in a variety of premalignant and malignant cell types in vitro and in a few animal models in vivo. Some chemopreventive agents such as non-steroidal anti-inflammatory drugs, tritepenoids, and retinoids increase the expression of death receptors. Thus, understanding the modulation of death receptors by chemopreventive agents and their implications in chemoprevention may provide a rational approach for using such agents alone or in combination with other agents to enhance death receptor-mediated apoptosis as a strategy for effective chemoprevention of cancer.     

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.     

Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines

Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.