Maleidride biosynthesis - construction of dimeric anhydrides - more than just heads or tails.

Covering: up to early 2022Maleidrides are a family of polyketide-based dimeric natural products isolated from fungi. Many maleidrides possess significant bioactivities, making them attractive pharmaceutical or agrochemical lead compounds. Their unusual biosynthetic pathways have fascinated scientists for decades, with recent advances in our bioinformatic and enzymatic understanding providing further insights into their construction. However, many intriguing questions remain, including exactly how the enzymatic dimerisation, which creates the diverse core structure of the maleidrides, is controlled. This review will explore the literature from the initial isolation of maleidride compounds in the 1930s, through the first full structural elucidation in the 1960s, to the most recent in vivo, in vitro, and in silico analyses.

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases.

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

Correction: Back et al. A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge. Mar. Drugs 2021, 19, 105.

After publication of this article [...].

The Role of Cytochrome†P450 AbyV in the Final Stages of Abyssomicin†C Biosynthesis.

Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the aby gene cluster, catalyses a key late-stage epoxidation required for the installation of the characteristic ether-bridged core of abyssomicin C. The X-ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon-13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme-catalysed reactions in vitro with no need for purification.

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.

Synthetic and biosynthetic methods for selective cyclisations of 4,5-epoxy alcohols to tetrahydropyrans.

Tetrahydropyrans (THPs) are common structural motifs found in natural products and synthetic therapeutic molecules. In Nature these 6-membered oxygen heterocycles are often assembled via intramolecular reactions involving either oxy-Michael additions or ring opening of epoxy-alcohols. Indeed, the polyether natural products have been particularly widely studied due to their fascinating structures and important biological properties; these are commonly formed via endo-selective epoxide-opening cascades. In this review we outline synthetic approaches for endo-selective intramolecular epoxide ring opening (IERO) of 4,5-epoxy-alcohols and their applications in natural product synthesis. In addition, the biosynthesis of THP-containing natural products which utilise IERO reactions are reviewed.

Cladobotric Acids: Metabolites from Cultures of Cladobotryum sp., Semisynthetic Analogues and Antibacterial Activity.

Three new polyketide-derived natural products, cladobotric acids G-I (1-3), and six known metabolites (4, 5, 8-11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12-20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity.

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

Core Steps to the Azaphilone Family of Fungal Natural Products.

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.

Ambruticins: tetrahydropyran ring formation and total synthesis.

The ambruticins are a family of polyketide natural products which exhibit potent antifungal activity. Gene knockout experiments are in accord with the proposal that the tetrahydropyran ring of the ambruticins is formed via the AmbJ catalysed epoxidation of the unsaturated 3,5-dihydroxy acid, ambruticin J, followed by regioselective cyclisation to ambruticin F. Herein, a convergent approach to the total synthesis of ambruticin J is described as well as model studies involving epoxidation and cyclisations of unsaturated hydroxy esters to give tetrahydropyrans and tetrahydrofurans. The total synthesis involves preparation of three key fragments which were united via a Suzuki-Miyaura cross-coupling and Julia-Kocienski olefination to generate the required carbon framework. Global deprotection to a triol and selective oxidation of the primary alcohol gave, after hydrolysis of the lactone, ambruticin J.

Synthesis and pharmacological characterisation of arctigenin analogues as antagonists of AMPA and kainate receptors.

(-)-Arctigenin and a series of new analogues have been synthesised and then tested for their potential as AMPA and kainate receptor antagonists of human homomeric GluA1 and GluK2 receptors expressed in HEK293 cells using a Ca2+ influx assay. In general, these compounds showed antagonist activity at both receptors with greater activity evident at AMPARs. Schild analysis indicates that a spirocyclic analogue 6c acts as a non-competitive antagonist. Molecular docking studies in which 6c was docked into the X-ray crystal structure of the GluA2 tetramer suggest that (-)-arctigenin and its analogues bind in the transmembrane domain in a similar manner to the known AMPA receptor non-competitive antagonists GYKI53655 and the antiepileptic drug perampanel. The arctigenin derivatives described herein may serve as novel leads for the development of drugs for the treatment of epilepsy.

Fusarochromene, a novel tryptophan-derived metabolite from Fusarium sacchari.

Fusarochromene isolated from the plant pathogenic fungus, Fusarium sacchari is closely related to a group of mycotoxins including fusarochromanone previously isolated from various Fusaria spp. Despite their assumed polyketide biogenesis, incorporation studies with 13C-labelled acetate, glycerol and tryptophans show that fusarochromene is unexpectedly derived via oxidative cleavage of the aromatic amino acid tryptophan. A putative biosynthetic gene cluster has been identified.

A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge.

To tackle the growing problem of antibiotic resistance, it is essential to identify new bioactive compounds that are effective against resistant microbes and safe to use. Natural products and their derivatives are, and will continue to be, an important source of these molecules. Sea sponges harbour a diverse microbiome that co-exists with the sponge, and these bacterial communities produce a rich array of bioactive metabolites for protection and resource competition. For these reasons, the sponge microbiota constitutes a potential source of clinically relevant natural products. To date, efforts in bioprospecting for these compounds have focused predominantly on sponge specimens isolated from shallow water, with much still to be learned about samples from the deep sea. Here we report the isolation of a new Micromonospora strain, designated 28ISP2-46T, recovered from the microbiome of a mid-Atlantic deep-sea sponge. Whole-genome sequencing reveals the capacity of this bacterium to produce a diverse array of natural products, including kosinostatin and isoquinocycline B, which exhibit both antibiotic and antitumour properties. Both compounds were isolated from 28ISP2-46T fermentation broths and were found to be effective against a plethora of multidrug-resistant clinical isolates. This study suggests that the marine production of isoquinocyclines may be more widespread than previously supposed and demonstrates the value of targeting the deep-sea sponge microbiome as a source of novel microbial life with exploitable biosynthetic potential.

Correction to: ACE2 activation protects against cognitive decline and reduces amyloid pathology in the Tg2576 mouse model of Alzheimer's disease.

Unfortunately, the acknowledgement section was not included in the original publication.

ACE2 activation protects against cognitive decline and reduces amyloid pathology in the Tg2576 mouse model of Alzheimer's disease.

Mid-life hypertension and cerebrovascular dysfunction are associated with increased risk of later life dementia, including Alzheimer's disease (AD). The classical renin-angiotensin system (cRAS), a physiological regulator of blood pressure, functions independently within the brain and is overactive in AD. cRAS-targeting anti-hypertensive drugs are associated with reduced incidence of AD, delayed onset of cognitive decline, and reduced levels of Aß and tau in both animal models and human pathological studies. cRAS activity is moderated by a downstream regulatory RAS pathway (rRAS), which is underactive in AD and is strongly associated with pathological hallmarks in human AD, and cognitive decline in animal models of CNS disease. We now show that enhancement of brain ACE2 activity, a major effector of rRAS, by intraperitoneal administration of diminazene aceturate (DIZE), an established activator of ACE2, lowered hippocampal Aß and restored cognition in mid-aged (13-14-month-old) symptomatic Tg2576 mice. We confirmed that the protective effects of DIZE were directly mediated through ACE2 and were associated with reduced hippocampal soluble Aß42 and IL1-ß levels. DIZE restored hippocampal MasR levels in conjunction with increased NMDA NR2B and downstream ERK signalling expression in hippocampal synaptosomes from Tg2576 mice. Chronic (10 weeks) administration of DIZE to pre-symptomatic 9-10-month-old Tg2576 mice, and acute (10 days) treatment in cognitively impaired 12-13-month-old mice, prevented the development of cognitive impairment. Together these data demonstrate that ACE2 enhancement protects against and reverses amyloid-related hippocampal pathology and cognitive impairment in a preclinical model of AD.

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI.

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.

Sequential Photochemical and Prins Reactions for the Diastereoselective Synthesis of Tricyclic Scaffolds.

Cyclobutene alcohols undergo Prins cyclisations to generate single diastereomers of novel tricyclic heterocycles with five contiguous stereocentres. The reaction times are significantly shorter (ca. 15 min) than with traditional alkene substrates. Stereoselective aza-Prins cyclisations of cyclobutene amine derivatives give fused aza-heterocyclic scaffolds. Computational studies provide insight into the observed stereocontrol. The modular approach is flexible, enabling the introduction of a variety of functional groups (including amides, nitriles, alkynes, and arenes) into the sp3 -rich heterocyclic scaffolds.

Control of ß-Branching in Kalimantacin Biosynthesis: Application of 13 C†NMR to Polyketide Programming.

The presence of ß-branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential ß-branching modifications. We use purified single and multi-domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of ß-branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of 13 C NMR of a single 13 C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl-CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific ß-branch.

An Esterase-like Lyase Catalyzes Acetate Elimination in Spirotetronate/Spirotetramate Biosynthesis.

Spirotetronate and spirotetramate natural products include a multitude of compounds with potent antimicrobial and antitumor activities. Their biosynthesis incorporates many unusual biocatalytic steps, including regio- and stereo-specific modifications, cyclizations promoted by Diels-Alderases, and acetylation-elimination reactions. Here we focus on the acetate elimination catalyzed by AbyA5, implicated in the formation of the key Diels-Alder substrate to give the spirocyclic system of the antibiotic abyssomicin C. Using synthetic substrate analogues, it is shown that AbyA5 catalyzes stereospecific acetate elimination, establishing the (R)-tetronate acetate as a biosynthetic intermediate. The X-ray crystal structure of AbyA5, the first of an acetate-eliminating enzyme, reveals a deviant acetyl esterase fold. Molecular dynamics simulations and enzyme assays show the use of a His-Ser dyad to catalyze either elimination or hydrolysis, via disparate mechanisms, under substrate control.

Fine Tuning of Antibiotic Activity by a Tailoring Hydroxylase in a Trans-AT Polyketide Synthase Pathway.

The addition or removal of hydroxy groups modulates the activity of many pharmacologically active biomolecules. It can be integral to the basic biosynthetic factory or result from associated tailoring steps. For the anti-MRSA antibiotic mupirocin, removal of a C8-hydroxy group late in the biosynthetic pathway gives the active pseudomonic acid A. An extra hydroxylation, at C4, occurs in the related but more potent antibiotic thiomarinol A. We report here in vivo and in vitro studies that show that the putative non-haem-iron(II)/α-ketoglutaratedependent dioxygenase TmuB, from the thiomarinol cluster, 4-hydroxylates various pseudomonic acids whereas C8-OH, and other substituents around the tetrahydropyran ring, block enzyme action but not substrate binding. Molecular modelling suggested a basis for selectivity, but mutation studies had a limited ability to rationally modify TmuB substrate specificity. 4-Hydroxylation had opposite effects on the potency of mupirocin and thiomarinol. Thus, TmuB can be added to the toolbox of polyketide tailoring technologies for the in vivo generation of new antibiotics in the future.