Shapeshifting bullvalene-linked vancomycin dimers as effective antibiotics against multidrug-resistant gram-positive bacteria.

The alarming rise in superbugs that are resistant to drugs of last resort, including vancomycin-resistant enterococci and staphylococci, has become a significant global health hazard. Here, we report the click chemistry synthesis of an unprecedented class of shapeshifting vancomycin dimers (SVDs) that display potent activity against bacteria that are resistant to the parent drug, including the ESKAPE pathogens, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-resistant S. aureus (VRSA). The shapeshifting modality of the dimers is powered by a triazole-linked bullvalene core, exploiting the dynamic covalent rearrangements of the fluxional carbon cage and creating ligands with the capacity to inhibit bacterial cell wall biosynthesis. The new shapeshifting antibiotics are not disadvantaged by the common mechanism of vancomycin resistance resulting from the alteration of the C-terminal dipeptide with the corresponding d-Ala-d-Lac depsipeptide. Further, evidence suggests that the shapeshifting ligands destabilize the complex formed between the flippase MurJ and lipid II, implying the potential for a new mode of action for polyvalent glycopeptides. The SVDs show little propensity for acquired resistance by enterococci, suggesting that this new class of shapeshifting antibiotic will display durable antimicrobial activity not prone to rapidly acquired clinical resistance.

Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase.

Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

Cage hydrocarbons as linkers in dimeric drug design: Case studies with trimethoprim and tedizolid.

The looming threat of a "post-antibiotic era" has been caused by a rapid rise in antibacterial resistance and subsequent depletion of effective antibiotic agents in the clinic. An efficient strategy to address this shortfall lies in the reengineering of pre-existing and commercially available antibiotic drugs. This is exemplified by dimerization, a design concept in which two pharmacophores are covalently linked to form a new chemical entity. The cage hydrocarbons cubane (1), bicyclo[2.2.2]octane (BCO) (2), adamantane (3), and bicyclo[1.1.1]pentane (BCP) (4) present themselves as an attractive family of linkers in this regard. In this report, all four hydrocarbon cages were employed as linkers in a series of dimers based on the commercially available antibiotics trimethoprim and tedizolid. A detailed synthetic roadmap for the protection and deprotection of each pharmacophore is outlined. Several members of the trimethoprim series showed activity on par with that of their trimethoprim progenitor, although this was not the case for the tedizolid series. The design strategy outlined herein highlights the utility of the group as a platform for the rapid and modular construction of future novel antibiotics.

Biosynthesis of uridine diphosphate N-Acetylglucosamine: An underexploited pathway in the search for novel antibiotics?

Although the prevalence of antibiotic resistance is increasing at an alarming rate, there are a dwindling number of effective antibiotics available. Thus, the development of novel antibacterial agents should be of utmost importance. Peptidoglycan biosynthesis has been and is still an attractive source for antibiotic targets; however, there are several components that remain underexploited. In this review, we examine the enzymes involved in the biosynthesis of one such component, UDP-N-acetylglucosamine, an essential building block and precursor of bacterial peptidoglycan. Furthermore, given the presence of a similar biosynthesis pathway in eukaryotes, we discuss the current knowledge on the differences and similarities between the bacterial and eukaryotic enzymes. Finally, this review also summarises the recent advances made in the development of inhibitors targeting the bacterial enzymes.

Synthesis and structure-activity relationship studies of 2,4-thiazolidinediones and analogous heterocycles as inhibitors of dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS), responsible for the first committed step of the diaminopimelate pathway for lysine biosynthesis, has become an attractive target for the development of new antibacterial and herbicidal agents. Herein, we report the discovery and exploration of the first inhibitors of E. coli DHDPS which have been identified from screening lead and are not based on substrates from the lysine biosynthesis pathway. Over 50 thiazolidinediones and related analogues have been prepared in order to thoroughly evaluate the structure-activity relationships against this enzyme of significant interest.

Heparanase: Cloning, Function and Regulation.

In 2019, we mark the 20th anniversary of the cloning of the human heparanase gene. Heparanase remains the only known enzyme to cleave heparan sulfate, which is an abundant component of the extracellular matrix. Thus, elucidating the mechanisms underlying heparanase expression and activity is critical to understanding its role in healthy and pathological settings. This chapter provides a historical account of the race to clone the human heparanase gene, describes the intracellular and extracellular function of the enzyme, and explores the various mechanisms regulating heparanase expression and activity at the gene, transcript, and protein level.

Metal-Free Synthesis of Functional 1-Substituted-1,2,3-Triazoles from Ethenesulfonyl Fluoride and Organic Azides.

The boom in growth of 1,4-disubstituted triazole products, in particular, since the early 2000's, can be largely attributed to the birth of click chemistry and the discovery of the CuI -catalyzed azide-alkyne cycloaddition (CuAAC). Yet the synthesis of relatively simple, albeit important, 1-substituted-1,2,3-triazoles has been surprisingly more challenging. Reported here is a straightforward and scalable click-inspired protocol for the synthesis of 1-substituted-1,2,3-triazoles from organic azides and the bench stable acetylene surrogate ethenesulfonyl fluoride (ESF). The new transformation tolerates a wide selection of substrates and proceeds smoothly under metal-free conditions to give the products in excellent yield. Under controlled acidic conditions, the 1-substituted-1,2,3-triazole products undergo a Michael addition reaction with a second equivalent of ESF to give the unprecedented 1-substituted triazolium sulfonyl fluoride salts.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ.

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/ß receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNß. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum.

MAIN CONCLUSION: Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a KM (pyruvate) of 0.45 mM, KM (l-aspartate-4-semialdehyde) of 0.07 mM, kcat of 56 s-1, and is inhibited by lysine (IC 50 LYS of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/ß structure and exists as a 7.5 S tetrameric species with a Rg of 33 Å and Dmax of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.

Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae.

Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, KM (pyruvate) of 0.14 mM and 0.15 mM, KM (L-aspartate-4-semialdehyde) of 0.08 mM and 0.09 mM, and kcat of 34 and 46 s-1, respectively. These results demonstrate there are no significant changes in the structure and function of Vc-DHDPS upon the addition of an N-terminal His tag and, hence, the tagged recombinant product is suitable for future studies, including screening for new inhibitors as potential anti-cholera agents. Additionally, a polyclonal antibody raised against untagged Vc-DHDPS is validated for specifically detecting recombinant and native forms of the enzyme.

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis.

Diaminopimelate decarboxylase (DAPDC) catalyzes the final step in the diaminopimelate biosynthesis pathway of bacteria. The product of the reaction is the essential amino acid l-lysine, which is an important precursor for the synthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Accordingly, the enzyme is a promising antibacterial target. Previous structural studies demonstrate that DAPDC exists as monomers, dimers, and tetramers in the crystal state. However, the active oligomeric form has not yet been determined. We show using analytical ultracentrifugation, small angle x-ray scattering, and enzyme kinetic analyses in solution that the active form of DAPDC from Bacillus anthracis, Escherichia coli, Mycobacterium tuberculosis, and Vibrio cholerae is a dimer. The importance of dimerization was probed further by generating dimerization interface mutants (N381A and R385A) of V. cholerae DAPDC. Our studies indicate that N381A and R385A are significantly attenuated in catalytic activity, thus confirming that dimerization of DAPDC is essential for function. These findings provide scope for the development of new antibacterial agents that prevent DAPDC dimerization.

Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a Gram-negative bacterium and causative agent of Crown Gall disease that infects a variety of economically important plants. The annotated A. tumefaciens genome contains 10 putative dapA genes, which code for dihydrodipicolinate synthase (DHDPS). However, we have recently demonstrated that only one of these genes (dapA7) encodes a functional DHDPS. The function of the other nine putative dapA genes is yet to be determined. Here, we demonstrate using bioinformatics that the product of the dapA5 gene (DapA5) possesses all the catalytic residues canonical to 2-keto-3-deoxygluconate (KDG) aldolase, which is a class I aldolase involved in glucose metabolism. We therefore expressed, purified, and characterized recombinant DapA5 using mass spectrometry, circular dichroism spectroscopy, analytical ultracentrifugation, and enzyme kinetics. The results show that DapA5 (1) adopts an α/ß structure consistent with the TIM-barrel fold of KDG aldolases, (2) possesses KDG aldolase enzyme activity, and (3) exists as a tight dimer in solution. This study shows for the first time that dapA5 from A. tumefaciens encodes a functional dimeric KDG aldolase.

Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus.

Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E. coli BPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in Ki values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.

Heterocyclic acyl-phosphate bioisostere-based inhibitors of Staphylococcus aureus biotin protein ligase.

Inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL) are generated by replacing the acyl phosphate group of biotinyl-5'-AMP with either a 1,2,3-triazole (see 5/10a/10b) or a 1,2,4-oxadiazole (see 7) bioisostere. Importantly, the inhibitors are inactive against the human BPL. The nature of the 5-substituent in the component benzoxazolone of the optimum 1,2,3-triazole series is critical to activity, where this group binds in the ATP binding pocket of the enzyme.

Selective inhibition of biotin protein ligase from Staphylococcus aureus.

There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.

Structural insight into the Scribble PDZ domains interaction with the oncogenic Human T-cell lymphotrophic virus-1 (HTLV-1) Tax1 PBM.

Scribble (Scrib) is a highly conserved cell polarity regulator that harbours potent tumour suppressor activity and plays an important role in cell migration. Dysregulation of polarity is associated with poor prognosis during viral infections. Human T-cell lymphotrophic virus-1 (HTLV-1) encodes for the oncogenic Tax1 protein, a modulator of the transcription of viral and human proteins that can cause cell cycle dysregulation as well as a loss of genomic integrity. Previous studies established that Scribble interacts with Tax1 via its C-terminal PDZ-binding motif (PBM), leading to aggregation of polarity regulators and subsequent perturbation of host cell adhesion, proliferation, and signalling. Using isothermal titration calorimetry, we now show that all four PDZ domains of Scribble bind to Tax1 PBM. We then determined crystal structures of Scribble PDZ1, PDZ2 and PDZ3 domains bound to Tax1 PBM. Our findings establish a structural basis for Tax1-mediated subversion of Scribble-mediated cell polarity signalling and provide the platform for mechanistic studies to examine Tax1 induced mislocalization of Scribble and the associated changes in cellular architecture and subsequent tumorigenesis.

Repurposed inhibitor of bacterial dihydrodipicolinate reductase exhibits effective herbicidal activity.

Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicide-resistant weeds. Here, we employed a novel strategy to repurpose a 'failed' antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana. We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana. We also showed that our lead compound is the first lysine biosynthesis inhibitor with activity against both monocotyledonous and dicotyledonous weed species, by demonstrating its effectiveness at reducing the germination and growth of Lolium rigidum (rigid ryegrass) and Raphanus raphanistrum (wild radish). These results provide proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing 'failed' antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes.

Enhancing allosteric inhibition of dihydrodipicolinate synthase through the design and synthesis of novel dimeric compounds.

The synthesis of the first dimeric inhibitor of E. coli dihydrodipicolinate synthase (DHDPS) is reported herein. Inspired by 2,4-thiazolidinedione based ligands previously shown to inhibit DHDPS, a series of dimeric inhibitors were designed and synthesised, incorporating various alkyl chain bridges between two 2,4-thiazolidinedione moieties. Aiming to exploit the multimeric nature of this enzyme and enhance potency, a dimeric compound with a single methylene bridge achieved the desired outcome with low micromolar inhibition of E. coli DHDPS observed. This work highlights the continued importance of investigation into DHDPS as an antibacterial target. Furthermore, we demonstrate the design of dimeric ligands can provide a promising strategy to improve potency in the search for novel bioactive compounds.

Antibacterial silver and gold complexes of imidazole and 1,2,4-triazole derived N-heterocyclic carbenes.

A series of gold(I) (4a-4h, 5a-5b) and silver(I) (3a-3h) complexes of 1,2,4-triazolylidene and imidazolylidene based N-heterocyclic carbene ligands were prepared and the antibacterial activities of these complexes have been evaluated. The complexes were characterised using 1H-NMR, 13C-NMR, HRMS and in the cases of 3a, 3c, 4b and 5b by X-ray crystallography. The gold(I) complexes with phenyl substituents (4a-4d) were found to have potent antibacterial activity against Gram-positive bacteria, with the complexes of the 1,2,4-triazolylidene ligands being more active (4c, MIC = 4-8 µg mL-1 against Enterococcus faecium and 2 µg mL-1 against Staphylococcus aureus) than the analogous imidazolylidene complexes 4a and 4b (4a, MIC = 64 µg mL-1 against E. faecium and 2-4 µg mL-1 against S. aureus). Two of the silver(I) complexes have promising antibacterial activity against Acinetobacter baumannii (3f, MIC = 2-4 µg mL-1 and 3g, MIC = 2 µg mL-1). Silver(I) complex 3f and gold(I) complex 4c were tested against multi-drug resistant bacterial strains and high levels of antibacterial activity were observed. The potential for antibacterial resistance to develop against these metal containing complexes was investigated and significantly, no resistance was observed upon continuous treatment, whilst resistance was developed against the widely used broad-spectrum antibiotic ciprofloxacin in the same bacterial strains, under the conditions tested. The solution and gas phase stabilities of the complexes have been investigated using a combination of 1H-NMR, HRMS and detailed computational mechanistic studies were undertaken to gain insights into the possible decomposition reactions for silver complexes in aqueous solution.

A dual-target herbicidal inhibitor of lysine biosynthesis.

Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.