Design and synthesis of fluorescent pilicides and curlicides: bioactive tools to study bacterial virulence mechanisms

Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure-activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores. Several of the resulting fluorescent compounds had improved activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing coumarin and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide central fragment. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated with the bacteria with a heterogeneous distribution.     

C-Terminal properties are important for ring-fused 2-pyridones that interfere with the chaperone function in uropathogenic E. coli

Virulence-associated organelles, termed pili or fimbriae, are assembled via the highly conserved chaperone-usher pathway in a vast number of pathogenic bacteria. Substituted bicyclic 2-pyridones, pilicides, inhibit pilus formation, possibly by interfering with the active site residues Arg8 and Lys112 of chaperones in uropathogenic E. coli. In this article we describe the synthesis and evaluation of nine analogues of a biologically active pilicide. Derivatization was performed with respect to its C-terminal features and the affinities for the chaperone PapD were studied with 1H relaxation-edited NMR spectroscopy. It could be concluded that the carboxylic acid functionality and also its spatial location was important for binding. In all cases, binding was significantly reduced or even abolished when the carboxylic acid was replaced by other substituents. In addition, in vivo results from a hemagglutination assay are presented where the derivatives have been evaluated for their ability to inhibit pilus formation in uropathogenic E. coli.     

NMR studies of interactions between periplasmic chaperones from uropathogenic E. coli and pilicides that interfere with chaperone function and pilus assembly

Adherence of uropathogenic Escherichia coli to host tissue is mediated by pili, which are hair-like protein structures extending from the outer cell membrane of the bacterium. The chaperones FimC and PapD are key components in pilus assembly since they catalyse folding of subunits that are incorporated in type 1 and P pili, respectively, and also transport the subunits across the periplasmic space. Recently, compounds that inhibit pilus biogenesis and interfere with chaperone-subunit interactions have been discovered and termed pilicides. In this paper NMR spectroscopy was used to study the interaction of different pilicides with PapD and FimC in order to gain structural knowledge that would explain the effect that some pilicides have on pilus assembly. First relaxation-edited NMR experiments revealed that the pilicides bound to the PapD chaperone with mM affinity. Then the pilicide-chaperone interaction surface was investigated through chemical shift mapping using 15N-labelled FimC. Principal component analysis performed on the chemical shift perturbation data revealed the presence of three binding sites on the surface of FimC, which interacted with three different classes of pilicides. Analysis of structure-activity relationships suggested that pilicides reduce pilus assembly in E. coli either by binding in the cleft of the chaperone, or by influencing the orientation of the flexible F1-G1 loop, both of which are part of the surface by which the chaperone forms complexes with pilus subunits. It is suggested that binding to either of these sites interferes with folding of the pilus subunits, which occurs during formation of the chaperone-subunit complexes. In addition, pilicides that influence the F1-G1 loop also appear to reduce pilus formation by their ability to dissociate chaperone-subunit complexes.     

Preparation of fluorinated linkers: use of 19F NMR spectroscopy to establish conditions for solid-phase synthesis of pilicide libraries

Three fluorinated linkers which are analogues of linkers commonly used in solid-phase peptide synthesis have been prepared. One of the linkers was used in combination with gel-phase 19F NMR spectroscopy to develop conditions for solid-phase synthesis of two libraries of pilicides, i.e. compounds designed to inhibit assembly of adhesive pili in uropathogenic Escherichia coli. Attachment to and cleavage from the linker could be monitored based on the chemical shift of the fluorine atom of the linker. In addition, use of the linker as internal standard allowed quantification and optimization of reactions occurring further away from the linker when fluorinated building blocks were employed. Importantly, high-quality 19F NMR spectra were obtained for compounds linked to a TentaGel resin in a standard NMR tube using an ordinary NMR instrument.     

New Found Hope for Antibiotic Discovery: Lipid II Inhibitors

Research into antibacterial agents has recently gathered pace in light of the disturbing crisis of antimicrobial resistance. The development of modern tools offers the opportunity of reviving the fallen era of antibacterial discovery through uncovering novel lead compounds that target vital bacterial cell components, such as lipid II. This paper provides a summary of the role of lipid II as well as an overview and insight into the structural features of macrocyclic peptides that inhibit this bacterial cell wall component. The recent discovery of teixobactin, a new class of lipid II inhibitor has generated substantial research interests. As such, the significant progress that has been achieved towards its development as a promising antibacterial agent is discussed.     

Traffic jam at the bacterial sec translocase: targeting the SecA nanomotor by small-molecule inhibitors

The rapid rise of drug-resistant bacteria is one of the most serious unmet medical needs facing the world. Despite this increasing problem of antibiotic resistance, the number of different antibiotics available for the treatment of serious infections is dwindling. Therefore, there is an urgent need for new antibacterial drugs, preferably with novel modes of action to potentially avoid cross-resistance with existing antibacterial agents. In recent years, increasing attention has been paid to bacterial protein secretion as a potential antibacterial target. Among the different protein secretion pathways that are present in bacterial pathogens, the general protein secretory (Sec) pathway is widely considered as an attractive target for antibacterial therapy. One of the key components of the Sec pathway is the peripheral membrane ATPase SecA, which provides the energy for the translocation of preproteins across the bacterial cytoplasmic membrane. In this review, we will provide an overview of research efforts on the discovery and development of small-molecule SecA inhibitors. Furthermore, recent advances on the structure and function of SecA and their potential impact on antibacterial drug discovery will be discussed.     

Functionalization of bicyclic 2-pyridones targeting pilus biogenesis in uropathogenic Escherichia coli

Substituted bicyclic 2-pyridones, termed pilicides, prevent pilus assembly in uropathogenic Escherichia coli. Based on the bioactive methyl ester protected 2-pyridone 4, further functionalization at C-6 has yielded a set of new compounds, which have been evaluated for their ability to inhibit pilus formation in uropathogenic E. coli. The key intermediate in the synthesis was formylated 2-pyridone 5, which could be obtained via a Vilsmeier reaction. This versatile intermediate was converted into secondary and tertiary amines via reductive amination and could also be converted to other interesting functionalities using simple chemical transformations.     

Synthesis and antibacterial evaluation of novel Schiff base derivatives containing 4(3<Emphasis Type="Italic">H</Emphasis>)-quinazolinone moiety

A series of novel Schiff base derivatives containing 4(3H)-quinazolinone moiety were synthesised and their antibacterial activities against tobacco and tomato bacterial wilts evaluated in vitro. Out of the synthesised compounds, 5g, 5j, 5n, 5m and 5p exhibited excellent antibacterial activities against tobacco bacterial wilt, with half maximal effective concentrations (EC50): 160.34, 158.03, 125.94, 148.09 and 133.67 (all in εg mL^?1), respectively, which were better than the EC50 of thiodiazole–copper (216.70 εg mL^?1). Compounds 5j, 5n and 5o also showed good antibacterial activities against tomato bacterial wilt, with EC50 of 95.20, 90.03 and 83.21 (all in εg mL^?1) respectively, which were better than the EC50of thiodiazole–copper (99.80 εg mL^?1). These compounds may prove to be useful as potential antibacterial agents.     

Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation

According to the latest report released by the World Health Organization, bacterial resistance to well-known and widely available antibacterial drugs has become a significant and severe global health concern and a grim challenge to tackle in order to cure infections associated with multidrug-resistant pathogenic microorganisms efficiently. Consequently, various strategies have been orchestrated to cure the severe complications related to multidrug-resistant bacteria effectively. Some approaches involved the retardation of biofilm formation and multidrug-resistance pumps in bacteria as well as the discovery of new antimicrobial agents demonstrating different mechanisms of action. In this regard, natural products namely alkaloids, terpenoids, steroids, anthraquinone, flavonoids, saponins, tannins, etc., have been suggested to tackle the multidrug-resistant bacterial strains owing to their versatile pharmacological effects. Amongst these, flavonoids, also known as polyphenolic compounds, have been widely evaluated for their antibacterial property due to their tendency to retard the growth of a wide range of pathogenic microorganisms, including multidrug-resistant bacteria. The hydroxylation of C5, C7, C3′, and C4′; and geranylation or prenylation at C6 have been extensively studied to increase bacterial inhibition of flavonoids. On the other hand, methoxylation at C3′ and C5 has been reported to decrease flavonoids’ antibacterial action. Hence, the latest information on the antibacterial activity of flavonoids is summarized in this review, with particular attention to the structure–activity relationship of this broad class of natural compounds to discover safe and potent antibacterial agents as natural products.     

Preparation of Some Thiazolyl Hydrazone Derivatives and Evaluation of Their Antibacterial Activities

The increasing clinical importance of drug-resistant fungal and bacterial pathogens has provided additional urgency to microbiological research and to the development of new antibacterial compounds. For this purpose, new tert-butyl [1-aryl/alkyl-2[(4-aryl-2-thiazolyl)hydrazono]ethyl]carbamate derivatives were synthesized and evaluated for antibacterial activity. The reaction of Boc-L-phenylalaninal, Boc-D-phenylalaninal, Boc-L-leucinal, and Boc-L-tryptophanal with thiosemicarbazide yielded the thiosemicarbazones, which furnished the title compounds on reaction with phenacyl bromides. The new compounds were screened for antibacterial activity. The results from the bioassay tests show that some of the compounds have notable activity against Gram-positive bacteria.     

The Synthesis and Evaluation of Diethyl Benzylphosphonates as Potential Antimicrobial Agents

The impact of substituent at phenyl ring of diethyl benzylphosphonate derivatives on cytotoxic activity was studied. The organophosphonates were obtained based on developed palladium-catalyzed α, β-homodiarylation of vinyl esters protocol. The new synthetic pathway toward 1,2-bis(4-((diethoxyphosphoryl)methyl)phenyl)ethyl acetate was proposed which significantly improves the overall yield of the final product (from 1% to 38%). Several newly synthesized organophosphonates were tested as new potential antimicrobial drugs on model Escherichia coli bacterial strains (K12 and R2-R3). All tested compounds show the highest selectivity and activity against K12 and R2 strains. Preliminary cellular studies using MIC and MBC tests and digestion of Fpg after modification of bacterial DNA suggest that selected benzylphosphonate derivatives may have greater potential as antibacterial agents than typically used antibiotics such as ciprofloxacin, bleomycin and cloxacillin. These compounds are highly specific for pathogenic E. coli strains based on the model strains used and may be engaged in the future as new substitutes for commonly used antibiotics, which is especially important due to the increasing resistance of bacteria to various drugs and antibiotics.     

Small organometallic compounds as antibacterial agents

The emergence of bacterial resistance to commercial antibiotics is an issue of global importance. During the last two decades, the number of antibacterial agents that have been discovered and introduced into the market has steadily declined and failed to meet the challenges posed by rapidly increasing resistance of the pathogens against common antibacterial drugs. The development of new classes of compounds to control the virulence of the pathogens is therefore urgently required. This perspective describes the historical development in brief and recent advances on the preparation of small organometallic compounds as new classes of antibacterial agents with potential for clinical development.     

Biosynthetic studies of Nocathiacin-I

Nocathiacin-I is one of the newest members of thiazolyl peptide class of antibiotics. It is a potent inhibitor of bacterial protein synthesis and showed potent in vitro and in vivo Gram-positive antibacterial activity. Understanding of the biosynthesis of natural products is important for improvement of titer and precursor directed biosynthesis for new compounds. Biosynthesis of nocathiacin-I in Amycolatopsis fastidiosa using stable isotope precursor incorporation is described.     

Merging Natural Products: Muraymycin–Sansanmycin Hybrid Structures as Novel Scaffolds for Potential Antibacterial Agents

To overcome bacterial resistances, the need for novel antimicrobial agents is urgent. The class of so-called nucleoside antibiotics furnishes promising candidates for the development of new antibiotics, as these compounds block a clinically unexploited bacterial target: the integral membrane protein MraY, a key enzyme in cell wall (peptidoglycan) biosynthesis. Nucleoside antibiotics exhibit remarkable structural diversity besides their uridine-derived core motifs. Some sub-classes also show specific selectivities towards different Gram-positive and Gram-negative bacteria, which are poorly understood so far. Herein, the synthesis of a novel hybrid structure is reported, derived from the 5′-defunctionalized uridine core moiety of muraymycins and the peptide chain of sansanmycin B, as a new scaffold for the development of antimicrobial agents. The reported muraymycin–sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, but displayed no significant antibacterial activity against S. aureus, E. coli, and P. aeruginosa.     

Synthesis and investigation of binding interactions of 1,4-benzoxazine derivatives on topoisomerase IV in Acinetobacter baumannii$

Abstract

Acinetobacter baumannii has emerged as an important pathogen for nosocomial infections having high morbidity and mortality. This pathogen is notorious for antimicrobial resistance to many common antimicrobial agents including fluoroquinolones, which have both intrinsic and acquired resistance mechanisms. Fluoroquinolones targeting the bacterial topoisomerase II (DNA gyrase and Topo IV) show potent broad-spectrum antibacterial activity by the stabilization of the covalent enzyme–DNA complex. However, their efficacy is now being threatened by an increasing prevalence of resistance. Fluoroquinolones cause stepwise mutations in DNA gyrase and Topo IV, having alterations of their binding sites. Furthermore, the water–Mg⁺² bridge, which provides enzyme–fluoroquinolone interactions, has a significant role in resistance. In this study, 13 compounds were synthesized as 1,4-benzoxazine derivatives which act as bacterial topoisomerase II inhibitors and their antibacterial activities were determined against multi-drug resistant Acinetobacter strains which have ciprofloxacin (CIP) resistant and GyrA mutation. Afterwards we performed docking studies with Topo IV (pdb:2XKK) of these compounds to comprehend their binding properties in Discovery Studio 3.5. The results of this study show significant conclusions to elucidate the resistance mechanism and lead to the design of new antibacterial agents as bacterial topoisomerase II inhibitors.     

Discovery of kibdelomycin, a potent new class of bacterial type II topoisomerase inhibitor by chemical-genetic profiling in Staphylococcus aureus

Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied?a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.     

Microwave-assisted rapid and efficient synthesis of chromene-fused pyrrole derivatives through multicomponent reaction and evaluation of antibacterial activity with molecular docking investigation

The current study aimed to identify a new strategy of FeCl₃ catalyzed multicomponent synthesis of substituted 2H-chromene–fused pyrrole derivatives. A series of chromene-based pyrrole prepared by employing an array of 3-nitro-2H-chromenes, aniline, and acetylacetone in toluene under microwave irradiation. Using FeCl₃ as a prompt catalyst and microwave irradiation to synthesize 2H-chromene–fused pyrrole motifs significantly reduces the reaction time and facilitates to high yields (83%-95%). Structure of all synthesized compounds analyzed by spectroscopic analysis. One-pot reaction, short reaction period, and simple experimental procedure are the fascinating properties associated with this protocol. The in vitro antibacterial activity of the entire series was assessed against Staphylococcus aureus and Escherichia coli. Out of all the compounds, 15b and 15h revealed most excellent potency against both the bacterial strains relative to the reference gentamicin. Docking study was employed to determine the possible binding orientation of DNA gyrase with the active sites of chromene-fused pyrrole analog. The docking results show that compounds 15b and 15h have higher binding affinity with energy −8.00 and −8.80 kcal/mol. These results illuminate the mode of binding progression and provide an esteemed pathway for the design and the structural modification of chromene-fused pyrroles as a newly advanced class of antibacterial agent.     

Molecular dynamics simulation and linear interaction energy study of d-Glu-based inhibitors of the MurD ligase

The biosynthetic pathway of the bacterial peptidoglycan, where MurD is an enzyme involved at the intracellular stage of its construction, represents a collection of highly selective macromolecular targets for novel antibacterial drug design. In this study as part of our investigation of the MurD bacterial target two recently discovered classes of the MurD ligase inhibitors were investigated resulting from the lead optimization phases of the N-sulfonamide d-Glu MurD inhibitors. Molecular dynamics simulations, based on novel structural data, in conjunction with the linear interaction energy (LIE) method suggested the transferability of our previously obtained LIE coefficients to further d-Glu based classes of MurD inhibitors. Analysis of the observed dynamical behavior of these compounds in the MurD active site was supported by static drug design techniques. These results complement the current knowledge of the MurD inhibitory mechanism and provide valuable support for the d-Glu paradigm of the inhibitor design.     

Antibacterial and antifungal ferrocene incorporated dithiothione and dithioketone compounds

Two antibacterial and antifungal ferrocene incorporated compounds have been synthesized, characterized and screened for their in vitro antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae, Bacillus cereus, Corynebacterium diphtheriae, Staphylococcus aureus and Streptococcus pyogenes bacterial strains and for in vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. Results show that these compounds have significant activity against tested bacterial and fungal strains and thus introduce a novel class of ferrocene incorporating antibacterial and antifungal compounds. Copyright © 2005 John Wiley & Sons, Ltd.