Hyper-Responsive Chemiluminescent Probe Reveals Distinct PYRase Activity in Pseudomonas aeruginosa.

Pyrrolidone carboxyl peptidase, commonly known as PYRase, is an exopeptidase that catalytically cleaves an N-terminal pyroglutamic acid from peptides or proteins. The diverse functions of PYRases in bacterial enzymology have prompted the development of various bacterial diagnostic techniques. However, the specific physiological role and activity of this enzyme across the bacterial kingdom remain unclear. Here, we present a functional phenoxy-1,2-dioxetane chemiluminescent probe (PyrCL) that can selectively detect PYRase activity in both Gram-positive and Gram-negative bacteria. The probe activation mechanism is based on the cleavage of a pyroglutamyl substrate, followed by a release of the phenoxy-dioxetane luminophore, which then undergoes efficient chemiexcitation to emit a green photon. Probe PyrCL exhibits an effective turn-on response with superior detection capability in terms of response time and sensitivity compared to existing fluorescence probes. The superior detection sensitivity of the chemiluminescent probe enables us to reveal previously undetected PYRase activity in Streptococcus mutans. Furthermore, it enables the discrimination of Pseudomonas aeruginosa from other Gram-negative bacteria in the tested panel, based on their distinct PYRase activity. We expect that probe PyrCL will have great value for PYRase-based bacteria diagnosis with use in basic research and clinical applications.

Poacic Acid, a Plant-Derived Stilbenoid, Augments Cell Wall Chitin Production, but Its Antifungal Activity Is Hindered by This Polysaccharide and by Fungal Essential Metals.

Climate and environmental changes have modified the habitats of fungal pathogens, inflicting devastating effects on livestock and crop production. Additionally, drug-resistant fungi are increasing worldwide, driving the urgent need to identify new molecular scaffolds for the development of antifungal agents for humans, animals, and plants. Poacic acid (PA), a plant-derived stilbenoid, was recently discovered to be a novel molecular scaffold that inhibits the growth of several fungi. Its antifungal activity has been associated with perturbation of the production/assembly of the fungal cell wall ß-1,3-glucan, but its mode of action is not resolved. In this study, we investigated the antifungal activity of PA and its derivatives on a panel of yeast. PA had a fungistatic effect on S. cerevisiae and a fungicidal effect on plasma membrane-damaged Candida albicans mutants. Live cell fluorescence microscopy experiments revealed that PA increases chitin production and modifies its cell wall distribution. Chitin production and cell growth returned to normal after prolonged incubation. The antifungal activity of PA was reduced in the presence of exogenous chitin, suggesting that the potentiation of chitin production is a stress response that helps the yeast cell overcome the effect of this antifungal stilbenoid. Growth inhibition was also reduced by metal ions, indicating that PA affects the metal homeostasis. These findings suggest that PA has a complex antifungal mechanism of action that involves perturbation of the cell wall ß-1,3-glucan production/assembly, chitin production, and metal homeostasis.

Enzymatic Activity Profiling Using an Ultrasensitive Array of Chemiluminescent Probes for Bacterial Classification and Characterization.

Identification and characterization of bacterial species in clinical and industrial settings necessitate the use of diverse, labor-intensive, and time-consuming protocols as well as the utilization of expensive and high-maintenance equipment. Furthermore, while cutting-edge identification technologies such as mass spectrometry and PCR are highly effective in identifying bacterial pathogens, they fall short in providing additional information for identifying bacteria not present in the databases upon which these methods rely. In response to these challenges, we present a robust and general approach to bacterial identification based on their unique enzymatic activity profiles. This method delivers results within 90 min, utilizing an array of highly sensitive and enzyme-selective chemiluminescent probes. Leveraging our recently developed technology of chemiluminescent luminophores, which emit light under physiological conditions, we have crafted an array of probes designed to rapidly detect various bacterial enzymatic activities. The array includes probes for detecting resistance to the important and large class of ß-lactam antibiotics. The analysis of chemiluminescent fingerprints from a diverse range of prominent bacterial pathogens unveiled distinct enzymatic activity profiles for each strain. The reported universally applicable identification procedure offers a highly sensitive and expeditious means to delineate bacterial enzymatic activity fingerprints. This opens new avenues for characterizing and identifying pathogens in research, clinical, and industrial applications.

Spirostrain-Accelerated Chemiexcitation of Dioxetanes Yields Unprecedented Detection Sensitivity in Chemiluminescence Bioassays.

Chemiluminescence is a fascinating phenomenon that involves the generation of light through chemical reactions. The light emission from adamantyl-phenoxy-1,2-dioxetanes can glow from minutes to hours depending on the specific substituent present on the dioxetane molecule. In order to improve the light emission properties produced by these chemiluminescent luminophores, it is necessary to induce the chemiexcitation rate to a flash mode, wherein the bulk of light is emitted instantly rather than slowly over time. We report the realization of this goal through the incorporation of spirostrain release into the decomposition of 1,2-dioxetane luminophores. DFT computational simulations provided support for the hypothesis that the spiro-cyclobutyl substituent accelerates chemiexcitation as compared to the unstrained adamantyl substituent. Spiro-linking of cyclobutane and oxetane units led to greater than 100-fold and 1000-fold emission enhancement, respectively. This accelerated chemiexcitation rate increases the detection sensitivity for known chemiluminescent probes to the highest signal-to-noise ratio documented to date. A turn-ON probe, containing a spiro-cyclobutyl unit, for detecting the enzyme ß-galactosidase exhibited a limit of detection value that is 125-fold more sensitive than that for the previously described adamantyl analogue. This probe was also able to instantly detect and image ß-gal activity with enhanced sensitivity in E. coli bacterial assays.

Reshaping Echinocandin Antifungal Drugs To Circumvent Glucan Synthase Point-Mutation-Mediated Resistance.

Echinocandins are a class of antifungal drugs that inhibit the activity of the ß-(1,3)-glucan synthase complex, which synthesizes fungal cell wall ß-(1,3)-glucan. Echinocandin resistance is linked to mutations in the FKS gene, which encodes the catalytic subunit of the glucan synthase complex. We present a molecular-docking-based model that provides insight into how echinocandins interact with the target Fks protein: echinocandins form a ternary complex with both Fks and membrane lipids. We used reductive dehydration of alcohols to generate dehydroxylated echinocandin derivatives and evaluated their potency against a panel of Candida pathogens constructed by introducing resistance-conferring mutations in the FKS gene. We found that removing the hemiaminal alcohol, which drives significant conformational alterations in the modified echinocandins, reduced their efficacy. Conversely, eliminating the benzylic alcohol of echinocandins enhanced potency by up to two orders of magnitude, in a manner dependent upon the resistance-conferring mutation. Strains that have developed resistance to either rezafungin, the most recently clinically approved echinocandin, or its dehydroxylated derivative RZF-1, exhibit high resistance to rezafungin while demonstrating moderate resistance to RZF-1. These findings provide valuable insight for combating echinocandin resistance through chemical modifications.

Introduction to the themed collection on antimicrobial resistance.

Guest editors Jayanta Haldar, Sylvie Garneau-Tsodikova and Micha Fridman introduce the RSC Medicinal Chemistry themed collection on 'Antibiotic microbial resistance'.

Chemiluminescent duplex analysis using phenoxy-1,2-dioxetane luminophores with color modulation.

Multiplex technology is an important emerging field, in diagnostic sciences, that enables the simultaneous detection of several analytes in a single sample. The light-emission spectrum of a chemiluminescent phenoxy-dioxetane luminophore can be accurately predicted by determining the fluorescence-emission spectrum of its corresponding benzoate species, which is generated during the chemiexcitation process. Based on this observation, we designed a library of chemiluminescent dioxetane luminophores with multicolor emission wavelengths. Two dioxetane luminophores that have different emission spectra, but similar quantum yield properties, were selected from the synthesized library for a duplex analysis. The selected dioxetane luminophores were equipped with two different enzymatic substrates to generate turn-ON chemiluminescent probes. This pair of probes exhibited a promising ability to act as a chemiluminescent duplex system for the simultaneous detection of two different enzymatic activities in a physiological solution. In addition, the pair of probes were also able to simultaneously detect the activities of the two enzymes in a bacterial assay, using a blue filter slit for one enzyme and a red filter slit for the other enzyme. As far as we know, this is the first successful demonstration of a chemiluminescent duplex system composed of two-color phenoxy-1,2-dioxetane luminophores. We believe that the library of dioxetanes presented here will be beneficial for developing chemiluminescence luminophores for multiplex analysis of enzymes and bioanalytes.

Dual Chemiexcitation by a Unique Dioxetane Scaffold Gated by an OR Logic Set of Triggers.

Chemiexcitation of phenoxy-1,2-dioxetane chemiluminescent luminophores is initiated by electron transfer from a meta-positioned phenolate ion to the peroxide-dioxetane bond. Here we report the development of a unique 1,2-dioxetane chemiluminescent scaffold with chemiexcitation gated by an OR logic dual-set of triggering events. This scaffold is composed of meta-dihydroxyphenyl-1,2-dioxetane-adamantyl molecules, equipped with acrylic acid and chlorine substituents, that chemiexcitation under physiological conditions. A dual-mode chemiluminescent probe, armed with two different triggering substrates designed for activation by the enzymes ß-galactosidase and alkaline phosphatase, was synthesized. The probe emitted intense light signals in the response to each enzyme, demonstrating its ability to serve as a single-component chemiluminescent sensor for dual-analyte detection. We also demonstrated the ability of the probe to detect ß-galactosidase and phosphatase activities in bacteria. This is the first 1,2-dioxetane scaffold capable of responding to two different chemiexcitation events from two different positions on the same dioxetane molecule. We anticipate that the OR-gated mode of chemiexcitation, described herein, will find utility in the preparation of chemiluminescent probes with a dual-analyte detection/imaging mode.

Deciphering the Biological Activities of Antifungal Agents with Chemical Probes.

The growing number of fungal infections caused by pathogens resistant to one or more classes of antifungal drugs emphasizes the threat that these microorganisms pose to animal and human health and global food security. Open questions remain regarding the mechanisms of action of the limited repertoire of antifungal agents, making it challenging to rationally develop more efficacious therapeutics. In recent years, the use of chemical biology approaches has resolved some of these questions and has provided new promising concepts to guide the design of antifungal agents. By focusing on examples from studies carried out in recent years, this minireview describes the key roles that probes based on antifungal agents and their derivatives have played in uncovering details about their activities, in detecting resistance, and in characterizing the interactions between these agents and their targets.

Cationic, Steroid-Based Imidazolium Amphiphiles Show Tunable Backbone-Dependent Membrane Selectivity in Fungi.

Cationic amphiphiles have been reported to show broad antimicrobial activity. The potential for antimicrobial resistance to these molecules is low owing to their general cell membrane permeabilizing mode of action. However, their applications are often limited by toxicity resulting from their low selectivity for microbial cell membranes. Herein, we report a library of cationic, steroid-based imidazolium amphiphiles that show tunable antifungal activity in a variety of fungal pathogens of the genus Candida. We show that adoption of an ergosterol-derived backbone increases antifungal activity while modestly affecting hemolytic activity, thereby increasing overall selectivity by more than 8-fold in comparison to cholesterol-derived imidazolium salts. We hypothesize that this effect is caused by a privileged integration of the ergosterol-derived salts into fungal membranes leading to increased membrane disorder. We propose that these findings offer a useful platform for the development of improved amphiphilic fungicides.

Echinocandins Localized to the Target-Harboring Cell Surface Are Not Degraded but Those Entering the Vacuole Are.

Echinocandin antifungal drugs have a broad spectrum of activities and excellent safety profiles. These agents noncompetitively inhibit the formation of the major polysaccharide component of the fungal cell wall, a reaction catalyzed by the membrane-bound ß-glucan synthase (GS) protein complex. We have developed fluorescent probes of three echinocandin drugs: caspofungin (CSF), anidulafungin (ANF), and rezafungin (RZF). Fluorescent echinocandins had the same spectrum of activities as the parent echinocandins, supporting the fact that conjugation of the dye did not alter their mode of action. Of the three echinocandins, ANF has the most potent in vitro activity. Investigation of the subcellular distribution of the fluorescent echinocandins in live Candida yeast cells revealed that despite their high structural similarity, each of the drug probes had a unique subcellular distribution pattern. Fluorescent CSF, which is the least potent of the three echinocandins, accumulated in Candida vacuoles; fluorescent ANF localized in the extracellular environment and on the yeast cell surface where the target GS resides; and fluorescent RZF was partitioned between the surface and the vacuole over time. Recovery of fluorescent CSF from Candida cells revealed substantial degradation over time; functional vacuoles were necessary for this degradation. Under the same conditions, fluorescent ANF was not degraded. This study supports the "target-oriented drug subcellular localization" principle. In the case of echinocandins, localization to the cell surface can contribute to improved potency and accumulation in vacuoles induces degradation leading to drug deactivation.

Benzylic Dehydroxylation of Echinocandin Antifungal Drugs Restores Efficacy against Resistance Conferred by Mutated Glucan Synthase.

Each year, infections caused by fungal pathogens claim the lives of about 1.6 million people and affect the health of over a billion people worldwide. Among the most recently developed antifungal drugs are the echinocandins, which noncompetitively inhibit ß-glucan synthase, a membrane-bound protein complex that catalyzes the formation of the main polysaccharide component of the fungal cell wall. Resistance to echinocandins is conferred by mutations in FKS genes, which encode the catalytic subunit of the ß-glucan synthase complex. Here, we report that selective removal of the benzylic alcohol of the nonproteinogenic amino acid 3S,4S-dihydroxy-l-homotyrosine of the echinocandins anidulafungin and rezafungin, restored their efficacy against a large panel of echinocandin-resistant Candida strains. The dehydroxylated compounds did not significantly affect the viability of human-derived cell culture lines. An analysis of the efficacy of the dehydroxylated echinocandins against resistant Candida strains, which contain mutations in the FKS1 and/or FKS2 genes of the parental strains, identified amino acids of the Fks proteins that are likely to reside in proximity to the l-homotyrosine residue of the bound drug. This study describes the first example of a chemical modification strategy to restore the efficacy of echinocandin drugs, which have a critical place in the arsenal of antifungal drugs, against resistant fungal pathogens.

Fluconazole-COX Inhibitor Hybrids: A Dual-Acting Class of Antifungal Azoles.

When used in combination with azole antifungal drugs, cyclooxygenase (COX) inhibitors such as ibuprofen improve antifungal efficacy. We report the conjugation of a chiral antifungal azole pharmacophore to COX inhibitors and the evaluation of activity of 24 hybrids. Hybrids derived from ibuprofen and flurbiprofen were considerably more potent than fluconazole and comparable to voriconazole against a panel of Candida species. The potencies of hybrids composed of an S-configured azole pharmacophore were higher than those with an R-configured pharmacophore. Tolerance, defined as the ability of a subpopulation of cells to grow in the presence of the drug, to the hybrids was lower than to fluconazole and voriconazole. The hybrids were active against a mutant lacking CYP51, the target of azole drugs, indicating that these agents act via a dual mode of action. This study established that azole-COX inhibitor hybrids are a novel class of potent antifungals with clinical potential.

Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin.

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin (CSP) is the second line of therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, CSP induces a tolerance phenotype with partial reestablishment of fungal growth called CSP paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high CSP concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in CSP tolerance and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzAAf293 mutant fails to activate cell wall remodeling genes upon CSP exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This study describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

Azide-Functionalized Derivatives of the Virulence-Associated Sugar Pseudaminic Acid: Chiral Pool Synthesis and Labeling of Bacteria.

Pseudaminic acid (Pse) is a significant prokaryotic monosaccharide found in important Gram-negative and Gram-positive bacteria. This unique sugar serves as a component of cell-surface-associated glycans or glycoproteins and is associated with their virulence. We report the synthesis of azidoacetamido-functionalized Pse derivatives as part of a search for Pse-derived metabolic labeling reagents. The synthesis was initiated with d-glucose (Glc), which served as a cost-effective chiral pool starting material. Key synthetic steps involve the conversion of C1 of Glc into the terminal methyl group of Pse, and inverting deoxyaminations at C3 and C5 of Glc followed by backbone elongation with a three-carbon unit using the Barbier reaction. Metabolic labeling experiments revealed that, of the four Pse derivatives, ester-protected C5 azidoacetamido-Pse successfully labeled cells of Pse-expressing Gram-positive and Gram-negative strains. No labeling was observed in cells of non-Pse-expressing strains. The ester-protected and C5 azidoacetamido-functionalized Pse is thus a useful reagent for the identification of bacteria expressing this unique virulence-associated nonulosonic acid.

Design Guidelines for Cationic Pillar[n]arenes that Prevent Biofilm Formation by Gram-Positive Pathogens.

Bacterial biofilms are a major threat to human health, causing persistent infections that lead to millions of fatalities worldwide every year. Biofilms also cause billions of dollars of damage annually by interfering with industrial processes. Recently, cationic pillararenes were found to be potent inhibitors of biofilm formation in Gram-positive bacteria. To identify the structural features of pillararenes that result in antibiofilm activity, we evaluated the activity of 16 cationic pillar[5]arene derivatives including that of the first cationic water-soluble pillar[5]arene-based rotaxane. Twelve of the derivatives were potent inhibitors of biofilm formation by Gram-positive pathogens. Structure activity analyses of our pillararene derivatives indicated that positively charged head groups are critical for the observed antibiofilm activity. Although certain changes in the lipophilicity of the substituents on the positively charged head groups are tolerated, dramatic elevation in the hydrophobicity of the substituents or an increase in steric bulk on these positive charges abolishes the antibiofilm activity. An increase in the overall positive charge from 10 to 20 did not affect the activity significantly, but pillararenes with 5 positive charges and 5 long alkyl chains had reduced activity. Surprisingly, the cavity of the pillar[n]arene is not essential for the observed activity, although the macrocyclic structure of the pillar[n]arene core, which facilitates the clustering of the positive charges, appears important. Interestingly, the compounds found to be efficient inhibitors of biofilm formation were nonhemolytic at concentrations that are ∼100-fold of their MBIC50 (the minimal concentration of a compound at which at least 50% inhibition of biofilm formation was observed compared to untreated cells). The structure-activity relationship guidelines established here pave the way for a rational design of potent cationic pillar[n]arene-based antibiofilm agents.

Fresh Molecular Concepts to Extend the Lifetimes of Old Antimicrobial Drugs.

Antimicrobial drug development generally initiates with target identification and mode of action studies. Often, emergence of resistance and/or undesired side effects that are discovered only after prolonged clinical use, result in discontinuation of clinical use. Since the cost and time required for improvement of existing drugs are considerably lower than those required for the development of novel drugs, academic and pharmaceutical company researchers pursue this direction. In this account we describe selected examples of how chemical probes generated from antimicrobial drugs and chemical and enzymatic modifications of these drugs have been used to modify modes of action, block mechanisms of resistance, or reduce side effects, improving performance. These examples demonstrate how new and comprehensive mechanistic insights can be translated into fresh concepts for development of next-generation antimicrobial agents.

Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality.

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Serum Prevents Interactions between Antimicrobial Amphiphilic Aminoglycosides and Plasma Membranes.

Antimicrobial cationic amphiphiles have broad-spectrum activity, and microbes do not readily develop resistance to these agents, highlighting their clinical and industrial potential. Cationic amphiphiles perturb the integrity of membranes leading to cell death, and the lack of discrimination between microbial and mammalian plasma membranes is thought to be one of the main barriers of using these agents for the treatment of systemic infections. Here, we describe the synthesis and study of 20 antimicrobial cationic amphiphiles that are derivatives of the aminoglycoside nebramine with different numbers of alkyl chain ethers that differ in length and degree of unsaturation. We determined antifungal activities and evaluated hemoglobin release from red blood cells as a measure of membrane selectivity and analyzed how serum influences these activities. Microscopic images revealed morphological transformations of red blood cells from the normal double-disc shape to empty ghost cells upon treatment with the cationic amphiphiles. Antifungal activity, hemolysis, and morphological changes in red blood cells decreased as the percentage of serum in the culture medium was increased. In images of red blood cells treated with fluorescently labeled amphiphilic nebramine probes, the accumulation of the cationic amphiphiles in the membranes decreased as serum concentration increased. This suggests that, in addition to its known effect of preventing the deformability of red blood cells, serum prevents interactions between cationic amphiphiles and the plasma membrane. The results of this study indicate that biological activities of cationic amphiphiles are abrogated in serum. Thus, these agents are suitable for external and industrial uses but probably not for effective treatment of systemic infections.