Adaptations to cationic biocide exposure differentially influence virulence factors and pathogenicity in Pseudomonas aeruginosa.

Cationic biocides (CBs), which include quaternary ammonium compounds (QACs), are employed to mitigate the spread of infectious bacteria, but resistance to such surface disinfectants is rising. CB exposure can have profound phenotypic implications that extend beyond allowing microorganisms to persist on surfaces. Pseudomonas aeruginosa is a deadly bacterial pathogen that is intrinsically tolerant to a wide variety of antimicrobials and is commonly spread in healthcare settings. In this study, we pursued resistance selection assays to the QAC benzalkonium chloride and quaternary phosphonium compound P6P-10,10 to assess the phenotypic effects of CB exposure in P. aeruginosa PAO1 and four genetically diverse, drug-resistant clinical isolates. In particular, we sought to examine how CB exposure affects defensive strategies and the virulence-associated "offensive" strategies in P. aeruginosa. We demonstrated that development of resistance to BAC is associated with increased production of virulence-associated pigments and alginate as well as pellicle formation. In an in vivo infection model, CB-resistant PAO1 exhibited a decreased level of virulence compared to wild type, potentially due to an observed fitness cost in these strains. Taken together, these results illustrate the significant consequence CB resistance exerts on the virulence-associated phenotypes of P. aeruginosa.

Leveraging Natural Product-inspired Antifungals to Investigate the Mechanism of Action of Peniciaculin A.

Ubiquinone mimics known as quinone outside inhibitors (QoIs) are one of the most prominent fungicides used to protect crops in the agricultural industry. Due to chemotype similarities with known QoIs, peniciaculin A, a triaryl natural product, was proposed to exhibit similar broad spectrum antifungal activity against phytopathogens. Instability of the tertiary alcohol and phenol motif, however, prompted exploration of the antifungal properties of simplified analogues to probe possible overlap in mechanism of action between the natural product and QoIs. Peniciaculin A inspired analogues mimicking known QoI scaffolds displayed broad spectrum antifungal activity while those containing scaffolds dissimilar to QoIs possessed negligible bioactivity. These activity profiles suggest peniciaculin A is likely acting as a QoI.

In Situ Biofilm Affinity-Based Protein Profiling Identifies the Streptococcal Hydrolase GbpB as the Target of a Carolacton-Inspired Chemical Probe.

Natural products are important precursors for antibiotic drug design. These chemical scaffolds serve as synthetic inspiration for chemists who leverage their structures to develop novel antibacterials and chemical probes. We have previously studied carolacton, a natural product macrolactone fromSorangium cellulosum, and discovered a simplified derivative, A2, that maintained apparent biofilm inhibitory activity, although the biological target was unknown. Herein, we utilize affinity-based protein profiling (AfBPP) in situ during biofilm formation to identify the protein target using a photoexcitable cross-linking derivative of A2. From these studies, we identified glucan binding protein B (GbpB), a peptidoglycan hydrolase, as the primary target of A2. Further characterization of the interaction between A2 and GbpB, as well as PcsB, a closely related homologue from the more pathogenic S. pneumoniae, revealed binding to the catalytic CHAP (cysteine, histidine, aminopeptidase) domain. To the best of our knowledge, this is the first report of a small-molecule binder of a conserved and essential bacterial CHAP hydrolase, revealing its potential as an antibiotic target. This work also highlights A2 as a useful tool compound for streptococci and as an initial scaffold for the design of more potent CHAP binders.

Siderophores: A Case Study in Translational Chemical Biology.

Siderophores are metal-binding secondary metabolites that assist in iron homeostasis and have been of interest to the scientific community for the last half century. Foundational siderophore research has enabled several translational applications including siderophore-antibiotic and siderophore-peptide conjugates, identification of new antimicrobial targets, advances in disease imaging, and novel therapeutics. This review aims to connect the basic science research (biosynthesis, cellular uptake, gene regulation, and effects on homeostasis) of well-known siderophores with the successive translational application that results. Intertwined throughout are connections to the career of Christopher T. Walsh, his impact on the field of chemical biology, and the legacy of his trainees who continue to innovate.

Mild Photochemical Reduction of Alkenes and Heterocycles via Thiol-Mediated Formate Activation.

The reduction of alkenes to their respective alkanes is one of the most important transformations in organic chemistry, given the abundance of natural and commercial olefins. Metal-catalyzed hydrogenation is the most common way to reduce alkenes; however, the use of H2 gas in combination with the precious metals required for these conditions can be impractical, dangerous, and expensive. More complex substrates often require extremely high pressures of H2, further emphasizing the safety concerns associated with these hydrogenation reactions. Here we report a safe, cheap, and practical photochemical alkene reduction using a readily available organophotocatalyst, catalytic thiol, and formate. These conditions reduce a variety of di-, tri-, and tetra-substituted alkenes in good yield as well as dearomatize pharmaceutically relevant heterocycles to generate sp3-rich isosteres of benzofurans and indoles. These formal-hydrogenation conditions tolerate a broad range of functionalities that would otherwise be sensitive to typical hydrogenations and are likely to be important for industry applications.

Bushy-Tailed QACs: The Development of Multicationic Quaternary Ammonium Compounds with a High Degree of Alkyl Chain Substitution.

Quaternary ammonium compounds have served as a first line of protection for human health as surface disinfectants and sanitizers for nearly a century. However, increasing levels of bacterial resistance have spurred the development of novel QAC architectures. In light of the observed reduction in eukaryotic cell toxicity when the alkyl chains on QACs are shorter in nature (≤10 C), we prepared 47 QAC architectures that bear multiple short alkyl chains appended to up to three cationic groups, thus rendering them "bushy-tailed" multiQACs. Antibacterial activity was strong (often ~1-4 µM) in a varied set of bushy-tailed architectures, though observed therapeutic indices were not significantly improved over QAC structures bearing fewer and longer alkyl chains.

Exploring the Correlation of Dynamic Surface Tension with Antimicrobial Activities of Quaternary Ammonium-Based Disinfectants.

Quaternary ammonium compound (QAC) disinfectants represent one of our first lines of defense against pathogens. Their inhibitory and bactericidal activities are usually tested through minimum inhibitory concentration (MIC) and time-kill assays, but these assays can become cumbersome when screening many compounds. We investigated how the dynamic surface tension (DST) measurements of QACs correlate with these antimicrobial activities by testing a panel of potent and structurally varied QACs against the gram-positive Staphylococcus aureus and the gram-negative Pseudomonas aeruginosa. We found that DST values correlated well with bactericidal activity in real-world disinfection conditions but not with MIC values. Moreover, no correlation between these two antimicrobial activities of QACs (bactericidal and inhibition) was observed. In addition, we observed that the bactericidal activity of our QAC panel against the gram-negative P. aeruginosa was severely affected in the presence of hard water. Interestingly, we found that the counterion of the QAC affects the killing of bacteria in these conditions, a phenomenon not observed in most MIC assessments. Moreover, some of our best-in-class QACs show enhanced bactericidal activity when combined with a commercially available QAC. In conclusion, we determined that an intrinsic physical property of QACs (DST) can be used as a technique to screen for bactericidal activity of QACs in conditions that mimic real-world disinfection conditions.

Chimeric Amphiphilic Disinfectants: Quaternary Ammonium/Quaternary Phosphonium Hybrid Structures.

Cationic biocides play a crucial role in the disinfection of domestic and healthcare surfaces. Due to the rise of bacterial resistance towards common cationic disinfectants like quaternary ammonium compounds (QACs), the development of novel actives is necessary for effective infection prevention and control. Toward this end, a series of 15 chimeric biscationic amphiphilic compounds, bearing both ammonium and phosphonium residues, were prepared to probe the structure and efficacy of mixed cationic ammonium-phosphonium structures. Compounds were obtained in two steps and good yields, with straightforward and chromatography-free purifications. Antibacterial activity evaluation of these compounds against a panel of seven bacterial strains, including two MRSA strains as well as opportunistic pathogen A. baumannii, were encouraging, as low micromolar inhibitory activity was observed for multiple structures. Alkyl chain length on the ammonium group was, as expected, a major determinant of bioactivity. In addition, high therapeutic indexes (up to 125-fold) for triphenyl phosphonium-bearing amphiphiles were observed when comparing antimicrobial activity to mammalian cell lysis activity.

Total Synthesis of the Reported Structure of Cahuitamycin A: Insights into an Elusive Natural Product Scaffold.

In a 2016 screen of natural product extracts, a new family of natural products, the cahuitamycins, was discovered and found to inhibit biofilm formation in the human pathogen Acinetobacter baumannii. The proposed molecular structures contained an unusual piperazic acid residue, which piqued interest related to their structure/function and biosynthesis. Herein we disclose the first total synthesis of the proposed structure of cahuitamycin A in a 12-step longest linear sequence and 18% overall yield. Comparison of spectral and biological data of the authentic natural product and synthetic compound revealed inconsistentancies with the isolated metabolite. We therefore executed the diverted total synthesis of three isomeric compounds, which were also found to be disparate from the isolated natural product. This work sets the stage for future synthetic and biochemical investigations of an important class of natural products.

Investigating the Role of Metabolism for Antibiotic Combination Therapies in Pseudomonas aeruginosa.

Antibacterial resistance poses a severe threat to public health; an anticipated 14-fold increase in multidrug-resistant (MDR) bacterial infections is expected to occur by 2050. Contrary to antibiotics, combination therapies are the standard of care for antiviral and anticancer treatments, as synergistic drug-drug interactions can decrease dosage and resistance development. In this study, we investigated combination treatments of a novel succinate dehydrogenase inhibitor (promysalin) with specific inhibitors of metabolism and efflux alongside a panel of clinically approved antibiotics in synergy studies. Through these investigations, we determined that promysalin can work synergistically with vancomycin and antagonistically with aminoglycosides and a glyoxylate shunt pathway inhibitor at subinhibitory concentrations; however, these cooperative effects do not reduce minimum inhibitory concentrations. The variability of these results underscores the complexity of targeting metabolism for combination therapies in antibiotic development.

Deriving Novel Quaternary Ammonium Compound Disinfectant Scaffolds from a Natural Product: Mechanistic Insights of the Quaternization of Ianthelliformisamine C.

In the search for novel quaternary ammonium compound (QAC) disinfectants that can evade bacterial resistance, we turned to natural products as a source of inspiration. Herein we used natural product ianthelliformisamine C as a scaffold to design a small library of QACs. We first synthesized ianthelliformisamine C via an amide coupling that allowed for facile purification without the need for protecting groups. We then alkylated and quaternized the internal amines to yield four novel QACs, but all but one demonstrated no antibacterial activity against the tested strains. Using a combination of membrane depolarization and permeabilization assays, we were able to demonstrate that ianthelliformisamine C and the active QAC analog enact cell death via membrane permeabilization, contrary to prior reports on ianthelliformisamine C's mechanism of action.

Di-berberine conjugates as chemical probes of Pseudomonas aeruginosa MexXY-OprM efflux function and inhibition.

The Resistance-Nodulation-Division (RND) efflux pump superfamily is pervasive among Gram-negative pathogens and contributes extensively to clinical antibiotic resistance. The opportunistic pathogen Pseudomonas aeruginosa contains 12 RND-type efflux systems, with four contributing to resistance including MexXY-OprM which is uniquely able to export aminoglycosides. At the site of initial substrate recognition, small molecule probes of the inner membrane transporter (e.g., MexY) have potential as important functional tools to understand substrate selectivity and a foundation for developing adjuvant efflux pump inhibitors (EPIs). Here, we optimized the scaffold of berberine, a known but weak MexY EPI, using an in-silico high-throughput screen to identify di-berberine conjugates with enhanced synergistic action with aminoglycosides. Further, docking and molecular dynamics simulations of di-berberine conjugates reveal unique contact residues and thus sensitivities of MexY from distinct P. aeruginosa strains. This work thereby reveals di-berberine conjugates to be useful probes of MexY transporter function and potential leads for EPI development.

Total Synthesis and Biological Investigation of Mindapyrroles A and B.

In the search for antibacterial compounds that can overcome drug resistant species, molecules that enact novel or polypharmacological mechanisms of action (MoA) are needed. As a preliminary foray into molecules of this background, the total synthesis of mindapyrroles A and B was undertaken leveraging a biomimetic approach. Following their synthesis, they and their monomer pyoluteorin were tested against a range of pathogenic bacteria in minimum inhibitory concentration assays to confirm their activity. These molecules were then tested for their ability to disrupt membrane potential in S. aureus. Our findings indicate that pyoluteorin acts as a protonophore but the mindapyrroles do not. This work encapsulates the first total synthesis of mindapyrrole B and the second total synthesis of mindapyrrole A in 11 % and 30 % overall yields, respectively. It also provides insights into the antibacterial properties and different MoAs between the monomer and dimers.

Visible-Light/Nickel-Catalyzed Carboxylation of C(sp2) Bromides via Formate Activation.

A new visible-light-driven method for the carboxylation of (hetero)aryl/vinyl bromides has been developed using catalytic 4CzIPN, nickel, phenyl triflimide, and sodium formate as a carboxylation agent. Interestingly, we found catalytic phenyl triflimide plays an essential role in promoting the reaction. While many C(sp2) carboxylation reactions require harsh reagents or gaseous carbon dioxide, we demonstrate the mild and facile construction of carboxylic acids from readily available starting materials.

Microbial Changes occurring during oronasal fistula wound healing.

The oral microbiome is a complex community that matures with dental development while oral health is also a recognized risk factor for systemic disease. Despite the oral cavity having a substantial microbial burden, healing of superficial oral wounds occurs quickly and with little scarring. By contrast, creation of an oro-nasal fistula (ONF), often occurring after surgery to correct a cleft palate, is a significant wound healing challenge that is further complicated by a connection of the oral and nasal microbiome. In this study, we characterized the changes in the oral microbiome of mice following a freshly inflicted wound in the oral palate that results in an open and unhealed ONF. Creation of an ONF in mice significantly lowered oral microbiome alpha diversity, with concurrent blooms of Enterococcus faecalis, Staphylococcus lentus, and Staphylococcus xylosus in the oral cavity. Treatment of mice with oral antibiotics one week prior to ONF infliction resulted in a reduction in the alpha diversity, prevented E. faecalis and S. lentus, and S. xylosus blooms, but did not impact ONF healing. Strikingly, delivery of the beneficial microbe Lactococcus lactis subsp. cremoris (LLC) to the wound bed of the freshly inflicted ONF via a PEG-MAL hydrogel vehicle resulted in rapid healing of the ONF. Healing of the ONF was associated with the maintenance of relatively high microbiome alpha diversity, and limited the abundance of E. faecalis and S. lentus, and S. xylosus in the oral cavity. These data demonstrate that a freshly inflicted ONF in the murine palate is associated with a dysbiotic oral microbiome state that may prevent ONF healing, and a bloom of opportunistic pathogens. The data also demonstrate that delivery of a specific beneficial microbe, LLC, to the ONF can boost wound healing, can restore and/or preserve oral microbiome diversity, and inhibit blooms of opportunistic pathogens.

Ribosome-targeting antibiotics and resistance via ribosomal RNA methylation.

The rise of multidrug-resistant bacterial infections is a cause of global concern. There is an urgent need to both revitalize antibacterial agents that are ineffective due to resistance while concurrently developing new antibiotics with novel targets and mechanisms of action. Pathogen associated resistance-conferring ribosomal RNA (rRNA) methyltransferases are a growing threat that, as a group, collectively render a total of seven clinically-relevant ribosome-targeting antibiotic classes ineffective. Increasing frequency of identification and their growing prevalence relative to other resistance mechanisms suggests that these resistance determinants are rapidly spreading among human pathogens and could contribute significantly to the increased likelihood of a post-antibiotic era. Herein, with a view toward stimulating future studies to counter the effects of these rRNA methyltransferases, we summarize their prevalence, the fitness cost(s) to bacteria of their acquisition and expression, and current efforts toward targeting clinically relevant enzymes of this class.

Structure-Based Design of Promysalin Analogues to Overcome Mechanisms of Bacterial Resistance.

The search for antibiotics that function through novel mechanisms of action is ongoing, and recent progress in our lab identified the tricarboxylic acid cycle as a viable option. Promysalin is a secondary metabolite capable of species-specific inhibition of Pseudomonas aeruginosa, a common opportunistic pathogen. Promysalin disrupts primary metabolism in this bacterium by competitively inhibiting succinate dehydrogenase at the ubiquinone binding site. However, the activity of promysalin in cellulo is marred potentially by its chemical instability and/or propensity for efflux. To assess the success of these novel analogues, a novel strain of P. aeruginosa harboring gene deletions of eight efflux pumps and porins was developed and implemented. Herein, we disclose the synthesis and biological investigation of six promysalin analogues to overcome these liabilities and demonstrate that efflux likely plays a significant role in tolerating the effect of the inhibitor.

Beyond Ergosterol: Strategies for Combatting Antifungal Resistance in Aspergillus fumigatus and Candida auris.

Aspergillus fumigatus and Candida auris are historically problematic fungal pathogens responsible for systemic infections and high mortality rates, especially in immunocompromised populations. The three antifungal classes that comprise our present day armamentarium have facilitated efficacious treatment of these fungal infections in past decades, but their potency has steadily declined over the years as resistance to these compounds has accumulated. Importantly, pan-resistant strains of Candida auris have been observed in clinical settings, leaving affected patients with no treatment options and a death sentence. Many compounds in the ongoing antifungal drug discovery pipeline, similar to those within our aforementioned trinity, are predicated on the binding or inhibition of ergosterol. Recurring accounts of resistance to antifungals targeting this pathway suggest optimization of ergosterol-dependent antifungals is likely not the best solution for the long-term. This review aims to present several natural products with novel or underexplored biological targets, as well as similarly underutilized drug discovery strategies to inspire future biological investigations and medicinal chemistry campaigns.

Soft QPCs: Biscationic Quaternary Phosphonium Compounds as Soft Antimicrobial Agents.

Quaternary ammonium compounds (QACs) serve as a first line of defense against infectious pathogens. As resistance to QACs emerges in the environment, the development of next-generation disinfectants is of utmost priority for human health. Balancing antibacterial potency with environmental considerations is required to effectively counter the development of bacterial resistance. To address this challenge, a series of 14 novel biscationic quaternary phosphonium compounds (bisQPCs) have been prepared as amphiphilic disinfectants through straightforward, high-yielding alkylation reactions. These compounds feature decomposable or "soft" amide moieties in their side chains, anticipated to promote decomposition under environmental conditions. Strong bioactivity against a panel of seven bacterial pathogens was observed, highlighted by single-digit micromolar activity for compounds P6P-12A,12A and P3P-12A,12A. Hydrolysis experiments in pure water and in buffers of varying pH revealed surprising decomposition of the soft QPCs under basic conditions at the phosphonium center, leading to inactive phosphine oxide products; QPC stability (>24 h) was maintained in neutral solutions. The results of this work unveil soft QPCs as a potent and environmentally conscious new class of bisQPC disinfectants.

Biological Evaluation of the Antibacterial Retinoid CD437 in Cutibacterium acnes Infection.

Acne vulgaris is a complex skin disease involving infection by Cutibacterium acnes, inflammation, and hyperkeratinization. We evaluated the activity of the retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and 16 other retinoid analogs as potential anti-C. acnes compounds and found that CD437 displayed the highest antimicrobial activity with an MIC against C. acnes (ATCC 6919 and HM-513) of 1 µg/mL. CD437 demonstrated an MBC of 2 µg/mL compared to up to 64 µg/mL for the retinoid adapalene and up to 16 µg/mL for tetracycline, which are commonly used clinically to treat acne. Membrane permeability assays demonstrated that exposure of C. acnes ATCC 6919 to CD437 damaged the integrity of C. acnes ATCC 6919 bacterial membranes, and this finding was confirmed with scanning electron microscopy. Additionally, CD437 downregulated the expression of C. acnes ATCC 6919 virulence factors, including the genes encoding Christie-Atkins-Munch-Petersen factor 1 (CAMP1), CAMP2, glycerol-ester hydrolase B (GehB), sialidase B, and neuraminidase. In a mouse skin infection model of C. acnes ATCC 6919, topical treatment with CD437 ameliorated skin lesions and reduced the bacterial burden in situ (P < 0.001). In human NHEK primary cells, CD437 reduced the transcriptional levels of the coding genes for inflammatory cytokines (interleukin-1α, ~10-fold; interleukin-6, ~20-fold; interleukin-8, ~30-fold; and tumor necrosis factor-alpha, ~6-fold) and downregulated the transcriptional levels of KRT10 (~10-fold), FLG (~4-fold), and TGM1 (~2-fold), indicating that CD437 can diminish inflammation and hyperkeratinization. In summary, CD437 deserves further attention for its dual function as a potential acne therapeutic that potentially acts on both the pathogen and the host.