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Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.

In the niches that Staphylococcus aureus and Pseudomonas aeruginosa coinhabit, the later pathogen produces phenazine antibiotics to inhibit the growth of S. aureus. Recently, a group of halogenated phenazines (HPs) has been shown to have potent antimicrobial activities against Staphylococci; however, no HP-resistant mutant has been reported. Here, we demonstrate that S. aureus develops HP-resistance via single amino acid change (Arg116Cys) in a transcriptional repressor TetR21. RNA-seq analysis showed that the TetR21R116C variation caused drastic up-regulation of an adjacent gene hprS (halogenated phenazine resistance protein of S. aureus). Deletion of the hprS in the TetR21R116C background restored bacterial susceptibility to HP, while hprS overexpression in S. aureus conferred HP-resistance. The expression of HprS is under tight transcriptional control of the TetR21 via direct binding to the promoter region of hprS. The R116C mutation in TetR21 significantly reduced its DNA binding affinity. Moreover, natural phenazine antibiotics (phenazine-1-carboxylic acid and pyocyanin) and a HP analog (HP-22) are ligands for the TetR21, regulating its repressor activity. Combining homology analysis and LC-MS/MS assay we demonstrated that HprS is a phenazine efflux pump. To the best of our knowledge, we provide the first report of phenazine efflux pump in S. aureus. Interestingly, the TetR21R116C variation has been found in some clinical S. aureus isolates, and a laboratory strain of S. aureus with TetR21R116C variation showed enhanced growth competitiveness toward P. aeruginosa and promoted coinfection with P. aeruginosa in the host environment, demonstrating significance of the mutation in host infections.

Antibiotic-resistant infections present significant challenges to patients. As a result, there is considerable need for new antibacterial therapies that eradicate pathogenic bacteria through non-conventional mechanisms. Our group has identified a series of halogenated phenazine (HP) agents that induce rapid iron starvation that leads to potent killing of methicillin-resistant Staphylococcus aureus biofilms. Here, we report the design, chemical synthesis and microbiological assessment of a HP-quinone ether prodrug model aimed to (1) eliminate general (off-target) iron chelation, and (2) release an active HP agent through the bioreduction of a quinone trigger. Here, we demonstrate prodrug analogue HP-29-Q to have a stable ether linkage that enables HP release and moderate to good antibacterial activities against lab strains and multi-drug resistant clinical isolates.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease that presents with clinical symptoms including myotonia, cardiac dysfunction and cognitive impairment. DM1 is caused by a CTG expansion in the 3’ UTR of the DMPK gene. The transcribed expanded CUG repeat RNA sequester the muscleblind-like (MBNL) and up-regulate the CUG-BP Elav-like (CELF) families of RNA-binding proteins leading to global mis-regulation of RNA processing and altered gene expression. Currently, there are no disease-targeting treatments for DM1. Given the multi-step pathogenic mechanism, combination therapies targeting different aspects of the disease mechanism may be a viable therapeutic approach. Here, as proof-of-concept, we studied a combination of two previously characterized small molecules, erythromycin and furamidine, in two DM1 models. In DM1 patient-derived myotubes, rescue of mis-splicing was observed with little to no cell toxicity. In a DM1 mouse model, a combination of erythromycin and the prodrug of furamidine (pafuramidine), administered orally, displayed both additive and synergistic mis-splicing rescue. Gene expression was only modestly affected and over 40 % of the genes showing significant expression changes were rescued back toward WT expression levels. Further, the combination treatment partially rescued the myotonia phenotype in the DM1 mouse. This combination treatment showed a high degree of mis-splicing rescue coupled with low off-target gene expression changes. These results indicate that combination therapies are a promising therapeutic approach for DM1.

There is a significant need for new antibacterial agents as pathogenic bacteria continue to threaten human health through the acquisition of resistance and tolerance towards existing antibiotics. Over the last several years, our group has been developing a novel series of halogenated phenazines that demonstrate potent antibacterial and biofilm eradication activities against critical Gram-positive pathogens, including: Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium. Here, we report the design, chemical synthesis and initial biological assessment of a halogenated phenazine-erythromycin conjugate prodrug 5 aimed at enhancing the translational potential for halogenated phenazines as a treatment of bacterial infections.

G protein-coupled receptors (GPCRs) constitute the largest protein superfamily in the human genome. GPCRs play key roles in mediating a wide variety of physiological events including proliferation and cancer metastasis. Given the major roles that GPCRs play in mediating cancer growth, they present promising targets for small molecule therapeutics. One of the principal goals of our lab is to identify complex natural products (NPs) suitable for ring distortion, or the dramatic altering of the inherently complex architectures of NPs, to rapidly generate an array of compounds with diverse molecular skeletal systems. The overarching goal of our ring distortion approach is to re-program the biological activity of select natural products and identify new compounds of importance to the treatment of disease, such as cancer. Described herein are the results from biological screens of diverse small molecules derived from the indole alkaloid yohimbine against a panel of GPCRs involved in various diseases. Several analogues displayed highly differential antagonistic activities across the GPCRs tested. We highlight the re-programmed profile of one analogue, Y7g, which exhibited selective antagonistic activities against AVPR2 (IC50 = 459 nM) and OXTR (IC50 = 1.16 µM). The activity profile of Y7g could correlate its HIF-dependent anti-cancer activity to its GPCR antagonism since these receptors are known to be upregulated in hypoxic cellular environments. Our findings demonstrate that the ring distortion of yohimbine can lead to the identification of new compounds capable of interacting with distinct cancer-relevant targets.

BackgroundMycoplasmas primarily cause respiratory or urogenital tract infections impacting avian, bovine, canine, caprine, murine, and reptilian hosts. In animal husbandry, mycoplasmas cause reduced feed-conversion, decreased egg production, arthritis, hypogalactia or agalactia, increased condemnations, culling, and mortality in some cases. Antibiotics reduce transmission and mitigate clinical signs; however, concerning levels of antibiotic resistance inMycoplasma gallisepticumandM. capricolumisolates exist. To address these issues, we evaluated the minimum inhibitory concentrations (MICs) of halogenated phenazine and quinoline compounds, anN-arylated NH125 analogue, and triclosan against six representative veterinary mycoplasmas via microbroth or agar dilution methods. Thereafter, we evaluated the minimum bactericidal concentration (MBC) of efficacious drugs.ResultsWe identified several compounds with MICs ≤25 μM againstM. pulmonis(n = 5),M. capricolum(n = 4),M. gallisepticum(n = 3),M. alligatoris(n = 3),M. agassizii(n = 2), andM. canis(n = 1). AnN-arylated NH125 analogue, compound 21, served as the most efficacious, having a MIC ≤25 μM against all mycoplasmas tested, followed by two quinolines, nitroxoline (compound 12) and compound 20, which were effective against four and three mycoplasma type strains, respectively. Nitroxoline exhibited bactericidal activity among all susceptible mycoplasmas, and compound 21 exhibited bactericidal activity when the MBC was able to be determined.ConclusionsThese findings highlight a number of promising agents from novel drug classes with potential applications to treat veterinary mycoplasma infections and present the opportunity to evaluate preliminary pharmacokinetic indices usingM. pulmonisin rodents as an animal model of human infection.

Bacterial pathogens that infect and kill plants are responsible for major crop damage (e.g. citrus canker). As is the case with human pathogens, bacterial species which are invasive to plants acquire resistance to common crop-bactericidal agents. Herein we report the identification of nitroxoline and simple halogenated quinoline (HQ) antibacterial agents that demonstrate antibacterial activities against a panel of plant pathogens, including Xanthomonas citri and Burkholderia andropogonis. A subset of active analogues was identified from an in-house library of 36 nitrogen-containing heterocycles which our group previously evaluated against multiple human pathogenic bacteria (e.g., Staphylococcus aureus). In this report, we also demonstrate that the phytochemical gallic acid (GA) potentiates the antibacterial activity of select halogenated quinolines against multiple Xanthomonas strains, including Xcc 306. We also show that nitroxoline and HQ 1 alone and in combination with GA inhibit Xcc 306 infection in a plant model, demonstrating the potential these compounds have as crop-bactericidal agents.

Bacterial biofilms are surface-attached communities of slow-growing or non-replicating bacteria tolerant to conventional antibiotic therapies. Although biofilms are known to occur in ca. 80% of all bacterial infections, no therapeutic agent has been developed to eradicate bacteria housed within biofilms. We have discovered that nitroxoline, an antibacterial agent used to treat urinary tract infections, displays broad-spectrum biofilm-eradicating activities against major human pathogens, including drug-resistant Staphylococcus aureus and Acinetobacter baumannii strains. In this study, the effectiveness of nitroxoline to eradicate biofilms was determined using an in vitro [minimum biofilm eradication concentration (MBEC) = 46.9 µM against A. baumannii] and ex vivo porcine skin model (2-3 log reduction in viable biofilm cells). Nitroxoline was also found to eradicate methicillin-resistant S. aureus (MRSA) persister cells in non-biofilm (stationary) cultures, whereas vancomycin and daptomycin were found to be inactive. These findings could lead to effective, nitroxoline-based therapies for biofilm-associated infections.

Bacterial biofilms housing dormant persister cells are innately tolerant to antibiotics and disinfectants, yet several membrane-active agents are known to eradicate tolerant bacterial cells. NH125, a membrane-active persister killer and starting point for development, led to the identification of two N-arylated analogues (1 and 2) that displayed improved biofilm eradication potencies compared to the parent compound and rapid persister-cell-killing activities in stationary cultures of methicillin-resistant Staphylococcus aureus (MRSA). We found 1 and 2 to be superior to other membrane-active agents in biofilm eradication assays, with 1 demonstrating minimum biofilm eradication concentrations (MBEC) of 23.5, 11.7, and 2.35 μm against MRSA, methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-resistant Enterococcus faecium (VRE) biofilms, respectively. We tested our panel of membrane-active agents against MRSA stationary cultures and found 1 to rapidly eradicate MRSA stationary cells by 4 log units (99.99 %) in 30 min. The potent biofilm eradication and rapid persister-cell-killing activities exhibited by N-arylated NH125 analogues could have significant impact in addressing biofilm-associated problems.

Bacterial biofilms are surface-attached communities of slow-growing and non-replicating persister cells that demonstrate high levels of antibiotic tolerance. Biofilms occur in nearly 80 % of infections and present unique challenges to our current arsenal of antibiotic therapies, all of which were initially discovered for their abilities to target rapidly dividing, free-floating planktonic bacteria. Bacterial biofilms are credited as the underlying cause of chronic and recurring bacterial infections. Innovative approaches are required to identify new small molecules that operate through bacterial growth-independent mechanisms to effectively eradicate biofilms. One source of inspiration comes from within the lungs of young cystic fibrosis (CF) patients, who often endure persistent Staphylococcus aureus infections. As these CF patients age, Pseudomonas aeruginosa co-infects the lungs and utilizes phenazine antibiotics to eradicate the established S. aureus infection. Our group has taken a special interest in this microbial competition strategy and we are investigating the potential of phenazine antibiotic-inspired compounds and synthetic analogues thereof to eradicate persistent bacterial biofilms. To discover new biofilm-eradicating agents, we have established an interdisciplinary research program involving synthetic medicinal chemistry, microbiology and molecular biology. From these efforts, we have identified a series of halogenated phenazines (HPs) that potently eradicate bacterial biofilms, and future work aims to translate these preliminary findings into ground-breaking clinical advances for the treatment of persistent biofilm infections.

Bacteria utilize multiple mechanisms that enable them to gain or acquire resistance to antibiotic therapies during the treatment of infections. In addition, bacteria form biofilms which are surface-attached communities of enriched populations containing persister cells encased within a protective extracellular matrix of biomolecules, leading to chronic and recurring antibiotic-tolerant infections. Antibiotic resistance and tolerance are major global problems that require innovative therapeutic strategies to address the challenges associated with pathogenic bacteria. Historically, natural products have played a critical role in bringing new therapies to the clinic to treat life-threatening bacterial infections. This Perspective provides an overview of antibiotic resistance and tolerance and highlights recent advances (chemistry, biology, drug discovery, and development) from various research programs involved in the discovery of new antibacterial agents inspired by a diverse series of natural product antibiotics.

Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in Mycoplasma pneumoniae and M. genitalium, have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value. To address this shortage, we screened nitroxoline, triclosan, and a library of 20 novel, halogenated phenazine, quinoline, and NH125 analogues against Ureaplasma species and M. hominis clinical isolates from urine. We tested a subset of these compounds (n = 9) against four mycoplasma type strains (M. pneumoniae, M. genitalium, M. hominis, and Ureaplasma urealyticum) using a validated broth microdilution or agar dilution method. Among 72 Ureaplasma species clinical isolates, nitroxoline proved most effective (MIC90, 6.25 µM), followed by an N-arylated NH125 analogue (MIC90, 12.5 µM). NH125 and its analogue had significantly higher MICs against U. urealyticum isolates than against U. parvum isolates, whereas nitroxoline did not. Nitroxoline exhibited bactericidal activity against U. parvum isolates but bacteriostatic activity against the majority of U. urealyticum isolates. Among the type strains, the compounds had the greatest activity against M. pneumoniae and M. genitalium, with 8 (80%) and 5 (71.4%) isolates demonstrating MICs of ≤12.5 µM, respectively. Triclosan also exhibited lower MICs against M. pneumoniae and M. genitalium Overall, we identified a promising range of quinoline, halogenated phenazine, and NH125 compounds that showed effectiveness against M. pneumoniae and M. genitalium and found that nitroxoline, approved for use outside the United States for the treatment of urinary tract infections, and an N-arylated NH125 analogue demonstrated low MICs against Ureaplasma species isolates.

Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm-associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2-position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2-position. We identified diverse analogues that are alkylated and aminated at the 2-position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μm) and biofilm eradication (MBEC 1.0-93.8 μm) activities against drug-resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating

Persistent bacteria, including persister cells within surface-attached biofilms and slow-growing pathogens lead to chronic infections that are tolerant to antibiotics. Here, we describe the structure-activity relationships of a series of halogenated phenazines (HP) inspired by 2-bromo-1-hydroxyphenazine 1. Using multiple synthetic pathways, we probed diverse substitutions of the HP scaffold in the 2-, 4-, 7-, and 8-positions, providing critical information regarding their antibacterial and bacterial eradication profiles. Halogenated phenazine 14 proved to be the most potent biofilm-eradicating agent (≥99.9% persister cell killing) against MRSA (MBEC < 10 μM), MRSE (MBEC = 2.35 μM), and VRE (MBEC = 0.20 μM) biofilms while 11 and 12 demonstrated excellent antibacterial activity against M. tuberculosis (MIC = 3.13 μM). Unlike antimicrobial peptide mimics that eradicate biofilms through the general lysing of membranes, HPs do not lyse red blood cells. HPs are promising agents that effectively target persistent bacteria while demonstrating negligible toxicity against mammalian cells.

Antibiotic-resistant bacteria and surface-attached bacterial biofilms play a significant role in human disease. Conventional antibiotics target actively replicating free-floating, planktonic cells. Unfortunately, biofilm communities are endowed with nonreplicating persister cells that are tolerant to antibiotics. Innovative approaches are necessary to identify new molecules able to eradicate resistant and tolerant bacterial cells. Our group has discovered that select halogenated quinolines (HQs) can eradicate drug-resistant, gram-positive bacterial pathogens and their corresponding biofilms. Interestingly, the HQ scaffold is synthetically tunable and we have discovered unique antibacterial profiles through extensive analogue synthesis and microbiologic studies. We recently reported the synthesis of 14 new HQs to investigate the impact of ClogP values on antibacterial and biofilm eradication activities. We conducted diverse synthetic modifications at the 2-position of the HQ scaffold in an attempt to enhance water solubility and found new compounds that display enhanced activities against Staphylococcus epidermidis. In particular, HQ 2 (ClogP = 3.44) demonstrated more potent antibacterial activities against methicillin-resistant S epidermidis (MRSE) 35984 planktonic cells (minimum inhibitory concentration = 0.59 µM) compared with methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus isolates while demonstrating potent MRSE biofilm eradication activities (minimum biofilm eradication concentration = 2.35 µM). We believe that HQ could play a critical role in the development of next-generation antibacterial therapeutics.

Bacterial biofilms are surface-attached communities comprised of nonreplicating persister cells housed within a protective extracellular matrix. Biofilms display tolerance toward conventional antibiotics, occur in ∼80% of infections, and lead to500000 deaths annually. We recently identified halogenated phenazine (HP) analogues which demonstrate biofilm-eradicating activities against priority pathogens; however, the synthesis of phenazines presents limitations. Herein, we report a refined HP synthesis which expedited the identification of improved biofilm-eradicating agents. 1-Methoxyphenazine scaffolds were generated through a Buchwald-Hartwig cross-coupling (70% average yield) and subsequent reductive cyclization (68% average yield), expediting the discovery of potent biofilm-eradicating HPs (e.g., 61: MRSA BAA-1707 MBEC = 4.69 μM). We also developed bacterial-selective prodrugs (reductively activated quinone-alkyloxycarbonyloxymethyl moiety) to afford HP 87, which demonstrated excellent antibacterial and biofilm eradication activities against MRSA BAA-1707 (MIC = 0.15 μM, MBEC = 12.5 μM). Furthermore, active HPs herein exhibit negligible cytotoxic or hemolytic effects, highlighting their potential to target biofilms.

Conventional antibiotics are ineffective against non-replicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1, that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC = 0.2-12.5 μM), as well as the effective killing of MRSA persister cells in non-biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non-biofilm persisters alongside 14. HP 13 displayed potent antibacterial activity against slow-growing M. tuberculosis (MIC = 3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non-toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections.

With the continued rise of drug-resistant bacterial infections coupled with the current discouraging state of the antibiotic pipeline, the need for new antibacterial agents that operate through unique mechanisms compared with conventional antibiotics and work in synergy with other agents is at an all-time high. We have discovered that gallic acid, a plant-derived phytochemical, dramatically potentiates the antibacterial activities of several halogenated quinolines (up to 11,800-fold potentiation against Staphylococcus aureus) against pathogenic bacteria, including drug-resistant clinical isolates. S. aureus demonstrated the highest sensitivity towards gallic acid-halogenated quinoline combinations, including one halogenated quinoline that demonstrated potentiation of biofilm eradication activity against a methicillin-resistant S. aureus (MRSA) clinical isolate. During our studies, we also demonstrated that these halogenated quionlines operate through an interesting metal(II) cation-dependent mechanism and display promising mammalian cytotoxicity.

Bacterial biofilms are surface-attached communities of bacteria that are: (1) highly prevalent in human infections, and (2) resistant to conventional antibiotic treatments and host immune responses. It has only been in the last ∼20 years that bacterial biofilms have been identified as a critical biomedical hurdle in infectious disease and human health. Staphylococcus aureus is a leading cause of nosocomial and community-acquired infections and is notorious for its ability to form drug-resistant biofilms. Despite the need for antibacterial agents that target S. aureus biofilms, few chemical scaffolds are known that are capable of inhibiting, dispersing or eradicating their biofilms. Here, we report the discovery of bromophenazine derivatives that display antibiofilm activities as either potent biofilm inhibitors (IC50 values 0.55–10.3 μM) or dispersal agents (EC50 values 1.4–29.3 μM) and biofilm eradicators (MBEC values 100–200 μM) against S. aureus strains, including a methicillin-resistant Staphylococcus aureus clinical isolate. These discoveries could lead to the development of new treatment options that target drug-resistant, biofilm-associated S. aureus infections.

Staphylococcus aureus and Staphylococcus epidermidis are recognized as the most frequent cause of biofilm-associated nosocomial and indwelling medical device infections. Biofilm-associated infections are known to be highly resistant to our current arsenal of clinically used antibiotics and antibacterial agents. To exacerbate this problem, no therapeutic option exists that targets biofilm-dependent machinery critical to Staphylococcal biofilm formation and maintenance. Here, we describe the discovery of a series of quinoline small molecules that demonstrate potent biofilm dispersal activity against methicillin-resistant S. aureus and S. epidermidis using a scaffold hopping strategy. This interesting class of quinolines also has select synthetic analogues that demonstrate potent antibacterial activity and biofilm inhibition against S. aureus and S. epidermidis.

During microbial infection, antimicrobial peptides are utilized by the immune response to rapidly eradicate microbial pathogens through the destruction of cellular membranes. Inspired by antimicrobial peptides, quaternary ammonium cationic (QAC) compounds have emerged as agents capable of destroying bacterial membranes leading to rapid bacterial death, including the eradication of persistent, surface-attached bacterial biofilms. NH125, an imidazolium cation with a sixteen membered fatty tail, was recently reported to eradicate persister cells and was our starting point for the development of novel antimicrobial agents. Here, we describe the design, chemical synthesis and biological investigations of a collection of 30 diverse NH125 analogues which provided critical insights into structural features that are important for antimicrobial activities in this class. From these studies, multiple NH125 analogues were identified to possess potent antibacterial and antifungal activities, eradicate both bacterial and fungal biofilms and rapidly eradicate MRSA persister cells in stationary phase. NH125 analogues also demonstrated more rapid persister cell killing activities against MRSA when tested alongside a panel of diverse membrane-active agents, including BAC-16 and daptomycin. NH125 analogues could have a significant impact on persister- and biofilm-related problems in numerous biomedical applications.

Unlike individual, free-floating planktonic bacteria, biofilms are surface-attached communities of slow- or non-replicating bacteria encased within a protective extracellular polymeric matrix enabling persistent bacterial populations to tolerate high concentrations of antimicrobials. Our current antibacterial arsenal is composed of growth-inhibiting agents that target rapidly-dividing planktonic bacteria but not metabolically dormant biofilm cells. We report the first modular synthesis of a library of 20 halogenated phenazines (HP), utilizing the Wohl-Aue reaction, that targets both planktonic and biofilm cells. New HPs, including 6-substituted analogues, demonstrate potent antibacterial activities against MRSA, MRSE and VRE (MIC = 0.003–0.78 µM). HPs bind metal(II) cations and demonstrate interesting activity profiles when co-treated in a panel of metal(II) cations in MIC assays. HP 1 inhibited RNA and protein biosynthesis while not inhibiting DNA biosynthesis using 3H-radiolabeled precursors in macromolecular synthesis inhibition assays against MRSA. New HPs reported here demonstrate potent eradication activities (MBEC = 0.59–9.38 µM) against MRSA, MRSE and VRE biofilms while showing minimal red blood cell lysis or cytotoxicity against HeLa cells. PEG-carbonate HPs 24 and 25 were found to have potent antibacterial activities with significantly improved water solubility. HP small molecules could have a dramatic impact on persistent, biofilm-associated bacterial infection treatments.

Nearly all clinically used antibiotics have been (1) discovered from microorganisms (2) using phenotype screens to identify inhibitors of bacterial growth. The effectiveness of these antibiotics is attributed to their endogenous roles as bacterial warfare agents against competing microorganisms. Unfortunately, every class of clinically used antibiotic has been met with drug resistant bacteria. In fact, the emergence of resistant bacterial infections coupled to the dismal pipeline of new antibacterial agents has resulted in a global health care crisis. There is an urgent need for innovative antibacterial strategies and treatment options to effectively combat drug resistant bacterial pathogens. Here, we describe the implementation of a Pseudomonas competition strategy, using redox-active phenazines, to identify novel antibacterial leads against Staphylococcus aureus and Staphylococcus epidermidis. In this report, we describe the chemical synthesis and evaluation of a diverse 27-membered phenazine library. Using this microbial warfare inspired approach, we have identified several bromophenazines with potent antibacterial activities against S. aureus and S. epidermidis. The most potent bromophenazine analogue from this focused library demonstrated a minimum inhibitory concentration (MIC) of 0.78-1.56 μM, or 0.31-0.62 μg mL(-1), against S. aureus and S. epidermidis and proved to be 32- to 64-fold more potent than the phenazine antibiotic pyocyanin in head-to-head MIC experiments. In addition to the discovery of potent antibacterial agents against S. aureus and S. epidermidis, we also report a detailed structure-activity relationship for this class of bromophenazine small molecules.

2-Aminoimidazoles are an emerging class of small molecules that possess the ability to inhibit and disperse biofilms across bacterial order, class, and phylum. Herein, we report the synergistic effect between a 2-aminoimidazole/triazole conjugate and antibiotics toward dispersing preestablished biofilms, culminating with a 3-orders-of-magnitude increase of biofilm dispersion toward Staphylococcus aureus biofilms. Furthermore, we document that the 2-aminoimidazole/triazole conjugate will also resensitize multidrug-resistant strains of bacteria to the effects of conventional antibiotics, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii.

Small molecules capable of eradicating non-replicating bacterial biofilms are of great importance to human health as conventional antibiotics are ineffective against these surface-attached bacterial communities. Here, we report the discovery of several halogenated quinolines (HQs) identified through a reductive amination reaction that demonstrated potent eradication of MRSA (HQ-6; MBEC = 125 μM), MRSE (HQ-3; MBEC = 3.0 μM) and VRE (HQ-4, HQ-5 and HQ-6; MBEC = 1.0 μM) biofilms. HQs were evaluated using the Calgary Biofilm Device (CBD) and demonstrated near equipotent killing activities against planktonic and biofilm cells based on MBC and MBEC values. When tested against red blood cells, these HQ analogues demonstrated low haemolytic activity (3 to 21% at 200 μM) thus we conclude that these HQ analogues do not operate primarily through the destruction of bacterial membranes, typical of other biofilm-eradicating agents (i.e., antimicrobial peptides). HQ antibacterial agents are potent biofilm-eradicating compounds and could lead to useful treatments for biofilm-associated bacterial infections.

The synthesis of a 20 member 2-aminoimidazole/triazole pilot library is reported. Each member of the library was screened for its ability to inhibit or promote biofilm development of either Escherichia coli and Acinetobacter baumannii. From this screen, E. coli-selective 2-aminoimidazoles were discovered, with the best inhibitor inhibiting biofilm development with an IC(50) of 13μM. The most potent promoter of E. coli biofilm formation promoted biofilm development by 321% at 400μM.

Glucose transporters and the glycolysis enzyme lactate dehydrogenase A (LDH-A) are both overexpressed in cancer cells, two proliferation tactics that underlie the phenomenon known as the Warburg effect. Herein we report the development and activity of a glucose-conjugated LDH-A inhibitor designed to target both of these tumor-promoting facets. In addition to the promise of this conjugate, dual targeting of the Warburg effect using glycoconjugation as an anticancer strategy could be applied to inhibitors of many of the enzymes involved in glycolysis or tumor metabolism.

Multidrug-resistant bacterial infections continue to be a rising global health concern. Herein is described the development of a class of novel 2-aminobenzimidazoles with antibiotic activity. These active 2-aminobenzimidazoles retain their antibiotic activity against several strains of multidrug-resistant Staphylococcus aureus and Acinetobacter baumannii when compared to susceptible strains.

Bacterial biofilms are defined as a community of surface-attached bacteria that are protected by an extracellular matrix of biomolecules. We have recently reported the synthesis of a small molecule, denoted TAGE, based on the natural product bromoageliferin and demonstrated that TAGE has anti-biofilm activity against Pseudomonas aeruginosa. Herein we demonstrate that TAGE: (1) does not have selective toxicity against cells within the biofilm state, (2) will inhibit biofilm development under flow conditions, indicating that the CV staining protocol correlates with the ability to be active under biomimetic conditions, and (3) will disperse preformed P. aeruginosa biofilms. We also present preliminary toxicity work that indicates that TAGE is devoid of cytotoxicity in rat and mice cell lines. Advanced derivatives of TAGE have generated compounds shown to be exceedingly effective as biofilm inhibitors against the gamma-proteobacteria in this study (P. aeruginosa strains PAO1, PA14, PDO300, and Acinetobacter baumannii). TAGE derivatives also possessed anti-biofilm activity against the beta-proteobacterium Bordetella bronchiseptica (Rb50) and the Gram-positive bacterium Staphylococcus aureus;TAGE derivatives inhibited the formation of biofilms, however, some of this activity is attributed to microbicidal activity. The TAGE derivatives presented in this study, however, do not disperse pre-formed biofilms with the same efficiency as TAGE.

A 50-compound library based on the marine natural product oroidin was synthesized and assayed for anti-biofilm activity against PAO1 and PA14, two strains of the medically relevant gamma-proteobacterium Pseudomonas aeruginosa. Through structure-activity relationship (SAR) analysis of analogues based on the oroidin template, several conclusions can be drawn as to what structural properties of the synthetic derivatives are necessary to elicit a biological response. Notably, the most active analogues identified were those that contained a 2-aminoimidazole (2-AI) motif and a dibrominated pyrrolecarboxamide subunit. Here we disclose the synthesis and subsequently determined biological activity of this unique class of compounds as inhibitors of biofilm formation that have no direct antibiotic effect.

Two analogues of the marine natural product bromoageliferin have been synthesized and subsequently assayed for the ability to inhibit the formation of Pseudomonas aeruginosa biofilms. Both compounds inhibited the ability of Pseudomonas aeruginosa strains PAO1 and PA14 to form biofilms; however, they displayed differential toxicity to planktonic bacteria. The cis analogue, CAGE, was found to possess greater toxicity than the trans analogue, TAGE. These compounds have no structural homology to any known inhibitors of Pseudomonas aeruginosa biofilms.

A chemically diverse library of TAGE-triazole conjugates was synthesized utilizing click chemistry on the TAGE scaffold. This library of small molecules was screened for anti-biofilm activity and found to possess the ability of inhibiting biofilm formation against Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa. One such compound in this library demonstrated the most potent inhibitory effect against Staphylococcus aureus biofilm formation that has been displayed by any 2-aminoimidazole derivative.