A dual-therapy approach for the treatment of biofilm-mediated Salmonella gallbladder carriage.

Asymptomatic carriage of Salmonella Typhi continues to facilitate the transmission of typhoid fever, resulting in 14 million new infections and 136,000 fatalities each year. Asymptomatic chronic carriage of S. Typhi is facilitated by the formation of biofilms on gallstones that protect the bacteria from environmental insults and immune system clearance. Here, we identified two unique small molecules capable of both inhibiting Salmonella biofilm growth and disrupting pre-formed biofilm structures without affecting bacterial viability. In a mouse model of chronic gallbladder Salmonella carriage, treatment with either compound reduced bacterial burden in the gallbladder by 1-2 logs resulting in bacterial dissemination to peripheral organs that was associated with increased mortality. Co-administration of either compound with ciprofloxacin not only enhanced compound efficacy in the gallbladder by a further 1-1.5 logs for a total of 3-4.5 log reduction, but also prevented bacterial dissemination to peripheral organs. These data suggest a dual-therapy approach targeting both biofilm and planktonic populations can be further developed as a safe and efficient treatment of biofilm-mediated chronic S. Typhi infections.

Natural products as inspiration for the development of bacterial antibiofilm agents.

Natural products have historically been a rich source of diverse chemical matter with numerous biological activities, and have played an important role in drug discovery in many areas including infectious disease. Synthetic and medicinal chemistry have been, and continue to be, important tools to realize the potential of natural products as therapeutics and as chemical probes. The formation of biofilms by bacteria in an infection setting is a significant factor in the recalcitrance of many bacterial infections, conferring increased tolerance to many antibiotics and to the host immune response, and as yet there are no approved therapeutics for combatting biofilm-based bacterial infections. Small molecules that interfere with the ability of bacteria to form and maintain biofilms can overcome antibiotic tolerance conferred by the biofilm phenotype, and have the potential to form combination therapies with conventional antibiotics. Many natural products with anti-biofilm activity have been identified from plants, microbes, and marine life, including: elligic acid glycosides, hamamelitannin, carolacton, skyllamycins, promysalin, phenazines, bromoageliferin, flustramine C, meridianin D, and brominated furanones. Total synthesis and medicinal chemistry programs have facilitated structure confirmation, identification of critical structural motifs, better understanding of mechanistic pathways, and the development of more potent, more accessible, or more pharmacologically favorable derivatives of anti-biofilm natural products.

Structure-Function Studies on IMD-0354 Identifies Highly Active Colistin Adjuvants.

Infections caused by multidrug-resistant (MDR) bacteria, particularly Gram-negative bacteria, are an escalating global health threat. Often clinicians are forced to administer the last-resort antibiotic colistin; however, colistin resistance is becoming increasingly prevalent, giving rise to the potential for a situation in which there are no treatment options for MDR Gram-negative infections. The development of adjuvants that circumvent bacterial resistance mechanisms is a promising orthogonal approach to the development of new antibiotics. We recently disclosed that the known IKK-ß inhibitor IMD-0354 potently suppresses colistin resistance in several Gram-negative strains. In this study, we explore the structure-activity relationship (SAR) between the IMD-0354 scaffold and colistin resistance suppression, and identify several compounds with more potent activity than the parent against highly colistin-resistant strains of Acinetobacter baumannii and Klebsiella pneumoniae.

Repurposing Eukaryotic Kinase Inhibitors as Colistin Adjuvants in Gram-Negative Bacteria.

Kinase inhibitors comprise a diverse cohort of chemical scaffolds that are active in multiple biological systems. Currently, thousands of eukaryotic kinase inhibitors are commercially available, have well-characterized targets, and often carry pharmaceutically favorable toxicity profiles. Recently, our group disclosed that derivatives of the natural product meridianin D, a known inhibitor of eukaryotic kinases, modulated behaviors of both Gram-positive and Gram-negative bacteria. Herein, we expand our exploration of kinase inhibitors in Gram-negative bacilli utilizing three commercially available kinase inhibitor libraries and, ultimately, identify two chemical structures that potentiate colistin (polymyxin E) in multiple strains. We report IMD-0354, an inhibitor of IKK-ß, as a markedly effective adjuvant in colistin-resistant bacteria and also describe AR-12 (OSU-03012), an inhibitor of pyruvate dehydrogenase kinase-1 (PDK-1), as a potentiator in colistin-sensitive strains. This report comprises the first description of the novel cross-reactivity of these molecules.

Halogenated quinolines bearing polar functionality at the 2-position: Identification of new antibacterial agents with enhanced activity against Staphylococcus epidermidis.

Antibiotic-resistant bacteria and surface-attached biofilms continue to play a significant role in human health and disease. Innovative strategies are needed to identify new therapeutic leads to tackle infections of drug-resistant and tolerant bacteria. We synthesized a focused library of 14 new halogenated quinolines to investigate the impact of ClogP values on antibacterial and biofilm-eradication activities. During these investigations, we found select polar appendages at the 2-position of the HQ scaffold were more well-tolerated than others. We were delighted to see multiple compounds display enhanced activities against the major human pathogen S. epidermidis. In particular, HQ 2 (ClogP = 3.44) demonstrated enhanced activities against MRSE 35984 planktonic cells (MIC = 0.59 µM) compared to MRSA and VRE strains in addition to potent MRSE biofilm eradication activities (MBEC = 2.35 µM). Several of the halogenated quinolines identified here reported low cytotoxicity against HeLa cells with minimal hemolytic activity against red blood cells. We believe that halogenated quinoline small molecules could play an important role in the development of next-generation antibacterial therapeutics capable of targeting and eradicating biofilm-associated infections.

Antimicrobial peptide-inspired NH125 analogues: bacterial and fungal biofilm-eradicating agents and rapid killers of MRSA persisters.

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.

Identification of N-Arylated NH125 Analogues as Rapid Eradicating Agents against MRSA Persister Cells and Potent Biofilm Killers of Gram-Positive Pathogens.

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.

In vitro antifungal and antibiofilm activities of halogenated quinoline analogues against Candida albicans and Cryptococcus neoformans.

With the increasing prevalence of fungal infections coupled with emerging drug resistance, there is an urgent need for new and effective antifungal agents. Here we report the antifungal activities of 19 diverse halogenated quinoline (HQ) small molecules against Candida albicans and Cryptococcus neoformans. Four HQ analogues inhibited C. albicans growth with a minimum inhibitory concentration (MIC) of 100 nM, whilst 16 analogues effectively inhibited C. neoformans at MICs of 50-780 nM. Remarkably, two HQ analogues eradicated mature C. albicans and C. neoformans biofilms [minimum biofilm eradication concentration (MBEC) = 6.25-62.5 µM]. Several active HQs were found to penetrate into fungal cells, whilst one inactive analogue was unable to, suggesting that HQs elicit their antifungal activities through an intracellular mode of action. HQs are a promising class of small molecules that may be useful in future antifungal treatments.

Synthetically Tuning the 2-Position of Halogenated Quinolines: Optimizing Antibacterial and Biofilm Eradication Activities via Alkylation and Reductive Amination Pathways.

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 <5 % haemolysis activity against human red blood cells (RBCs) at 200 µm. In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram-positive pathogens that could lead to useful treatments against persistent bacterial infections.

Halogenated quinolines discovered through reductive amination with potent eradication activities against MRSA, MRSE and VRE biofilms.

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.

Photophysics of novel coumarin-labeled depsipeptides in solution: sensing interactions with SDS micelle via TICT model.

N-Acylbenzotriazoles enable the synthesis (69-92% yield) of blue to green fluorescent coumarin-labeled depsidipeptides 8a-f (quantum yields 0.004-0.97) and depsitripeptides 12a-d (quantum yields 0.02-0.96). Detailed photophysical studies of fluorescent coumarin-labeled depsipeptides 8a-f and 12a-d are reported for both polar protic and polar aprotic solvents. 7-Methoxy and 7-diethylaminocoumarin-3-ylcarbonyl depsipeptides 8c,f and 12d are highly solvent sensitive. These highly fluorescent compounds could be useful for peptide assays. Further photophysical studies of 7-diethylaminocoumarin-labeled depsipeptides 8c,12d within the micellar microenvironment of SDS reflect their ability to bind with the biological membrane, suggesting potential applications in the fields of bio- and medicinal chemistry.

Benzotriazole-mediated synthesis of aza-peptides: en route to an aza-leuenkephalin analogue.

Novel N-(N-Pg-azadipeptidoyl)benzotriazoles 20a-e couple efficiently with α-amino acids 21a-e, dipeptides 22a-c, aminoxyacetic acid 23a, depsidipeptide 23b, and α-hydroxy-ß-phenylpropionic acid 27 yielding, respectively, azatripeptides 24a-g, azatetrapeptides 25a,b, a hybrid azatripeptide with an oxyamide bond 26a, a hybrid azatetrapeptide with an ester bond 26b, and a hybrid azatripeptide with an ester bond 28. A new protocol for the synthesis of N-Pg-azatripeptides 33a,b and 35a,b, each containing a natural amino acid at the N-terminus, avoids the low coupling rates of the aza-amino acid residue and enables the solution-phase synthesis of an azaphenylalanine analogue of Leu-enkephalin 40.