A small-molecule membrane fluidizer re-sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics.

Novel antibacterial agents and strategies are urgently needed to fight against the ongoing global antibiotic resistance problem. While natural products remain the main source in antibiotic discovery, synthetic antibacterials provide an attractive alternative and may evade the ancient antibiotic resistance. Herein, we report a small molecule that re-sensitizes methicillin-resistant Staphylococcus aureus to ß-lactam antibiotics with extremely low potential for resistance development. It belongs to a new class of broad-spectrum antibacterials, trypyricins, which share similar structural characteristics and mechanism of action to the cationic antimicrobial peptides. Mechanistic studies indicated that trypyricins fluidize and disrupt bacterial cytoplasmic membrane. These results suggested that trypyricins represent a promising new class of antibacterials and may be further developed as antibiotic adjuvants to fight against resistant bacteria in the clinic.

A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load.

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAßN (Phe-Arg ß-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.

Bio-inspired synthesis yields a tricyclic indoline that selectively resensitizes methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics.

The continuous emergence of resistant bacteria has become a major worldwide health threat. The current development of new antibacterials has lagged far behind. To discover reagents to fight against resistant bacteria, we initiated a chemical approach by synthesizing and screening a small molecule library, reminiscent of the polycyclic indole alkaloids. Indole alkaloids are a class of structurally diverse natural products, many of which were isolated from plants that have been used as traditional medicine for millennia. Specifically, we adapted an evolutionarily conserved biosynthetic strategy and developed a concise and unified diversity synthesis pathway. Using this pathway, we synthesized 120 polycyclic indolines that contain 26 distinct skeletons and a wide variety of functional groups. A tricyclic indoline, Of1, was discovered to selectively potentiate the activity of ß-lactam antibiotics in multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA), but not in methicillin-sensitive S. aureus. In addition, we found that Of1 itself does not have antiproliferative activity but can resensitize several MRSA strains to the ß-lactam antibiotics that are widely used in the clinic, such as an extended-spectrum ß-lactam antibiotic amoxicillin/clavulanic acid and a first-generation cephalosporin cefazolin. These data suggest that Of1 is a unique selective resistance-modifying agent for ß-lactam antibiotics, and it may be further developed to fight against resistant bacteria in the clinic.

Discovery and initial structure-activity relationships of N-benzyl tricyclic indolines as antibacterials for methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent drug resistant bacteria. In 2012, over 11,000 fatalities in the United States were directly attributable to MRSA. In an effort to develop novel structural and mechanistic classes of antibacterial agents to fight against MRSA, we have optimized a hit compound, Of4, previously discovered in a screening campaign of a bio-inspired polycyclic indoline library previously developed in our lab. We took advantage of our concise and versatile synthetic strategy to conduct initial structure-activity relationship studies of Of4, and we now report the discovery of compound 4k as a more potent antibacterial agent against S. aureus. Compound 4k also displayed equivalent activity in four MRSA and a methicillin-susceptible strains while demonstrating an improved mammalian cytotoxicity profile compared to Of4. Interestingly, 4k shares the same tricyclic indoline core as Of1, a ß-lactam-selective resistance-modifying agent, but harbors a distinct modification pattern conferring unique bioactivity. This phenomenon is reminiscent of many bioactive natural products.

Increase in cerebral blood flow indicated by increased cerebral arterial area and pixel intensity on brain magnetic resonance angiogram following correction of cervical lordosis.

CONTEXT: Loss of cervical lordosis is associated with decreased vertebral artery hemodynamics. AIM: The aim of this study is to evaluate cerebral blood flow changes on brain magnetic resonance angiogram (MRA) in patients with loss of cervical lordosis before and following correction of cervical lordosis. SETTINGS AND DESIGN: This study is a retrospective consecutive case series of patients in a private practice. MATERIALS AND METHODS: Cervical lordosis of seven patients (five females and two males, 28-58 years) was measured on lateral cervical radiographs ranging from -13.1° to 19.0° (ideal is -42.0°). Brain MRAs were analyzed for pixel intensities representing blood flow. Pixel intensity of the cerebral vasculature was quantified, and percentage change was determined. STATISTICAL ANALYSIS USED: A Student's t-test established significance of the percentage change in cerebral blood flow between pre- and postcervical lordosis adjustment images. Regression analysis was performed. An a priori analysis determined correlation between cervical lordosis and change in MRA pixel intensity. The statistician was blinded to the cervical lordosis. RESULTS: Pixel intensity increased 23.0%-225.9%, and a Student's t-test determined that the increase was significant (P < 0.001). Regression analysis of the change in pixel intensity versus the cervical lordosis showed that as the deviation from a normal cervical lordosis increases, percentage change in pixel intensity on MRA decreases. CONCLUSION: These results indicate that correction of cervical lordosis may be associated with an immediate increase in cerebral blood flow. Further studies are needed to confirm these findings and understand clinical implications.

Tetracyclic indolines as a novel class of ß-lactam-selective resistance-modifying agent for MRSA.

Antibiotic-resistant bacterial infections have seen a marked increase in recent years, while antibiotic discovery has waned. Resistance-modifying agents (RMA) offer an intriguing alternative strategy to fight against resistant bacteria. Here we report the discovery, antibiotic profiling, and structure-activity relationships of a novel class of RMAs, tetracyclic indolines. These selectively potentiate ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) without antibacterial or ß-lactamase inhibitory activity on their own. The most potent analogue, 6a, showed strong potentiation of amoxicillin/clavulanic acid in a variety of hospital-acquired and community-acquired MRSA strains with low mammalian toxicity. These compounds may be further developed to extend the clinic life span of ß-lactam antibiotics.

Structure-activity relationship studies of the tricyclic indoline resistance-modifying agent.

Previously we discovered a tricyclic indoline, N-[2-(6-bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]-4-chlorobenzene-1-sulfonamide (1, Of1), from bioinspired synthesis of a highly diverse polycyclic indoline alkaloid library, that selectively resensitizes methicillin-resistant Staphylococcus aureus strains to ß-lactam antibiotics. Herein, we report a thorough structure-activity relationship investigation of 1, which identified regions of 1 that tolerate modifications without compromising activity and afforded the discovery of a more potent analogue with reduced mammalian toxicity.

Quantitative analysis of histone demethylase probes using fluorescence polarization.

We previously reported methylstat as a selective inhibitor of jumonji C domain-containing histone demethylases (JHDMs). Herein, we describe the synthesis of a fluorescent analogue of methylstat and its application as a tracer in fluorescence polarization assays. Using this format, we have evaluated the binding affinities of several known JHDM probes, as well as the native cofactor and substrate of JHDM1A. This fluorophore allowed a highly robust and miniaturized competition assay sufficient for high-throughput screening.