A Gram-negative-selective antibiotic that spares the gut microbiome.

Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.

Correction to "An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections".

[This corrects the article DOI: 10.1021/acscentsci.2c00598.].

An Iterative Approach Guides Discovery of the FabI Inhibitor Fabimycin, a Late-Stage Antibiotic Candidate with In Vivo Efficacy against Drug-Resistant Gram-Negative Infections.

Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens.

Synthesis of Fusidic Acid Derivatives Yields a Potent Antibiotic with an Improved Resistance Profile.

Fusidic acid (FA) is a potent steroidal antibiotic that has been used in Europe for more than 60 years to treat a variety of infections caused by Gram-positive pathogens. Despite its clinical success, FA requires significantly elevated dosing (3 g on the first day, 1.2 g on subsequent days) to minimize resistance, as FA displays a high resistance frequency, and a large shift in minimum inhibitory concentration is observed for resistant bacteria. Despite efforts to improve on these aspects, all previously constructed derivatives of FA have worse antibacterial activity against Gram-positive bacteria than the parent natural product. Here, we report the creation of a novel FA analogue that has equivalent potency against clinical isolates of Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium) as well as an improved resistance profile in vitro when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant strain of S. aureus in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.

Gram-Negative Antibiotic Active Through Inhibition of an Essential Riboswitch.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Implementation of permeation rules leads to a FabI inhibitor with activity against Gram-negative pathogens.

Gram-negative bacterial infections are a significant public health concern, and the lack of new drug classes for these pathogens is linked to the inability of most drug leads to accumulate inside Gram-negative bacteria1-7. Here, we report the development of a web application-eNTRyway-that predicts compound accumulation (in Escherichia coli) from its structure. In conjunction with structure-activity relationships and X-ray data, eNTRyway was utilized to re-design Debio-1452-a Gram-positive-only antibiotic8-into versions that accumulate in E. coli and possess antibacterial activity against high-priority Gram-negative pathogens. The lead compound Debio-1452-NH3 operates as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of the enoyl-acyl carrier protein reductase FabI, as validated by in vitro enzyme assays and the generation of bacterial isolates with spontaneous target mutations. Debio-1452-NH3 is well tolerated in vivo, reduces bacterial burden in mice and rescues mice from lethal infections with clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae and E. coli. This work provides tools for the facile discovery and development of high-accumulating compounds in E. coli, and a general blueprint for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.

Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis.

The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.

Characterization of the Saffron Derivative Crocetin as an Inhibitor of Human Lactate Dehydrogenase 5 in the Antiglycolytic Approach against Cancer.

Inhibition of lactate dehydrogenase (LDH) represents an innovative approach to tackle cancer because this peculiar glycolytic metabolism is characteristic of most invasive tumor cells. An investigation into the biological properties of saffron extracts led to the discover of their LDH-inhibition properties. In particular, the most important saffron components, crocetin, was found to inhibit LDH (IC50 = 54.9 ± 4.7 µM). This carotenoid was independently produced by chemical synthesis, and its LDH-inhibition properties manifested via its antiproliferative activity against two glycolytic cancer cell lines (A549 and HeLa, IC50 = 114.0 ± 8.0 and 113.0 ± 11.1 µM, respectively). The results described in this article suggest that saffron may be a helpful alimentary component in the prevention of cancer that potentially contributes to the efficacy of approved cancer therapies.

Reactive Oxygen Species Synergize To Potently and Selectively Induce Cancer Cell Death.

A distinctive feature of cancer cells is their elevated levels of reactive oxygen species (ROS), a trait that can cause cancer cells to be more sensitive to ROS-inducing agents than normal cells. ROS take several forms, each with different reactivity and downstream consequence. Here we show that simultaneous generation of superoxide and hydrogen peroxide within cancer cells results in significant synergy, potently and selectively causing cancer cell death. In these experiments superoxide is generated using the NAD(P)H quinone oxidoreductase 1 (NQO1) substrate deoxynyboquinone (DNQ), and hydrogen peroxide is generated using the lactate dehydrogenase A (LDH-A) inhibitor NHI-Glc-2. This combination reduces tumor burden and prolongs survival in a mouse model of lung cancer. These data suggest that simultaneous induction of superoxide and hydrogen peroxide can be a powerful and selective anticancer strategy.

A Potent Inhibitor of Protein Sequestration by Expanded Triplet (CUG) Repeats that Shows Phenotypic Improvements in a Drosophila Model of Myotonic Dystrophy.

Myotonic dystrophy is the most common form of adult-onset muscular dystrophy, originating in a CTG repeat expansion in the DMPK gene. The expanded CUG transcript sequesters MBNL1, a key regulator of alternative splicing, leading to the misregulation of numerous pre-mRNAs. We report an RNA-targeted agent as a possible lead compound for the treatment of myotonic dystrophy type 1 (DM1) that reveals both the promise and challenges for this type of small-molecule approach. The agent is a potent inhibitor of the MBNL1-rCUG complex with an inhibition constant (Ki ) of 25±8 nm, and is also relatively nontoxic to HeLa cells, able to dissolve nuclear foci, and correct the insulin receptor splicing defect in DM1 model cells. Moreover, treatment with this compound improves two separate disease phenotypes in a Drosophila model of DM1: adult external eye degeneration and larval crawling defect. However, the compound has a relatively low maximum tolerated dose in mice, and its cell uptake may be limited, providing insight into directions for future development.

Salicylketoximes That Target Glucose Transporter†1 Restrict Energy Supply to Lung Cancer Cells.

The glucose transporter GLUT1 is frequently overexpressed in most tumor tissues because rapidly proliferating cancer cells rely primarily on glycolysis, a low-efficiency metabolic pathway that necessitates a very high rate of glucose consumption. Because blocking GLUT1 is a promising anticancer strategy, we developed a novel class of GLUT1 inhibitors based on the 4-aryl-substituted salicylketoxime scaffold. Some of these compounds are efficient inhibitors of glucose uptake in lung cancer cells and have a notable antiproliferative effect. In contrast to their 5-aryl-substituted regioisomers, the newly synthesized compounds reported herein do not display significant binding to the estrogen receptors. The inhibition of glucose uptake in cancer cells by these compounds was further observed by fluorescence microscopy imaging using a fluorescent analogue of glucose. Therefore, blocking the ability of tumor cells to take up glucose by means of these small molecules, or by further optimized derivatives, may be a successful approach in the development of novel anticancer drugs.

Dual targeting of the Warburg effect with a glucose-conjugated lactate dehydrogenase inhibitor.

Effective glucose diet: We report the development and activity of glucose-conjugated LDH-A inhibitors designed for dual targeting of the Warburg effect (elevated glucose uptake and glycolysis) in cancer cells. Glycoconjugation could be applied to inhibitors of many enzymes involved in glycolysis or tumor metabolism.

A study on Korean nursing students' educational outcomes.

The purpose of this study was to describe outcome indicators of nursing education including critical thinking, professionalism, leadership, and communication and to evaluate differences among nursing programs and academic years. A descriptive research design was employed. A total of 454 students from four year baccalaureate (BS) nursing programs and two three-year associate degree (AD) programs consented to complete self-administered questionnaires. The variables were critical thinking, professionalism, leadership and communication. Descriptive statistics, χ(2)-test, t-tests, ANOVA, and the Tukey test were utilized for the data analysis. All the mean scores of the variables were above average for the test instruments utilized. Among the BS students, those in the upper classes tended to attain higher scores, but this tendency was not identified in AD students. There were significant differences between BS students and AD students for the mean scores of leadership and communication. These findings suggested the need for further research to define properties of nursing educational outcomes, and to develop standardized instruments for research replication and verification.

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases.

Pharmacophore-based strategy for the development of general and specific scFv biosensors for abused antibiotics.

We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFv's bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.

An international comparison of Korean and Chinese nursing students with nursing curricula and educational outcomes.

AIM: The aim of this study was to compare Korean and Chinese nursing students with respect to their nursing curricula and educational outcomes including critical thinking, professionalism, leadership, communication skills, and nursing practice skills. METHODS: Data were collected from 762 nursing college students (355 in Korea and 407 in China) using the validated self-report questionnaires. The instruments were translated into Chinese for the Chinese students. FINDINGS: Korea offered various nursing courses more focused on specific nursing compared to China. With respect to critical thinking skills, the Korean students had significantly higher scores than the Chinese students. The Chinese students had significantly higher scores than the Korean students on the professionalism and communication skills. There were no differences between the groups in scores of leadership and nursing practice skills. CONCLUSION AND RECOMMENDATIONS: This study provides preliminary information on cross-national nursing educational outcomes. A comparison of educational outcomes among nursing students of other countries as well as China will determine differences in nursing educational outcomes that the nursing program is located in, and an international flow of students through the nursing educational should develop a general direction for nursing education.

Surface modification for small-molecule microarrays and its application to the discovery of a tyrosinase inhibitor.

Small-molecule microarrays are powerful, high-throughput tools for gathering information about direct binding events between proteins of interest and small molecules. However, nonspecific binding on modified glass slides is the major factor reducing the quality of information obtained in proteomic screening with small-molecule microarrays. To improve the signal-to-noise ratio by suppressing the background signal, we tested several surface modification methods for glass slides. Jeffamine-coated slides showed a high fluorescence signal and a significantly enhanced signal-to-noise ratio. We applied this surface modification to proteomic screening of potential tyrosinase inhibitors with a small-molecule microarray and identified 2,4,4'-trihydroxychalcone as a new small-molecule binder to tyrosinase. Its actual binding and inhibitory effects on tyrosinase were validated using an SPR binding assay and an enzyme-based inhibition assay, respectively. Thus, we successfully demonstrate the application of Jeffamine-based modification to proteomics screening with small-molecule microarrays.

Small molecule microarray: functional-group specific immobilization of small molecules.

Proteomic screening with small molecule microarrays can be a powerful tool in conjunction with various forward chemical genetics screening and high-throughput phenotype assays. Small molecule microarray screening can provide high quality information from the direct binding interaction between proteins of interest and a collection of small molecules in a high-throughput fashion. To realize this potential of small molecule microarray in the postgenomic era, the immobilization of small molecules on the surface of microscope glass slides has been a critical step, to apply small molecule library in protein screening assays and dissecting the protein network. In this chapter, we would like to focus on the protocol for the systematic immobilization of synthetic drug-like small molecules containing either specific functional handles or common functional groups.

Antidiabetic and antiobesity effects of Ampkinone (6f), a novel small molecule activator of AMP-activated protein kinase.

Adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK) has emerged as an attractive target molecule for the treatment of metabolic disorders, including obesity and type 2 diabetes. In this study, we identified a novel small molecule, ampkinone (6f), as an indirect AMPK activator, which was derived from the small molecule library constructed by diversity-oriented synthesis. Ampkinone stimulated the phosphorylation of AMPK via the indirect activation of AMPK in various cell lines. Ampkinone-mediated activation of AMPK required the activity of LKB1 and resulted in increased glucose uptake in muscle cells. In addition, ampkinone-treated DIO mice significantly reduced total body weight and overall fat mass. Histological examination and measurement of lipid parameters showed that ampkinone effectively improved metabolic abnormalities in the DIO mice model. Our results demonstrate that ampkinone, a small molecule with a privileged benzopyran substructure, has a potential as a new class of therapeutic agent for antidiabetic and antiobesity treatment via the indirect stimulation of AMPK.

Effect of the Auricular Acupressure Therapy on Dysmenorrhea of Puberty Girls.

PURPOSE: The purpose of this study was to identify the effect of auricular acupressure therapy on dysmenorrhea of puberty girls. METHODS: This study was a pretest-posttest design with a nonequivalent control group. The subjects of this study were 61 girls who were middle and high school students in Seoul and the experiencing dysmenorrhea; 31 for the experimental group and 30 for the control group. The obtained data were analyzed by SPSS/WIN 12.0 program. RESULTS: The experimental group which applied the auricular acupressure therapy scored lower for low abdominal pain (F=136.13, p=.000), low back pain (F=62.79, p=.000). autonomic nervous system reactions (F=102.31, p=.000) and negative emotions on the menstruation (F=102.31, p=.000) compared to the control group. There were significant interaction effects of auricular acupressure on low abdominal pain, low back pain, autonomic nervous system reactions, and negative emotions on the menstruation between time and group. CONCLUSION: We observed that auricular acupressure therapy is effective for reliving pain and the symptom of dysmenorrhea. Based on these result, auricular acupuncture is useful by individual nursing intervention and improves the quality of life for dysmenorrhea of puberty girls.