Small Molecule Benzothiophene with In Vivo Efficacy in a Mouse Model of Drug-Resistant Enterococcus faecium Infection.

Hospital-acquired infections, caused by ESKAPE bacteria, are a challenging global public health concern, in part due to the emergence of drug-resistant strains. While profiling a diverse set of compounds for in vitro activity versus this class of bacteria, we noted that the benzothiophene JSF-2827 exhibited promising antibacterial activity against Enterococcus faecium. A hit evolution campaign ensued, involving the design, synthesis, and biological assay of analogues designed to address early issues such as a short mouse liver microsome half-life and a modest mouse pharmacokinetic profile. Among these derivatives, JSF-3269 was found to exhibit an enhanced profile and in vivo efficacy in an immunocompetent mouse model of acute, drug-resistant E. faecium infection. The findings suggest a rationale for the further evolution of this promising series to afford a novel therapeutic strategy to treat drug-resistant E. faecium infection.

A novel drug-like water-soluble small molecule Focal Adhesion Kinase (FAK) activator promotes intestinal mucosal healing.

Non-steroidal anti-inflammatory drugs (NSAIDs) injure the proximal and distal gut by different mechanisms. While many drugs reduce gastrointestinal injury, no drug directly stimulates mucosal wound healing. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, induces epithelial sheet migration. We synthesized and evaluated a water-soluble FAK-activating small molecule, M64HCl, with drug-like properties. Monolayer wound closure and Western blots measured migration and FAK phosphorylation in Caco-2 â€‹cells, in vitro kinase assays established FAK activation, and pharmacologic tests assessed drug-like properties. 30 â€‹mg/kg/day M64HCl was administered in two murine small intestine injury models for 4 days. M64HCl (0.1-1000 â€‹nM) dose-dependently increased Caco-2 FAK-Tyr 397 phosphorylation, without activating Pyk2 and accelerated Caco-2 monolayer wound closure. M64HCl dose-responsively activates the FAK kinase domain vs. the non-salt M64, increasing the Vmax of ATP-binding. Pharmacologic tests suggested M64HCl has drug-like properties and is enterally absorbed. M64HCl 25 â€‹mg/kg/day continuous infusion promoted healing of ischemic jejunal ulcers and indomethacin-induced small intestinal injury in C57Bl/6 mice. M64HCl-treated mice exhibited smaller ulcers 4 days after ischemic ulcer induction or indomethacin injury. Renal histology and plasma creatinine were normal. Mild hepatic inflammatory changes and ALT elevation were similar among M64HCl-treated mice and controls. M64HCl was concentrated in kidney and gastrointestinal mucosa and functional nephrectomy studies suggested predominantly urinary excretion. Little toxicity was observed in vitro or in single-dose mouse toxicity studies until >1000x higher than effective concentrations. M64HCl, a water-soluble FAK activator, promotes epithelial restitution and intestinal mucosal healing and may be useful to treat gut mucosal injury.

Mycobacterium tuberculosis KasA as a drug target: Structure-based inhibitor design.

Recent studies have reported the ß-ketoacyl-acyl carrier protein KasA as a druggable target for Mycobacterium tuberculosis. This review summarizes the current status of major classes of KasA inhibitors with an emphasis on significant contributions from structure-based design methods leveraging X-ray crystal structures of KasA alone and in complex with inhibitors. The issues addressed within each inhibitor class are discussed while detailing the characterized interactions with KasA and structure-activity relationships. A critical analysis of these findings should lay the foundation for new KasA inhibitors to study the basic biology of M. tuberculosis and to form the basis of new antitubercular molecules of clinical significance with activity against drug-sensitive and drug-resistant infections.

Assessment of carbapenems in a mouse model of Mycobacterium tuberculosis infection.

We present further study of a subset of carbapenems, arising from a previously reported machine learning approach, with regard to their mouse pharmacokinetic profiling and subsequent study in a mouse model of sub-acute Mycobacterium tuberculosis infection. Pharmacokinetic metrics for such small molecules were compared to those for meropenem and biapenem, resulting in the selection of two carbapenems to be assessed for their ability to reduce M. tuberculosis bacterial loads in the lungs of infected mice. The original syntheses of these two carbapenems were optimized to provide multigram quantities of each compound. One of the two experimental carbapenems, JSF-2204, exhibited efficacy equivalent to that of meropenem, while both were inferior to rifampin. The lessons learned in this study point toward the need to further enhance the pharmacokinetic profiles of experimental carbapenems to positively impact in vivo efficacy performance.

Discovery of Novel Small-Molecule FAK Activators Promoting Mucosal Healing.

Gastrointestinal mucosal wounds are common to patients injured by factors as diverse as drugs, inflammatory bowel disease, peptic ulcers, and necrotizing enterocolitis. However, although many drugs are used to ameliorate injurious factors, there is no drug available to actually stimulate mucosal wound healing. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, induces epithelial sheet migration and wound healing, making FAK a potential pharmacological target in this regard. In our previous research, we found a lead compound with drug-like properties, ZINC40099027, which promotes FAK phosphorylation, inducing mucosal healing in murine models. Herein we describe the design and optimization of a small library of novel FAK activators based on ZINC40099027 and their applications toward human intestinal epithelial wound closure and mouse ulcer healing.

Optimization of N-benzyl-5-nitrofuran-2-carboxamide as an antitubercular agent.

The optimization campaign for a nitrofuran antitubercular hit (N-benzyl-5-nitrofuran-2-carboxamide; JSF-3449) led to the design, synthesis, and biological profiling of a family of analogs. These compounds exhibited potent in vitro antitubercular activity (MIC = 0.019-0.20 µM) against the Mycobacterium tuberculosis H37Rv strain and low in vitro cytotoxicity (CC50 = 40->120 µM) towards Vero cells. Significant improvements in mouse liver microsomal stability and mouse pharmacokinetic profile were realized by introduction of an α, α-dimethylbenzyl moiety. Among these compounds, JSF-4088 is highlighted due to its in vitro antitubercular potency (MIC = 0.019 µM) and Vero cell cytotoxicity (CC50 > 120 µM). The findings suggest a rationale for the continued evolution of this promising series of antitubercular small molecules.

Human Microbiome Inspired Antibiotics with Improved ß-Lactam Synergy against MDR Staphylococcus aureus.

The flippase MurJ is responsible for transporting the cell wall intermediate lipid II from the cytoplasm to the outside of the cell. While essential for the survival of bacteria, it remains an underexploited target for antibacterial therapy. The humimycin antibiotics are lipid II flippase (MurJ) inhibitors that were synthesized on the basis of bioinformatic predictions derived from secondary metabolite gene clusters found in the human microbiome. Here, we describe an SAR campaign around humimycin A that produced humimycin 17S. Compared to humimycin A, 17S is a more potent ß-lactam potentiator, has a broader spectrum of activity, which now includes both methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE), and did not lead to any detectable resistance when used in combination with a ß-lactam. Combinations of ß-lactam and humimycin 17S provide a potentially useful long-term MRSA regimen.

Discovery of MRSA active antibiotics using primary sequence from the human microbiome.

Here we present a natural product discovery approach, whereby structures are bioinformatically predicted from primary sequence and produced by chemical synthesis (synthetic-bioinformatic natural products, syn-BNPs), circumventing the need for bacterial culture and gene expression. When we applied the approach to nonribosomal peptide synthetase gene clusters from human-associated bacteria, we identified the humimycins. These antibiotics inhibit lipid II flippase and potentiate ß-lactam activity against methicillin-resistant Staphylococcus aureus in mice, potentially providing a new treatment regimen.

Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

Picomolar Inhibitors of HIV-1 Reverse Transcriptase: Design and Crystallography of Naphthyl Phenyl Ethers.

Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Promising compounds are reported that show midpicomolar activity against the wild-type virus and sub-20 nM activity against viral variants bearing Tyr181Cys and Lys103Asn mutations in HIV-RT. An X-ray crystal structure at 2.49 Å resolution is also reported for the key compound 6e with HIV-RT.

Prevalence of leisure-time sedentary behaviour and sociodemographic correlates: a cross-sectional study in Spanish adults.

BACKGROUND: Being physically inactive has been linked to a higher mortality and poorer quality of life. This cross-sectional study examines the prevalence of leisure-time sedentary behaviour in a population of Spanish adults and its correlates with several sociodemographic variables. METHODS: Data were collected from 1,330 subjects living in Madrid (age: 18-65 years, 51.6% women) by telephone interview. The sampling error was ±2.7% for a 95.5% confidence level. Leisure-time sedentary behaviour was assessed using the Global Physical Activity Questionnaire (version 2). Further factors examined were: country of birth, sex, age, civil state, education level, employment and economic status and physical activity of parents. RESULTS: 76.3% of the subjects interviewed reported a mostly sedentary leisure-time lifestyle. The remaining subjects (23.7%) reported a moderate to high level of physical activity, meeting minimum recommendations. Logistic regression adjusted for all variables identified the following population subsets as showing a greater risk of sedentary behaviour: women (odds ratio (OR) = 2.14; 95% confidence interval (CI): 1.64, 2.79), participants aged 41-50 years (OR = 1.64; 95%CI:1.05, 2.51), those with a middle economic status (OR = 1.48; 95% CI: 1.04, 2.10) or not providing information about their income (OR = 1.97; 95% CI: 1.05, 3.67), and those whose father (OR = 1.53; 95% CI: 1.13, 2.07) and/or mother (OR = 1.41; 95% CI: 1.01, 1.97) were never physically active during leisure-time. CONCLUSIONS: The high prevalence of self-reported sedentary behaviour recorded suggests the need for public health policies targeted at increasing leisure-time physical activity levels. Our data identified several population subsets as priority candidates for possible interventions pursuing this goal.

Structure-based evaluation of C5 derivatives in the catechol diether series targeting HIV-1 reverse transcriptase.

Using a computationally driven approach, a class of inhibitors with picomolar potency known as the catechol diethers were developed targeting the non-nucleoside-binding pocket of HIV-1 reverse transcriptase. Computational studies suggested that halogen-bonding interactions between the C5 substituent of the inhibitor and backbone carbonyl of conserved residue Pro95 might be important. While the recently reported crystal structures of the reverse transcriptase complexes confirmed the interactions with the non-nucleoside-binding pocket, they revealed the lack of a halogen-bonding interaction with Pro95. To understand the effects of substituents at the C5 position, we determined additional crystal structures with 5-Br and 5-H derivatives. Using comparative structural analysis, we identified several conformations of the ethoxy uracil dependent on the strength of a van der Waals interaction with the Cγ of Pro95 and the C5 substitution. The 5-Cl and 5-F derivatives position the ethoxy uracil to make more hydrogen bonds, whereas the larger 5-Br and smaller 5-H position the ethoxy uracil to make fewer hydrogen bonds. EC50 values correlate with the trends observed in the crystal structures. The influence of C5 substitutions on the ethoxy uracil conformation may have strategic value, as future derivatives can possibly be modulated to gain additional hydrogen-bonding interactions with resistant variants of reverse transcriptase.

Picomolar inhibitors of HIV reverse transcriptase featuring bicyclic replacement of a cyanovinylphenyl group.

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC50 values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds such as rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4 nM activity against the wild-type virus. The compounds also performed well with EC50 values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogues were also investigated; the most potent compounds in these cases have EC50 values toward wild-type HIV-1 near 10 nM and high-nanomolar activities toward the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88 Å resolution for the complex of the parent 2-cyanoindolizine 10b and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogues were also measured to be ~40 µg/mL, which is similar to that for the approved drug efavirenz and ~1000-fold greater than for rilpivirine.

Optimization of benzyloxazoles as non-nucleoside inhibitors of HIV-1 reverse transcriptase to enhance Y181C potency.

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved activity towards Tyr181Cys containing variants was pursued with the assistance of free energy perturbation (FEP) calculations. Optimization of the 4-R substituent in 1 led to ethyl and isopropyl analogs 1e and 1f with 1-7 nM potency towards both the wild-type virus and a Tyr181C variant.

Crystal structures of HIV-1 reverse transcriptase with picomolar inhibitors reveal key interactions for drug design.

X-ray crystal structures at 2.9 Å resolution are reported for two complexes of catechol diethers with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.

Computationally-guided optimization of a docking hit to yield catechol diethers as potent anti-HIV agents.

A 5-µM docking hit has been optimized to an extraordinarily potent (55 pM) non-nucleoside inhibitor of HIV reverse transcriptase. Use of free energy perturbation (FEP) calculations to predict relative free energies of binding aided the optimizations by identifying optimal substitution patterns for phenyl rings and a linker. The most potent resultant catechol diethers feature terminal uracil and cyanovinylphenyl groups. A halogen bond with Pro95 likely contributes to the extreme potency of compound 42. In addition, several examples are provided illustrating failures of attempted grafting of a substructure from a very active compound onto a seemingly related scaffold to improve its activity.

Influencia de determinantes socio-demográficos en la adherencia a las recomendaciones de actividad física en personas de entre 15 y 74 años de Madrid / Influence of socio-demographic correlates on the adherence to physical activity recommendations in adults aged from 15-to 74 years. Madrid, Spain

Fundamento: Conocer el nivel de adherencia de las personas a las recomendaciones de actividad física (AF) es de interés socio-sanitario. El objetivo del presente estudio fue describir el nivel de adherencia a las recomendaciones de AF por los adultos de la Comunidad de Madrid, así como analizar su asociación con posibles determinantes socio-demográfi-cos. Métodos: En el presente estudio transversal se realizó una encuesta telefónica a una muestra de 1.500 personas de 15 a 74 años de la Comunidad de Madrid. La AF se evaluó con la versión 2 del Global Physical Activity Questionnaire (GPAQv2) y se clasificó en tres niveles de intensidad (bajo, moderado y alto), según procedimiento de análisis del GPAQv2. Las variables socio-demográficas estudiadas fueron: sexo, edad, nivel de estudios, ocupación, estado civil, consumo de tabaco y salud percibida. Para analizar la asociación entre las características socio-demográficas y la AF, se realizaron análisis de regresión logística multinomial. Resultados: El 82% de los varones y 78% de las mujeres (80% en total) tenían un nivel global de AF moderado o alto, y el 40,1% y el 22,6% (31% en total) de los varones y mujeres, respectivamente, alcanzó las recomendaciones de AF en el tiempo libre. Tenían más probabilidades de no alcanzar las recomendaciones de AF los participantes con estudios superiores (OR: 2,05; 95%IC: 1,48-2,86), los fumadores habituales (OR: 1,41; 95%IC: 1,04-1,90) y los que percibían su salud como mala (OR: 3,58; 95%IC: 2,39-5,38). En cuanto a la edad fueron las personas del grupo entre 35-44 años las que tenían menos probabilidades de no cumplirlas (OR: 0,61; 95%IC: 0,39-0,95) y 45-54 (OR: 0,52; 95%IC: 0,32-0,83). Conclusiones: El 20% de las personas de la Comunidad de Madrid que tienen entre 15 y 74 años no cumplen con las recomendaciones mínimas de AF, y cuando se considera la AF realizada exclusivamente durante el tiempo libre se llega al 69% de los participantes que no alcanzan las recomendaciones de AF. El nivel de estudios, el hábito de fumar y la percepción de tener mala salud influyen en los niveles de AF(AU)

Background: To know the adherence to physical activity recommendations of the population is of clinical and social interest. The aim of this study was to estimate the proportion of Spanish adults adhering to the physical activity recommendations, and to examine the influences of socio-demographic correlates. Methods: In the present cross-sectional study we conducted a telephone survey of 1,500 Spanish adults (15-74 years old) from Madrid (Spain). Physical activity (work place, transport and leisure time) was assessed with the version 2 of the Global Physical Activity Questionnaire (GPAQv2). Participants were categorized in three physical activity levels (low, moderate and high). The socio-demographic correlates included: gender, age, educational level, employment status, marital status, smoking status, and self-perceived health. The association between socio-demographic factors and physical activity was examined with multinomial logistic regression analysis. Results: A total of 82% of men and 78% of women (total 80%) had moderate to high levels of physical activity, yet, when considering the leisure time physical activity, only 40,1% of mean and 22,6% of women (total 31.1%) reach the recommendations. Participants with university degree (OR: 2.05; 95%IC: 1.48-2.86), those who were smokers (OR: 1.41; 95%IC: 1.04-1.90), and those who perceived their health as bad (OR: 3.58; 95%IC: 2.39-5.38) were more likely to not to reach the recommendations. In contrast, those participants aged 35-44 years (OR: 0.61; 95%IC: 0.39-0.95) and 45-54 years (OR: 0.52; 95%IC: 0.32-0.83) were less likely not to reach the recommendations. Conclusions: The 20% of adults from Madrid did not reach the physical activity recommendations, and when considering only leisure time physical activity, only 69% reached the recommendations. The findings suggest that the educational level, smoking status, and the self-perceived health seem to be key determinants. There is a large diversity in the physical activity levels in the population subgroup; therefore, there is a need of developing socialecological approaches to physical activity promotion(AU)

[Influence of socio-demographic correlates on the adherence to physical activity recommendations in adults aged from 15-to 74 years]. / Influencia de determinantes socio-demográficos en la adherencia a las recomendaciones de actividad física en personas de entre 15 y 74 años de Madrid: Madrid, Spain.

BACKGROUND: To know the adherence to physical activity recommendations of the population is of clinical and social interest. The aim of this study was to estimate the proportion of Spanish adults adhering to the physical activity recommendations, and to examine the influences of socio-demographic correlates. METHODS: In the present cross-sectional study we conducted a telephone survey of 1,500 Spanish adults (15-74 years old) from Madrid (Spain). Physical activity (work place, transport and leisure time) was assessed with the version 2 of the Global Physical Activity Questionnaire (GPAQv2). Participants were categorized in three physical activity levels (low, moderate and high). The socio-demographic correlates included: gender, age, educational level, employment status, marital status, smoking status, and self-perceived health. The association between socio-demographic factors and physical activity was examined with multinomial logistic regression analysis. RESULTS: A total of 82% of men and 78% of women (total 80%) had moderate to high levels of physical activity, yet, when considering the leisure time physical activity, only 40,1% of mean and 22,6% of women (total 31.1%) reach the recommendations. Participants with university degree (OR: 2.05; 95%IC: 1.48-2.86), those who were smokers (OR: 1.41; 95%IC: 1.04-1.90), and those who perceived their health as bad (OR: 3.58; 95%IC: 2.39-5.38) were more likely to not to reach the recommendations. In contrast, those participants aged 35-44 years (OR: 0.61; 95%IC: 0.39-0.95) and 45-54 years (OR: 0.52; 95%IC: 0.32-0.83) were less likely not to reach the recommendations. CONCLUSIONS: The 20% of adults from Madrid did not reach the physical activity recommendations, and when considering only leisure time physical activity, only 69% reached the recommendations. The findings suggest that the educational level, smoking status, and the self-perceived health seem to be key determinants. There is a large diversity in the physical activity levels in the population subgroup; therefore, there is a need of developing social-ecological approaches to physical activity promotion.