Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA.

IMPORTANCE: Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

Macrocyclic Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonists.

Macrocyclic retinoic acid receptor-related orphan receptor C2 (RORC2) inverse agonists have been designed with favorable properties for topical administration. Inspired by the unanticipated bound conformation of an acyclic sulfonamide-based RORC2 ligand from cocrystal structure analysis, macrocyclic linker connections between the halves of the molecule were explored. Further optimization of analogues was accomplished to maximize potency and refine physiochemical properties (MW, lipophilicity) best suited for topical application. Compound 14 demonstrated potent inhibition of interleukin-17A (IL-17A) production by human Th17 cells and in vitro permeation through healthy human skin achieving high total compound concentration in both skin epidermis and dermis layers.

Dismantling the Component-Specific Effects of Yogic Breathing: Feasibility of a Fully Remote Three-Arm RCT with Virtual Laboratory Visits and Wearable Physiology.

Despite the growing research base examining the benefits and physiological mechanisms of slow-paced breathing (SPB), mindfulness (M), and their combination (as yogic breathing, SPB + M), no studies have directly compared these in a "dismantling" framework. To address this gap, we conducted a fully remote three-armed feasibility study with wearable devices and video-based laboratory visits. Eighteen healthy participants (age 18-30 years, 12 female) were randomized to one of three 8-week interventions: slow-paced breathing (SPB, N = 5), mindfulness (M, N = 6), or yogic breathing (SPB + M, N = 7). The participants began a 24-h heart rate recording with a chest-worn device prior to the first virtual laboratory visit, consisting of a 60-min intervention-specific training with guided practice and experimental stress induction using a Stroop test. The participants were then instructed to repeat their assigned intervention practice daily with a guided audio, while concurrently recording their heart rate data and completing a detailed practice log. The feasibility was determined using the rates of overall study completion (100%), daily practice adherence (73%), and the rate of fully analyzable data from virtual laboratory visits (92%). These results demonstrate feasibility for conducting larger trial studies with a similar fully remote framework, enhancing the ecological validity and sample size that could be possible with such research designs.

"So, like, it's all a mix of one": Intersecting contexts of adolescents' ethnic-racial socialization.

Cultural-ecological theories posit that ethnic-racial identity (ERI) development is shaped by transactions between contexts of ethnic-racial socialization, yet research considering intersections among multiple contexts is limited. In this study, Black, Latino, White, and Asian American adolescents (N = 98; Mage  = 16.26, SD = 1.09; 55.1% female identifying) participated in surveys and focus group discussions (2013-2014) to share insights into ERI development in context. Using consensual qualitative research, results indicated: (a) family ethnic-racial socialization intersects with community-based, peer, media, and school socialization; (b) ethnic-racial socialization occurs outside family through intersections between peer, school, community-based, and media settings; and (c) ethnic-racial socialization is embedded within systems of racial oppression across contexts. Discussion includes implications for future research and interventions supporting youth ERI.

C9orf72 deficiency promotes microglial-mediated synaptic loss in aging and amyloid accumulation.

C9orf72 repeat expansions cause inherited amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) and result in both loss of C9orf72 protein expression and production of potentially toxic RNA and dipeptide repeat proteins. In addition to ALS/FTD, C9orf72 repeat expansions have been reported in a broad array of neurodegenerative syndromes, including Alzheimer's disease. Here we show that C9orf72 deficiency promotes a change in the homeostatic signature in microglia and a transition to an inflammatory state characterized by an enhanced type I IFN signature. Furthermore, C9orf72-depleted microglia trigger age-dependent neuronal defects, in particular enhanced cortical synaptic pruning, leading to altered learning and memory behaviors in mice. Interestingly, C9orf72-deficient microglia promote enhanced synapse loss and neuronal deficits in a mouse model of amyloid accumulation while paradoxically improving plaque clearance. These findings suggest that altered microglial function due to decreased C9orf72 expression directly contributes to neurodegeneration in repeat expansion carriers independent of gain-of-function toxicities.

Restoring mitofusin balance prevents axonal degeneration in a Charcot-Marie-Tooth type 2A model.

Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.

Evaluation of a Janus kinase 1 inhibitor, PF-04965842, in healthy subjects: A phase 1, randomized, placebo-controlled, dose-escalation study.

AIMS: To determine the safety, tolerability, pharmacokinetics and pharmacodynamics of the Janus kinase 1-selective inhibitor, PF-04965842. METHODS: This was a phase 1, first-in-human, randomized, double-blind, placebo-controlled, combination single- and multiple-dose escalation, parallel design study in healthy subjects (http://clinicaltrials.gov, NCT01835197). Subjects received a single dose of placebo or 3, 10, 30, 100, 200, 400 or 800 mg PF-04965842 (single ascending dose phase) and placebo or 30 mg once daily (QD), 100 mg QD, 200 mg QD, 400 mg QD, 100 mg twice daily (BID) or 200 mg BID PF-04965842 for 10 consecutive days (multiple ascending dose phase). The primary objective was to determine the safety and tolerability of PF-04965842. RESULTS: Seventy-nine subjects were randomized and received study treatments. There were no deaths or serious adverse events. The most frequent treatment-emergent adverse events were headache (n = 13), diarrhoea (n = 11) and nausea (n = 11). PF-04965842 was absorbed rapidly (median time at which maximum plasma concentration occurred generally ≤1 h following either single- or multiple-dose administration) and eliminated rapidly (mean t½ 2.8-5.2 h after 10 days of QD or BID administration in the multiple ascending dose phase). Increases in maximum plasma concentration and area under the concentration-time curve were dose proportional up to 200 mg (single or total daily doses) with an apparent trend towards greater than proportional increases with higher doses. Less than 4.4% of the dose was recovered unchanged in urine. Changes in pharmacodynamic biomarkers were consistent with the known effects of Janus kinase signalling inhibition. CONCLUSIONS: These results support further evaluation of PF-04965842 for clinical use in patients with inflammatory diseases.

Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases.

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.

Microfluidic-Enabled Intracellular Delivery of Membrane Impermeable Inhibitors to Study Target Engagement in Human Primary Cells.

Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.

Development of a high-throughput crystal structure-determination platform for JAK1 using a novel metal-chelator soaking system.

Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg2+-ADP at the ATP-binding site proved recalcitrant to ligand displacement. Addition of a molar excess of EDTA helped to dislodge the divalent metal ion, promoting the release of ADP and allowing facile exchange with ATP-competitive small-molecule ligands. Many kinases require the presence of a stabilizing ligand in the ATP site for crystallization. This procedure could be useful for developing co-crystallization systems with an exchangeable ligand to enable structure-based drug design of other protein kinases.

Renal effects induced by prolonged mPGES1 inhibition.

The importance of membrane-bound PGE synthase 1 (mPGES1) in the regulation of renal function has been examined in mPGES1-deficient mice or by evaluating changes in its expression. However, it is unknown whether prolonged mPGES1 inhibition induces significant changes of renal function when Na(+) intake is normal or low. This study examined the renal effects elicited by a selective mPGES1 inhibitor (PF-458) during 7 days in conscious chronically instrumented dogs with normal Na(+) intake (NSI) or low Na(+) intake (LSI). Results obtained in both in vitro and in vivo studies have strongly suggested that PF-458 is a selective mPGES1 inhibitor. The administration of 2.4 mg·kg(-1)·day(-1) PF-458 to dogs with LSI did not induce significant changes in renal blood flow (RBF) and glomerular filtration rate (GFR). A larger dose of PF-458 (9.6 mg·kg(-1)·day(-1)) reduced RBF (P < 0.05) but not GFR in dogs with LSI and did not induce changes of renal hemodynamic in dogs with NSI. Both doses of PF-458 elicited a decrease (P < 0.05) in PGE2 and an increase (P < 0.05) in 6-keto-PGF1α. The administration of PF-458 did not induce significant changes in renal excretory function, plasma renin activity, and plasma aldosterone and thromboxane B2 concentrations in dogs with LSI or NSI. The results obtained suggest that mPGES1 is involved in the regulation of RBF when Na(+) intake is low and that the renal effects elicited by mPGES1 inhibition are modulated by a compensatory increment in PGI2. These results may have some therapeutical implications since it has been shown that prolonged mPGES1 inhibition has lower renal effects than those elicited by nonsteroidal anti-inflammatory drugs or selective cyclooxygenase-2 inhibitors.

Synthesis and biological evaluation of substituted benzoxazoles as inhibitors of mPGES-1: use of a conformation-based hypothesis to facilitate compound design.

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) is a novel therapeutic target for the treatment of inflammation and pain. In the preceding letter, we detailed the discovery of clinical candidate PF-04693627, a potent mPGES-1 inhibitor possessing a novel benzoxazole structure. While PF-04693627 was undergoing further preclinical profiling, we sought to identify a back-up mPGES-1 inhibitor that differentiated itself from PF-04693627. The design, synthesis, mPGES-1 activity and in vivo PK of a novel set of substituted benzoxazoles are described herein. Also described is a conformation-based hypothesis for mPGES-1 activity based on the preferred conformation of the cyclohexane ring within this class of inhibitors.

Discovery and SAR of PF-4693627, a potent, selective and orally bioavailable mPGES-1 inhibitor for the potential treatment of inflammation.

Inhibition of mPGES-1, the terminal enzyme in the arachidonic acid/COX pathway to regulate the production of pro-inflammatory prostaglandin PGE2, is considered an attractive new therapeutic target for safe and effective anti-inflammatory drugs. The discovery of a novel series of orally active, selective benzoxazole piperidinecarboxamides as mPGES-1 inhibitors is described. Structure-activity optimization of lead 5 with cyclohexyl carbinols resulted in compound 12, which showed excellent in vitro potency and selectivity against COX-2, and reasonable pharmacokinetic properties. Further SAR studies of the benzoxazole ring substituents lead to a novel series of highly potent compounds with improved PK profile, including 23, 26, and 29, which were effective in a carrageenan-stimulated guinea pig air pouch model of inflammation. Based on its excellent in vitro and in vivo pharmacological, pharmacokinetic and safety profile and ease of synthesis, compound 26 (PF-4693627) was advanced to clinical studies.

Novel benzoxazole inhibitors of mPGES-1.

A novel series of potent benzoxazole mPGES-1 inhibitors has been derived from a hit from a high throughput screen. Compound 37 displays mPGES-1 inhibition in an enzyme assay (0.018 µM) and PGE-2 inhibition in a cell-based assay (0.034 µM). It demonstrates 500- and 2500-fold selectivity for mPGES-1 over COX-2 and 6-keto PGF-1α, respectively. In vivo PK studies in dogs demonstrate 55% oral bioavailability and an 7 h half-life.

Selective inducible microsomal prostaglandin E(2) synthase-1 (mPGES-1) inhibitors derived from an oxicam template.

Here we describe the SAR of a series of potent and selective mPGES-1 inhibitors based on an oxicam template. Compound 13j demonstrated low nanomolar mPGES-1 inhibition in an enzyme assay. In addition, it displayed PGE(2) inhibition in a cell-based assay (0.42microM) and had over 238-fold selectivity for mPGES-1 over COX-2 and over 200-fold selectivity for mPGES-1 over 6-keto PGF(1alpha).

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor.

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E(2) (PGE(2)) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE(2) synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC(50)=16.5+/-3.8nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE(2) synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE(2) synthesis (IC(50) in the range of 0.5-5 microM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF(1alpha) (PGF(1alpha)) and PGF(2alpha). In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE(2), PGF(1alpha) and PGF(2alpha) synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.

Comparison of radiographic fracture healing in the distal radius for patients on and off bisphosphonate therapy.

PURPOSE: To compare healing rates of distal radius fractures in patients on bisphosphonate therapy at the time of injury to rates in those not on bisphosphonate therapy. METHODS: A total of 196 consecutive patients treated for distal radius fractures were included in this study. Patients currently on bisphosphonate therapy at the time of injury (bisphosphonate group, n = 43) were compared to the remaining patient group (control group, n = 153). Demographic information was recorded from the patients' medical records, and radiographs were reviewed to determine fracture healing. Patients were further stratified according to age, gender, fracture complexity, type of treatment, and comorbidities. Univariate and multivariate regression were used to identify factors associated with time to radiographic fracture union. RESULTS: The mean time to union was 55 (+/-17) days in the bisphosphonate group versus 49 (+/-14) days in the control group. Bisphosphonate use and surgical treatment were associated with a longer time to radiographic union. Bisphosphonate use was associated with increased healing times when individually controlling for age, gender, fracture complexity, or comorbidities. Bisphosphonate use was also associated with longer time to healing after adjusting for age, gender, and treatment type. Surgical fracture fixation was associated with a longer time to healing after adjusting for bisphosphonate use. CONCLUSIONS: Current bisphosphonate use and surgical treatment were both associated with longer times to radiographic union of distal radius fractures. However, the small differences in healing times (<1 week) are not considered clinically relevant. Although further studies are needed to better define the effects of bisphosphonate therapy on fracture healing, our results suggest that bisphosphonate therapy can be continued after distal radius fractures without notable deleterious effects. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic III.

Homo-timeric structural model of human microsomal prostaglandin E synthase-1 and characterization of its substrate/inhibitor binding interactions.

Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic pi-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H(2) (PGH(2)), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH(2) in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.

Synthesis, crystal structure, and activity of pyrazole-based inhibitors of p38 kinase.

A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.