Structural basis for dual-mode inhibition of the ABC transporter MsbA.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Prevalence, Clinical and Imaging Characteristics of Superior Ophthalmic Vein Periphlebitis in Thyroid Eye Disease.

PURPOSE: To determine the prevalence, clinical features, and radiographic findings of superior ophthalmic vein periphlebitis (SOVP) in thyroid eye disease (TED). METHODS: Patients with a clinical diagnosis of thyroid eye disease and contrast-enhanced imaging were included. Imaging was reviewed for the presence of SOVP, and patients with SOVP were compared to those without. A random eye was determined to be the affected eye in patients without SOVP. RESULTS: A total of 212 patients met the inclusion criteria. Unilateral SOVP was identified in 4.7% of cases. There was no significant difference in age ( p = 0.22), gender ( p = 0.09), or disease duration ( p = 0.14) between patients with and without SOVP. There was a significant ( p < 0.05) difference in stage classification and clinical activity core between the groups. The affected eye in patients with SOVP had significantly ( p < 0.05) greater margin reflex distance 1, degree of relative proptosis, horizontal motility restriction, and vertical motility restriction than in patients without SOVP. There was no significant difference in horizontal strabismus ( p = 1.0), vertical strabismus ( p = 0.87), or relative intraocular pressure ( p = 0.77). On imaging, the maximal diameter of the SR and IR were found to be significantly ( p < 0.05) larger in the affected eye of patients with periphlebitis; however, there was no difference in measured diameter of the medial rectus and ( p = 0.30) or lateral rectus ( p = 0.78). CONCLUSIONS: SOVP is an under-reported imaging finding of thyroid eye disease. It is associated with significantly greater margin reflex distance 1, relative proptosis, and motility restriction on exam as well as larger superior rectus and inferior rectus diameter on imaging. These patients tend to present in the active stage of disease with greater clinical activity score.

Orbital indeterminate cell histiocytosis.

An 8-year-old female presented to the oculoplastics clinic with 3 months of left upper eyelid fullness and edema. Examination showed a mass in the left anterior superior orbit with erythema. Imaging demonstrated a well-circumscribed superolateral orbital mass that was T1 hypointense and T2 hypo-to-iso intense with contrast enhancement. An incisional biopsy was performed via an upper lid crease incision. Histopathology showed aggregates of histiocytic cells with fibrosis and infiltration of eosinophils. Immunohistochemistry revealed positive CD68 and CD163 staining and negative langerin staining, confirming the diagnosis of indeterminate cell histiocytosis. There was no systemic involvement or associated dermatologic findings. Repeat exam 3 months later showed no change in the size of the lesion and the patient was referred to hematology-oncology for treatment. On most recent exam, the patient had no new symptoms or side effects following 3 months of oral hydroxyurea (25 mg/kg/day). Repeat orbital imaging showed no progression of the lesion and the patient will be monitored closely. Here, we report a rare case of isolated orbital indeterminate cell histiocytosis in a young child.

Recent Fluctuations in Public Searches for Cosmetic Procedures as Shown by Google Trends.

PURPOSE: The goal of this study is to understand the dynamic trends of 20 cosmetic procedures in the United States using Google Trends. METHODS: This was a descriptive cross-sectional study using Google Trends to measure changes in search volumes for 20 cosmetic procedures from a 5-year period (2018 to 2023) in the United States. Outcome measures included total and weekly changes in relative search volumes and variance levels for the following 20 search terms related to cosmetic surgeries: blepharoplasty, botox, breast augmentation, breast lift, brow lift, buccal fat removal, chemical peel, dermal filler, face lift, hair removal, hair transplant, lip filler, liposuction, masseter botox, microneedling, otoplasty, photofacial, rhinoplasty, tear trough filler, and tummy tuck. Search terms were divided into 3 categories, a) those with a change of public interest over a single week of 25% or less, b) 26%-50%, and c) greater than 50%. RESULTS: There was an overall 5-year increase of public interest in all but 5 search terms: breast augmentation, breast lift, liposuction, photofacial, and tummy tuck. Within a single week, lip filler, masseter botox, buccal fat removal, and tear trough filler exhibited greater than 50% change in public interest, while public interest in microneedling, hair removal, rhinoplasty, tummy tuck, liposuction, breast lift, and augmentation were more stable with less than 26% change. CONCLUSIONS: While certain procedures may experience rapid accelerations in public interest, not all procedures will consistently increase in popularity. Physicians must recognize the shifting popularity of cosmetic procedures to provide informed cosmetic care to patients.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

Low-grade fibromyxoid sarcoma of the orbit.

A 70-year-old male presented with diplopia and painless proptosis of the left eye for 5 months. Examination showed 6 mm of axial proptosis and restriction of supraduction, abduction and adduction, and mild limitation of infraduction of the left eye. Magnetic resonance imaging demonstrated a large, moderately well-circumscribed intraconal mass in the left lateral orbit, and excisional biopsy was performed. Histopathologic features of mixed fibrous and myxoid areas in a whorl-like pattern and immunohistochemical staining for MUC4 confirmed the diagnosis of low-grade fibromyxoid sarcoma (LGFMS). Next-generation sequencing revealed genetic fusion of EWSR1-CREB3L1. LGFMS is an extremely rare neoplasm with only two prior documented cases of orbital involvement. Here, we report the third case of orbital LGFMS.

The efficacy of 'high in' warning labels, health star and traffic light front-of-package labelling: an online randomised control trial.

OBJECTIVE: To examine the impact of front-of-package (FOP) labels on perceived healthfulness, purchasing intentions and understanding of common FOP systems. DESIGN: A parallel, open-label design randomised participants to different FOP labelling conditions: 'high in' warning labels (WL), multiple traffic light labelling (TLL), health star ratings (HSR) (all displayed per serving) or control with no interpretive FOP labelling. Participants completed a brief educational session via a smartphone application and two experimental tasks. In Task 1, participants viewed healthy or unhealthy versions of four products and rated healthiness and purchasing intention on a seven-point Likert-type scale. In Task 2, participants ranked three sets of five products from healthiest to least healthy. SETTING: Online commercial panel. PARTICIPANTS: Canadian residents ≥ 18 years who were involved in household grocery shopping, owned a smartphone and met minimum screen requirements. RESULTS: Data from 1997 participants (n 500/condition) were analysed. Task 1: across most product categories, the TLL and HSR increased perceived healthiness of healthier products. All FOP systems decreased perceived healthiness of less healthy products. Similar, albeit dampened, effects were seen regarding purchasing intentions. Task 2: participants performed best in the HSR, followed by the TLL, WL and control conditions. Lower health literacy was associated with higher perceived healthiness and purchasing intentions and poorer ranking task performance across all conditions. CONCLUSIONS: All FOP labelling systems, after a brief educational session, improved task performance across a wide spectrum of foods. This effect differed depending on the nutritional quality of the products and the information communicated on labels.Trial Registration: NCT03290118.

A randomized controlled trial examining consumers' perceptions and opinions on using different versions of a FoodFlip© smartphone application for delivery of nutrition information.

BACKGROUND: Food labelling is a common intervention to improve diets, where the back-of-pack Nutrition Information Panel (or Nutrition Facts table (NFt)) provides comprehensive nutrition information on food packages. However, many consumers find it difficult and time-consuming to identify healthier foods using the NFt. As a result, different interpretative nutrition rating systems (INRS) may enable healthier food choices and it is essential that consumers have the tools to allow for easily accessible nutrition information. The objective of this study was to examine consumers' perceptions of different (INRS) for delivery of nutrition information using different versions of a smartphone app, FoodFlip©. METHODS: This study was part of a larger randomized controlled trial examining consumer perceptions of different INRS on food products. A nationally representative commercial sample of 2008 Canadians were randomized to one of four INRS intervention groups: 1) traffic light, 2) health star rating, 3) 'high-in' warning labels or 4) no INRS (NFt only; control) and asked to scan or enter 20 products into FoodFlip© from a list of food products provided to them with varying levels of healthfulness. After completing the app task, participants were asked a series of 7-point Likert-scale and open-ended questions to provide opinions on the usability and functionality of the app. RESULTS: Of the survey sample of 1997 participants, 95% (n = 1907) completed the app task, with similar number of participants in each treatment group. The mean age was 40 ± 12 years with no differences in sociodemographic characteristics between treatment groups. The health star rating ranked significantly lower in comparison to the other treatment groups in terms of usefulness (OR, 95% CI -0.67, 0.52-0.85), believability (0.59, 0.46-0.75), and understanding (0.55, 0.44-0.71) (p < 0.001). The health star rating (1.20, 0.94-1.53) and control (NFt) (1,1,1) ranked significantly lower than the traffic light or the 'high-in' warning labels for their ability to compare the healthfulness of products (p < 0.001). CONCLUSION: This study demonstrated Canadian consumers' preference for a nutrient-specific system (i.e. traffic light or 'high-in' warning labels). The app, which was liked by majority of the participants for its functionality and usability, has the potential to support healthy dietary decision making and may also encourage reformulation. TRIAL REGISTRATION: NCT03290118 (Clinicaltrials.gov).

Influence of front-of-pack labelling and regulated nutrition claims on consumers' perceptions of product healthfulness and purchase intentions: A randomized controlled trial.

Mandatory front-of-pack (FOP) labelling was proposed in Canada to highlight foods with high contents of sugars, sodium and/or saturated fats, which would be displayed on labels along with the mandatory Nutrition Facts table and voluntary nutrition claims. In an online survey, participants (n = 1997) were randomized to one of four FOP labelling conditions: 1) control, 2) warning label, 3) health star rating or 4) traffic light labelling. Participants were shown four drinks (a healthier drink with or without a disease risk reduction claim, a healthier drink with or without a nutrient content claim, a less healthy drink with or without a disease risk reduction claim and a less healthy drink with or without a nutrient content claim) in random order and one at a time. Participants rated perceived product healthfulness and purchase intentions using a 7-point Likert scale. Participants could access the Nutrition Facts table while viewing labels. Results showed less healthy drinks displaying any FOP labelling were perceived as less healthy compared to the control. In healthier drinks, health star rating and traffic light labelling created a 'halo' effect, which was not observed with warning labels. Similar results were observed with purchase intentions. Drinks displaying a disease risk reduction claim were perceived as healthier than those without (p < 0.001) regardless of product's healthfulness. The effect of a nutrient content claim was not significantly different. The effect of FOP labelling and claims was mitigated for those who used the Nutrition Facts table. FOP labelling was likely helpful for consumers with different levels of health literacy. Overall, FOP labelling had significantly stronger influence than nutrition claims on consumers' perceptions; however, the effect of each FOP label varied on healthier and less healthy drinks.

Quantitative Determination of Protein-Ligand Affinity by Size Exclusion Chromatography Directly Coupled to High-Resolution Native Mass Spectrometry.

High throughput protein-ligand interaction screening assays employing mass spectrometric detection are widely used in early stage drug discovery. Mass spectrometry-based screening approaches employ a target protein added to a pool of small-molecule compounds, and binding is assessed by measuring ligands denatured from the complexes. Direct analysis of protein-ligand interactions using native mass spectrometry has been demonstrated but is not widely used due to the detection limit on protein size, the requirement of volatile buffers, and the necessity for specialized instrumentation to preserve weak interactions under native conditions. Here we present a robust, quantitative, and automated online size-exclusion chromatography-native mass spectrometry (SEC-nMS) platform for measuring affinities of noncovalent protein-small-molecule interactions on an Orbitrap mass spectrometer. Indoleamine 2,3-dioxygenase 1, a catabolic enzyme, and inhibitory ligands were employed as a demonstration of the method. Efficient separation and elution enabled preservation of protein-ligand complexes and increased throughput. The high sensitivity and intra charge state resolution at high m/ z offered by the Exactive Plus EMR Orbitrap allowed for protein ligand affinity quantitation and resolved individual compounds close in mass. Vc50 values determined via collision-induced dissociation experiments enabled the evaluation of complex stability in the gas phase and were found to be independent of the extent of complex formation. For the first time, Vc50 determinations were achieved on an inline SEC-nMS platform. Systematic comparison of our method with optimized chip-based nanoelectrospray infusion served as a reference for ligand screening and affinity quantitation and further revealed the advantages of SEC-MS.

Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA.

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor.

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins.

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Negative reinforcement impairs overnight memory consolidation.

Post-learning sleep is beneficial for human memory. However, it may be that not all memories benefit equally from sleep. Here, we manipulated a spatial learning task using monetary reward and performance feedback, asking whether enhancing the salience of the task would augment overnight memory consolidation and alter its incorporation into dreaming. Contrary to our hypothesis, we found that the addition of reward impaired overnight consolidation of spatial memory. Our findings seemingly contradict prior reports that enhancing the reward value of learned information augments sleep-dependent memory processing. Given that the reward followed a negative reinforcement paradigm, consolidation may have been impaired via a stress-related mechanism.

Structural and biochemical analyses of the catalysis and potency impact of inhibitor phosphoribosylation by human nicotinamide phosphoribosyltransferase.

Prolonged inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is a strategy for targeting cancer metabolism. Many NAMPT inhibitors undergo NAMPT-catalyzed phosphoribosylation (pRib), a property often correlated with their cellular potency. To understand this phenomenon and facilitate drug design, we analyzed a potent cellularly active NAMPT inhibitor (GNE-617). A crystal structure of pRib-GNE-617 in complex with NAMPT protein revealed a relaxed binding mode. Consistently, the adduct formation resulted in tight binding and strong product inhibition. In contrast, a biochemically equipotent isomer of GNE-617 (GNE-643) also formed pRib adducts but displayed significantly weaker cytotoxicity. Structural analysis revealed an altered ligand conformation of GNE-643, thus suggesting weak association of the adducts with NAMPT. Our data support a model for cellularly active NAMPT inhibitors that undergo NAMPT-catalyzed phosphoribosylation to produce pRib adducts that retain efficient binding to the enzyme.

Discovery of potent and efficacious cyanoguanidine-containing nicotinamide phosphoribosyltransferase (Nampt) inhibitors.

A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50=2.5nM, A2780 cell proliferation IC50=9.7nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined.