Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Robust Strategy for Hit-to-Lead Discovery: NMR for SAR.

Establishing robust structure-activity relationships (SARs) is key to successful drug discovery campaigns, yet it often remains elusive due to screening and hit validation artifacts (false positives and false negatives), which frequently result in unproductive downstream expenditures of time and resources. To address this issue, we developed an integrative biophysics-driven strategy that expedites hit-to-lead discovery, mitigates false positives/negatives and common hit validation errors, and provides a robust approach to obtaining accurate binding and affinity measurements. The advantage of this method is that it vastly improves the clarity and reproducibility for affinity-driven SAR by monitoring and eliminating confounding factors. We demonstrate the ease at which high-quality micromolar binders can be generated from the initial millimolar fragment screening hits against an "undruggable" protein target, HRas.

Small Molecule Inhibition of CPS1 Activity through an Allosteric Pocket.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

Exposing Small-Molecule Nanoentities by a Nuclear Magnetic Resonance Relaxation Assay.

Small molecules can self-assemble in aqueous solution into a wide range of nanoentity types and sizes (dimers, n-mers, micelles, colloids, etc.), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to nonspecific binding, off-target effects, and false positives and negatives. Here, we demonstrate the use of the spin-spin relaxation Carr-Purcell-Meiboom-Gill NMR experiment, which is sensitive to molecular tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone-tumbling molecules versus nanoentities of various sizes. The technique is highly sensitive to chemical exchange between single-molecule and aggregate states and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in solution behavior for compounds within structurally related series, demonstrating structure-nanoentity relationships. This practical experiment is a valuable tool to support drug discovery efforts.

Self-Monitoring of Blood Glucose: A Complementary Method Beyond the Oral Glucose Tolerance Test to Identify Hyperglycemia During Pregnancy.

OBJECTIVES: To compare: 1) 75 g oral glucose tolerance test (OGTT) and self-monitoring of blood glucose (SMBG) in identifying gestational diabetes mellitus (GDM) and other hyperglycemic statuses in pregnant women; 2) pregnancy outcomes according to glycemic status; and 3) participants' opinions regarding both methods. METHODS: A prospective study in women with a 50 g glucose load test ≥7.2 mmol/L at 24 to 28 weeks' gestation and singleton pregnancy. Women underwent OGTT (blinded) at day 1, followed by 7 days of SMBG (4 daily measurements: fasting and 2 h postprandially) without modifying diet or lifestyle. GDM (OGTT+) was diagnosed using the criteria of the International Association of the Diabetes and Pregnancy Study Groups, while pregnancy hyperglycemia (SMBG+) was defined as ≥4/7 glucose values ≥5.3 after fasting or ≥6.7 mmol/L 2 h postprandially for any meal of the day. Equivalent management was provided to women with GDM and/or pregnancy-related hyperglycemia. RESULTS: We divided 103 participants (age: 29.5±5.0 years; prepregnancy body mass index: 25.3±5.4 kg/m2) into 4 groups according to test results: OGTT+/SMBG+ (n=12, 11.7%); OGTT+/SMBG- (n=14, 13.6%); OGTT-/SMBG+ (n=9, 8.7%); and OGTT-/SMBG- (n=68, 66.0%). Clinical characteristics and maternal outcomes were statistically similar between groups. Neonatal complication rates were greater in groups with hyperglycemia than in the OGTT-/SMBG- group, notably neonatal hypoglycemia (9/12, 7/14, 5/9 vs. 6/68; p<0.001). Participants reported no convenience difference between methods but would prefer OGTT for a future pregnancy. CONCLUSIONS: More than half of the women with OGTT+ were normoglycemic in daily life. Conversely, 11.7% of women with OGTT- had pregnancy hyperglycemia. OGTT+ and/or SMBG+ were equally associated with greater neonatal complications. This study suggests that alongside OGTT, SMBG could improve the care of pregnant women.

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.

Structural insights into substrate recognition by the Neurospora Varkud satellite ribozyme: importance of U-turns at the kissing-loop junction.

Substrate recognition by the Neurospora Varkud satellite ribozyme depends on the formation of a magnesium-dependent kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain. From mutagenesis studies, it has been established that this I/V kissing-loop interaction involves three Watson-Crick base pairs and is associated with a structural rearrangement of the SLI substrate that facilitates catalysis. Here, we report the NMR structural characterization of this I/V kissing-loop using isolated stem-loops. NMR studies were performed on different SLI/SLV complexes containing a common SLV and shiftable, preshifted, or double-stranded SLI variants. These studies confirm the presence of three Watson-Crick base pairs at the kissing-loop junction and provide evidence for the structural rearrangement of shiftable SLI variants upon SLV binding. NMR structure determination of an SLI/SLV complex demonstrates that both the SLI and SLV loops adopt U-turn structures, which facilitates intermolecular Watson-Crick base pairing. Several other interactions at the I/V interface, including base triples and base stacking, help create a continuously stacked structure. These NMR studies provide a structural basis to understand the stability of the I/V kissing-loop interaction and lead us to propose a kinetic model for substrate activation in the VS ribozyme.

Riboswitch structure: an internal residue mimicking the purine ligand.

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson-Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39-C65 and A39-U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

A 10-year analysis of venous thromboembolism on the surgical service: the effect of practice guidelines for prophylaxis.

BACKGROUND: There is a national effort to decrease the incidence of venous thromboembolism (VTE) in surgical patients by encouraging compliance with established guidelines for prophylaxis. Reported compliance with these guidelines has been poor. The outcome of noncompliance in terms of morbidity and mortality in surgical patients is unknown. We sought to determine if there has been a decrease in the incidence of symptomatic VTE since implementation of the guidelines and whether there has been compliance with the guidelines in individual patients; we also analyzed the outcome of a cohort with VTE. METHODS: We reviewed the records of all patients with symptomatic VTE on 3 surgery services over the 10-year period since initial publication of the guidelines. We determined in each patient whether there was compliance with the guidelines. We weighted the morbidity of each episode of VTE based on the likelihood of short-term mortality and long-term morbidity to determine the disease burden. RESULTS: Of 37,615 patients, 172 developed a VTE (0.46%), and the incidence increased gradually over the years of the study. There was partial or complete compliance with the guidelines in 84% of the patients, but 37% of the VTEs were considered to be preventable. The disease burden was greatest in the higher-risk patients-there were 20 deaths (6%), 4 of which were caused by a pulmonary embolus. CONCLUSIONS: Despite one of the highest published rates of compliance with the guidelines for prophylaxis, the rate of symptomatic VTE is increasing.

Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme.

Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual (31)P and (15)N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface.

NMR structure of varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites.

In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.

Self-care activities for postdischarge nausea and vomiting.

Postdischarge nausea and vomiting (PDNV) is a serious threat to the recovery of ambulatory surgical patients. Pharmacological interventions available before discharge are not readily available to the patient after discharge; therefore, the activities that patients perform to manage new-onset PDNV may affect their surgical recovery. Assembled from a comprehensive study of PDNV, this paper describes the self-care activities that ambulatory patients use to manage new-onset nausea and vomiting after discharge. The findings have implications for patient education and future research.