Rosetta's Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery.

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da which weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in in silico screening. Here, we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF (UniProtKB Q9X0C6). We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability using RosettaLigand and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigand's ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 µM. The same library of fragments was blindly screened in silico. RosettaLigand was able to rank binders before non-binders with an area under the curve of the receiver operating characteristics of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.

Computational design of protein-small molecule interfaces.

The computational design of proteins that bind small molecule ligands is one of the unsolved challenges in protein engineering. It is complicated by the relatively small size of the ligand which limits the number of intermolecular interactions. Furthermore, near-perfect geometries between interacting partners are required to achieve high binding affinities. For apolar, rigid small molecules the interactions are dominated by short-range van der Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cation-π, and π-π interactions gain importance. These partial covalent interactions are longer ranged, and additionally, their strength depends on the environment (e.g. solvent exposure). To assess the current state of protein-small molecule interface design, we benchmark the popular computer algorithm Rosetta on a diverse set of 43 protein-ligand complexes. On average, we achieve sequence recoveries in the binding site of 59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full ligand reorientation was allowed. As expected, sequence recovery correlates with ligand displacement.

Factors Influencing Use of Continuous Physiologic Monitors for Hospitalized Pediatric Patients.

OBJECTIVES: Continuous physiologic monitors (CPMs) generate frequent alarms and are used for up to 50% of children who are hospitalized outside of the ICU. Our objective was to assess factors that influence the decision to use CPMs. METHODS: In this qualitative study, we used group-level assessment, a structured method designed to engage diverse stakeholder groups. We recruited clinicians and other staff who work on a 48-bed hospital medicine unit at a freestanding children's hospital. We developed a list of open-ended prompts used to address CPM use on inpatient units. Demographic data were collected from each participant. We conducted 6 sessions to permit maximum participation among all groups, and themes from all sessions were merged and distilled. RESULTS: Participants (n = 78) included nurses (37%), attending physicians (17%), pediatric residents (32%), and unit staff (eg, unit coordinator; 14%). Participants identified several themes. First, there are patient factors (eg, complexity and instability) for which CPMs are useful. Second, participants perceived that alarms have negative effects on families (eg, anxiety and sleep deprivation). Third, CPMs are often used as surrogates for clinical assessments. Fourth, CPM alarms cause anxiety and fatigue for frontline staff. Fifth, the decision to use CPMs should be, but is not often, a team decision. Sixth, and finally, there are issues related to the monitor system's setup that reduces its utility. CONCLUSIONS: Hospital medicine staff identified patient-, staff-, and system-level factors relevant to CPM use for children who were hospitalized. These data will inform the development of system-level interventions to improve CPM use and address high alarm rates.

Nurse Responses to Physiologic Monitor Alarms on a General Pediatric Unit.

BACKGROUND: Hospitalized children generate up to 152 alarms per patient per day outside of the intensive care unit. In that setting, as few as 1% of alarms are clinically important. How nurses make decisions about responding to alarms, given an alarm's low specificity for detecting clinical deterioration, remains unclear. OBJECTIVE: Our objective was to describe how bedside nurses think about and act upon monitor alarms for hospitalized children. DESIGN, SETTING, PARTICIPANTS: This was a qualitative study that involved the direct observation of nurses working on a general pediatric unit at a large children's hospital. MEASUREMENTS: We used a structured tool that included predetermined categories to assess nurse responses to monitor alarms. Data on alarm frequency and type were pulled from bedside monitors. RESULTS: We conducted 61.3 patient-hours of observation with nine nurses, in which we documented 207 nurse responses to patient alarms. For 67% of alarms heard outside of the room, the nurse decided not to respond without further assessment. Nurses most commonly cited reassuring clinical context (eg, medical team in room), as the rationale for alarm nonresponse. The nurse deemed clinical intervention necessary in only 14 (7%) of the observed responses. CONCLUSION: Nurses rely on clinical and contextual details to determine how to respond to alarms. Few of the alarm responses in our study resulted in a clinical intervention. These findings suggest that multiple system-level and educational interventions may be necessary to improve the efficacy and safety of continuous monitoring.

Obtaining Multiple Coproducts from Red Grape Pomace via Anthocyanin Extraction and Biogas Production.

Red grape pomace (RGP), a byproduct of red wine production, is an abundant food processing waste stream in California, rich in both anthocyanins, a class of red-blue pigments, and lignocellulose. Extraction of anthocyanins and biofuel production from RGP have been investigated independently, but no research has examined employing both strategies together for maximal valorization. In this study, anthocyanins were most effectively extracted from RGP at 80 °C. Convection- and vacuum-oven drying of the pomace were found to decrease anthocyanin yield, whereas lyophilization did not significantly affect yield. Fermentable sugars were successfully separated from the crude extract via solid-phase extraction. Ionic liquid pretreatment of RGP was determined to be a nonviable option for application to anaerobic digestion. Extraction reduced biomethane output, but supplementation with the aqueous fraction of the extract mitigated much of this difference, indicating sequential extraction and fractionation of anthocyanins from RGP can minimize the impact on biofuel yields.

Immunomodulatory and Antibacterial Properties of the Chumash Medicinal Plant Trichostema lanatum.

Background: The woody shrub Trichostema lanatum Benth. (Lamiaceae) is native to Southern California and was reportedly used by the Chumash people as a disinfectant and for the treatment of rheumatism. Based on its traditional uses, this study investigated the antibacterial and immunomodulatory properties of extracts from T. lanatum.Methods: A methanolic extract of the leaves and stems of T. lanatum was tested for immunomodulatory activity by measuring the proliferation of murine macrophage cell cultures and the production of the pro-inflammatory cytokine TNF-α by the cells after treatment with T. lanatum. Antibacterial activity of the extract against a panel of six Gram-positive bacteria and two Gram-negative bacteria was evaluated using a disc-diffusion assay. Results: The T. lanatum extract inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria. Treatment of activated macrophage cell cultures with T. lanatum extract resulted in decreased proliferation of the activated macrophages and a decrease in the production of TNF-α. Conclusions: These results provide the first pharmacological support for the traditional use of T. lanatum by the Chumash people of Southern California as a disinfectant and treatment for rheumatism.

Antibacterial activity of fractions from three Chumash medicinal plant extracts and in vitro inhibition of the enzyme enoyl reductase by the flavonoid jaceosidin.

We have investigated the in vitro antibacterial bioactivity of dichloromethane-soluble fractions of Artemisia californica, Trichostema lanatum, Salvia apiana, Sambucus nigra ssp. cerulea and Quercus agrifolia Née against a ΔtolC mutant strain of Escherichia coli. These plants are traditional medicinal plants of the Chumash American Indians of Southern California. Bioassay-guided fractionation led to the isolation of three flavonoid compounds from A. californica: jaceosidin (1), jaceidin (2), and chrysoplenol B (3). Compounds 1 and 2 exhibited antibacterial activity against E. coli ΔtolC in liquid cultures. The in vitro activity of 1 against the enoyl reductase enzyme (FabI) was measured using a spectrophotometric assay and found to completely inhibit FabI activity at a concentration of 100 µM. However, comparison of minimum inhibitory concentration values for 1-3 against E. coli ΔtolC and an equivalent strain containing a plasmid constitutively expressing fabI did not reveal any selectivity for FabI in vivo.

Rosetta and the Design of Ligand Binding Sites.

Proteins that bind small molecules (ligands) can be used as biosensors, signal modulators, and sequestering agents. When naturally occurring proteins for a particular target ligand are not available, artificial proteins can be computationally designed. We present a protocol based on RosettaLigand to redesign an existing protein pocket to bind a target ligand. Starting with a protein structure and the structure of the ligand, Rosetta can optimize both the placement of the ligand in the pocket and the identity and conformation of the surrounding sidechains, yielding proteins that bind the target compound.

The Effect of Ionic Liquid Pretreatment on the Bioconversion of Tomato Processing Waste to Fermentable Sugars and Biogas.

Tomato pomace is an abundant lignocellulosic waste stream from industrial tomato processing and therefore a potential feedstock for production of renewable biofuels. However, little research has been conducted to determine if pretreatment can enhance release of fermentable sugars from tomato pomace. Ionic liquids (ILs) are an emerging pretreatment technology for lignocellulosic biomass to increase enzymatic digestibility and biofuel yield while utilizing recyclable chemicals with low toxicity. In this study, pretreatment of tomato pomace with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was investigated. Changes in pomace enzymatic digestibility were affected by pretreatment time and temperature. Certain pretreatment conditions significantly improved reducing sugar yield and hydrolysis time compared to untreated pomace. Compositional analyses suggested that pretreatment primarily removed water-soluble compounds and enriched for lignocellulose in pomace, with only subtle changes to the composition of the lignocellulose. While tomato pomace was effectively pretreated with [C2mim][OAc] to improve enzymatic digestibility, as of yet, unknown factors in the pomace caused ionic liquid pretreatment to negatively affect anaerobic digestion of pretreated material. This result, which is unique compared to similar studies on IL pretreatment of grasses and woody biomass, highlights the need for additional research to determine how the unique chemical composition of tomato pomace and other lignocellulosic fruit residues may interact with ionic liquids to generate inhibitors for downstream fermentation to biofuels.