7196 research outputs found
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Discovery of novel Fe(II)/α-ketoglutarate-dependent dioxygenases for oxidation of L-proline
No full textGenome-mining for novel Fe(II)/α-ketoglutarate-dependent dioxygenases (αKGDs) to expand the enzymatic repertoire in the oxidation of L-proline is reported. Through clustering of genes, we predicted regio- and stereoselectivity in hydroxylation reaction and validated this hypothesis experimentally. Two novel by-products in reactions with BcePH and Ssp5PH were observed, isolated and structure was determined as an epoxide and a 3,4-diol, respectively. Mechanism for formation of epoxide is suggested and validated by using 18O-labelling experiment, that proceeds via cis-3-hydroxylation step first, followed by ring closure. A Biocatalytic step was performed on sub-gram quantities of starting material without any significant condition optimization. The substrate concentration, however, is already up to 40-fold higher than the usually reported titers for P450-mediated hydroxylations, showing the synthetic potential of αKGDs on preparative scal
Polar/Apolar Interfaces Modulate the Conformational Behavior of Cyclic Peptides with Impact on Their Passive Membrane Permeability
No full textCyclic peptides have the potential to vastly extend the scope of druggable proteins
and lead to new therapeutics for currently untreatable diseases. However, cyclic pep-
tides often su�er from poor bioavailability. To uncover design principles for permeable
cyclic peptides, a promising strategy is to analyze the conformational dynamics of the
peptides using molecular dynamics (MD) and Markov state models (MSMs). Previous
MD studies have focused on the conformational dynamics in pure aqueous or apolar
environments to rationalize membrane permeability. However, during the key steps of
the permeation through the membrane, cyclic peptides are exposed to interfaces be-
tween polar and apolar regions. Recent studies revealed that these interfaces constitute
the free energy minima of the permeation process. Thus, a deeper understanding of the
behavior of cyclic peptides at polar/apolar interfaces is desired. Here, we investigate
the conformational and kinetic behavior of cyclic decapeptides at a water/chloroform
interface using unbiased MD simulations and MSMs. The distinct environments at the
interface alter the conformational equilibrium as well as the interconversion kinetics of
cyclic peptide conformations. For peptides with low population of the permeable con-
formation in aqueous solution, the polar/apolar interface facilitates the interconversion
to the closed conformation, which is required for membrane permeation. Comparison
to unbiased MD simulations with a POPC bilayer reveals that not only the conforma-
tions but also the orientations are relevant in a membrane system. These �ndings allow
us to propose a permeability model that includes both 'prefolding' and 'non-prefolding'
cyclic peptides - an extension that can lead to new design considerations for permeable
cyclic peptides
Cultural Diversity Drives Innovation: Does Institutional Residence Time Impact Behaviors?
No full textnnovation activities in large organizations are typically conducted by
teams. Previous research noted the positive correlation between innovation
performance and the cultural diversity of teams, wherein people from different
backgrounds approach problems differently and have differing tolerances for
risk. In a long term extension of these studies we aim to determine if these
proclivities attenuate over time, as members modify & harmonize their behaviors
driven by cultural norms of the organization. In an early read out from this effort,
cohorts of innovation team members across several continents and representing
six of the ten global cultural clusters completed a series of team analytics and
questionnaires. The analytics were derived from cross-cultural communication
frameworks which have been utilized to assess how culturally associated values
influence behavioral traits. The respondents invited to participate were directly
involved in innovation projects either as part of their main function or through
membership of a specific innovation team and represented a range of experience
levels. Subjects were also invited to offer written commentary on team and
organizational culture as it applies to innovation. A definitive trend was
uncovered wherein employee service time (in years) correlated with moves from
cultural group norms towards more moderated, centrist decision making traits
and lowered risk taking appetite. Further, specific indicators which correlate to
disruptive ideation and innovation performance softened as a function of service
time, independent of cultural origins. Together, this may signal a need for
innovation teams to be mindful that balance is maintained with respect to
members service time and new team entrants are supported to pursue high-risk
high-reward ideas
SomaScan Proteomics Profiling - An Overview for (potential) External Collaborators
No full textno abstrac
Discovery of small molecules that target a tertiary-structured RNA.
No full textThere is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs
Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy.
No full textGlycation is a non-enzymatic and spontaneous post-translational modification (PTM) generated by the reaction between reducing sugars and primary amine groups within proteins. Because glycation can alter the properties of proteins, it is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) and should therefore be carefully monitored. The most abundant product of glycation is formed by glucose and lysine side chains resulting in fructoselysine after Amadori rearrangement. In proteomics, which routinely uses a combination of chromatography and mass spectrometry to analyze PTMs, there is no straight-forward way to distinguish between glycation products of a reducing monosaccharide and an additional hexose within a glycan, since both lead to a mass difference of 162 Da.To verify that the observed mass change is indeed a glycation product, we developed an approach based on 2D NMR spectroscopy spectroscopy and full-length protein samples denatured using high concentrations of deuterated urea.The dominating β-pyranose form of the Amadori product shows a characteristic chemical shift correlation pattern in 1H-13C HSQC spectra suited to identify glucose-induced glycation. The same pattern was observed in spectra of a variety of artificially glycated proteins, including two mAbs, as well as natural proteins.Based on this unique correlation pattern, 2D NMR spectroscopy can be used to unambiguously identify glucose-induced glycation in any protein of interest. We provide a robust method that is orthogonal to MS-based methods and can also be used for cross-validation
When to Extend Monitoring of Anti-drug Antibodies for High-risk Biotherapeutics in Clinical Trials: an Opinion from the European Immunogenicity Platform.
No full textThe determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit
Polyester-based long acting injectables: Advancements in molecular dynamics simulation and technological insights.
No full textLong-acting injectable (LAI) delivery technologies have enabled the development of several pharmaceutical products that improve patient health by delivering therapeutics from weeks to months. Over the last decade, due to its good biocompatibility, formulation tunability, wide range of degradation rates, and extensive clinical studies, polyester-based LAI technologies including poly(lactic-co-glycolic acid) (PLGA) have made substantial progress. Herein, we discuss PLGA properties with seminal approaches in the development of LAIs, the role of molecular dynamic simulations of polymer-drug interactions, and their effects on quality attributes. We also outline the landscape of various advanced PLGA-based and a few non-PLGA LAI technologies; their design, delivery, and challenges from laboratory scale to preclinical and clinical use; and commercial products incorporating the importance of end-user preferences
Evaluation of Ambient Sound, Vibration, and Light in Rodent Housing Rooms
No full textExcessive sound, vibration, and light are detrimental to rodent welfare, yet these parameters are rarely recorded in vivaria. Whether housing environments exceed the suggested thresholds and which specific factors may alter these parameters is generally unknown. The goal of this study was to determine how environmental factors may alter sound, vibration, and light at the room and cage levels. Measurements were made using an ultrasonic microphone, accelerometer, and light sensor. Measurement sites were 1) in open air at a central location in 64 rooms located in 9 buildings, and 2) inside an empty mouse or rat cage containing chow, water, and bedding and located on an animal transfer station (n = 51) or housing rack (n = 102). Information collected for each transfer station and rack measurement included the year of manufacture, the species on the rack, and the number of cages on the rack. For each location, a baseline measurement was taken with the transfer station turned off, followed by another measurement after the transfer station was turned on. In general, many factors influenced ambient sound, vibration, and light, indicating that values are not uniform across rodent rooms in the same institution or across cages in a single room. Sound peaks capable of startling rodents were measured in association with hallway ultrasonic motion sensors and during cage change. Vibration and light intensity were generally low when cages were located on the rack. In contrast, active transfer stations had more vibration and light intensity, reaching levels that were potentially stressful for rodents. These data reflect the ambient sound, vibration, and light that rodents experience during normal facility operations. These patterns may extend to other locations, but given the variability in all parameters, the data highlight the need for institutions to conduct their own monitoring
Contribution of machine learning to tumor growth inhibition modeling for hepatocellular carcinoma patients under Roblitinib (FGF401) drug treatment.
No full textMachine learning (ML) opens new perspectives in identifying predictive factors of efficacy among a large number of patients' characteristics in oncology studies. The objective of this work was to combine ML with population pharmacokinetic/pharmacodynamic (PK/PD) modeling of tumor growth inhibition to understand the sources of variability between patients and therefore improve model predictions to support drug development decisions. Data from 127 patients with hepatocellular carcinoma enrolled in a phase I/II study evaluating once-daily oral doses of the fibroblast growth factor receptor FGFR4 kinase inhibitor, Roblitinib (FGF401), were used. Roblitinib PKs was best described by a two-compartment model with a delayed zero-order absorption and linear elimination. Clinical efficacy using the longitudinal sum of the longest lesion diameter data was described with a population PK/PD model of tumor growth inhibition including resistance to treatment. ML, applying elastic net modeling of time to progression data, was associated with cross-validation, and allowed to derive a composite predictive risk score from a set of 75 patients' baseline characteristics. The two approaches were combined by testing the inclusion of the continuous risk score as a covariate on PD model parameters. The score was found as a significant covariate on the resistance parameter and resulted in 19% reduction of its variability, and 32% variability reduction on the average dose for stasis. The final PK/PD model was used to simulate effect of patients' characteristics on tumor growth inhibition profiles. The proposed methodology can be used to support drug development decisions, especially when large interpatient variability is observed