7196 research outputs found
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Ligandability of E3 Ligases for Targeted Protein Degradation Applications.
No full textTargeted protein degradation (TPD) using proteolysis targeting chimeras (PROTACs) and molecular glue degraders has arisen as a powerful therapeutic modality for eliminating disease-causing proteins from cells. PROTACs and molecular glue degraders employ heterobifunctional or monovalent small molecules, respectively, to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade specific proteins via the proteasome. Whereas TPD is an attractive therapeutic strategy for expanding the druggable proteome, only a relatively small number of E3 ligases out of the >600 E3 ligases encoded by the human genome have been exploited by small molecules for TPD applications. Here we review the existing E3 ligases that have thus far been successfully exploited for TPD and discuss chemoproteomics-enabled covalent screening strategies for discovering new E3 ligase recruiters. We also provide a chemoproteomic map of reactive cysteines within hundreds of E3 ligases that may represent potential ligandable sites that can be pharmacologically interrogated to uncover additional E3 ligase recruiters
Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation.
No full textThe RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma. Although mechanisms and structures of MEKi bound to MEK have been described for many of these compounds, recent studies suggest that RAF/MEK complexes, rather than free MEK, should be evaluated as the target of MEKi. Here, we describe structural and biochemical studies of eight structurally diverse, clinical-stage MEKi to better understand their mechanism of action on BRAF/MEK complexes. We find that all of these agents bind in the MEK allosteric site in BRAF/MEK complexes, in which they stabilize the MEK activation loop in a conformation that is resistant to BRAF-mediated dual phosphorylation required for full activation of MEK. We also show that allosteric MEK inhibitors act most potently on BRAF/MEK complexes rather than on free active MEK, further supporting the notion that a BRAF/MEK complex is the physiologically relevant pharmacologic target for this class of compounds. Our findings provide a conceptual and structural framework for rational development of RAF-selective MEK inhibitors as an avenue to more effective and better-tolerated agents targeting this pathway
5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice
No full textAntibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Escherichia coli, and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection
Proteomic Analysis of Non-human Primate Peripheral Blood Mononuclear Cells During Burkholderia mallei Infection Reveals a Role of Ezrin in Glanders Pathogenesis
No full textBurkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1β (IL-1β). On the contrary, the expression of IL-1β receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1β production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model
Inverse probability of censoring weighting for visual predictive checks of time-to-event models with time-varying covariates
No full textWhen constructing models to summarize clinical data to be used for simulations, it is good prac-tice to evaluate the model for its capacity to reproduce the data. This can be done by means of Visual Predictive Checks (VPC), which consist of (1) several reproductions of the original study by simulation from the model under evaluation, (2) calculating estimates of interest for each simulated study and (3) comparing the distribution of those estimates with the estimate from the original study. This procedure is a generic method that is straightforward to apply, in general. Here we consider the application of the method to time to event data and consider the case when a time-varying covariate is not known or cannot be approximated after event time. In this case, simulations cannot be conducted beyond the end of the follow-up time (event or censoring time) in the original study. Thus, the simulations must be censored at the end of the follow-up time. Since this censoring is not random, the standard KM estimate from the simulated study will be biased. We propose to use inverse probability of censoring weighting (IPoC) method to cor-rect the KM estimator. For analyzing the Cantos study, the IPoC weighting as described here proved valuable and enabled qualification of PKPD models for simulations. Here, we use a gen-erated data set, which allows illustration of the different situations and checking of the implemen-tation against the known truth
Human plasma proteomic profiles indicative of cardiorespiratory fitness
No full textMaximal oxygen uptake (VO2max) is a direct measure of human cardiorespiratory fitness and is associated with health. However, the molecular determinants of interindividual differences in baseline (intrinsic) VO2max, and of increases of VO2max in response to exercise training (ΔVO2max), are largely unknown. Here, we measure ~5,000 plasma proteins using an affinity-based platform in over 650 sedentary adults before and after a 20-week endurance-exercise intervention and identify 147 proteins and 102 proteins whose plasma levels are associated with baseline VO2max and ΔVO2max, respectively. Addition of a protein biomarker score derived from these proteins to a score based on clinical traits improves the prediction of an individual’s ΔVO2max. We validate findings in a separate exercise cohort, further link 21 proteins to incident all-cause mortality in a community-based cohort and reproduce the specificity of ~75% of our key findings using antibody-based assays. Taken together, our data shed light on biological pathways relevant to cardiorespiratory fitness and highlight the potential additive value of protein biomarkers in identifying exercise responsiveness in humans
Guidelines for FAIR sharing of preclinical safety and off-target pharmacology data
No full textPre-competitive data sharing can offer the pharmaceutical industry significant benefits in terms of reducing the time and costs involved in getting a new drug to market through more informed testing strategies and knowledge gained by pooling data. Data sharing benefits can be further enhanced by applying the FAIR Guiding Principles to make data more Findable, Accessible, Interoperable and Reusable, reducing time spent curating, transforming and aggregating datasets and allowing more time for data mining and analysis. The Enhancing TRANslational SAFEty Assessment through Integrative Knowledge Management (eTRANSAFE) project is an Innovative Medicines Initiative (IMI) which aims to integrate publicly available data sources with proprietary preclinical and clinical data donated by pharmaceutical organisations participating in the consortium. A key deliverable which supports the sustainability of the project output is the development of policies and guidelines for the safe sharing of proprietary data. Beyond developing guidelines that can be practically implemented within the project and used to overcome company internal resistance to data sharing, the eTRANSAFE project also aims to publicise them in order to gain widespread adoption and reuse by other data sharing initiatives, regulatory bodies and international standard-setting organisations. This article is focussed on how the FAIR Guiding Principles can be applied to the general framework for data sharing guidelines which were developed in eTRANSAFE
Calculating qualified non-mutagenic impurity levels: Harmonization of approaches
No full textThe presence of impurities in drugs is unavoidable. As impurities offer no direct benefit to the patient, it is critical that impurities do not compromise patient safety. Current guidelines on the derivation of acceptable impurity levels leave aspects of calculations open for interpretation, resulting in inconsistencies across industry and regulators. To understand current impurity qualification practices from a safety standpoint, regulatory expectations and the safety risk that impurities pose, the IQ DruSafe Impurities Working Group (WG) conducted a pharmaceutical industry-wide survey. Survey results highlighted areas that could benefit from harmonization, including nonclinical species/sex selection and the application of adjustment factors (i.e., body surface area). Recommendations for alignment on these topics is included in this publication. Additionally, the WG collated repeat-dose toxicity information for 181 starting materials and intermediates, reflective of pharmaceutical impurities, to understand the toxicological risks they generally pose in relation to the drug substance (DS) and the assumptions surrounding the calculation of qualified impurity levels. An evaluation of this dataset and the survey were used to harmonize how to calculate a safe limit for an impurity based on toxicology testing of the impurity when present within the DS
Scientific and Regulatory Policy Committee Points to Consider*: Fixation, Trimming and Sectioning of Non-Rodent Eyes and Ocular Tissues for Examination in Ocular and General Toxicity Studies
No full textA Working Group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee conducted a technical and scientific review of current practices relating to the fixation, trimming, and sectioning of the non-rodent eye, to identify key points and species-specific anatomical landmarks to consider when preparing and evaluating eyes of rabbits, dogs, minipigs and nonhuman primates from ocular and general toxicity studies. The topics addressed in this Points to Consider article include determination of situations when more comprehensive evaluation of the globe and/or associated periocular tissues should be implemented (expanded ocular sampling), and what constitutes expanded ocular sampling. In addition, this manuscript highlights the practical aspects of trimming and sectioning the eye to ensure adequate histopathological evaluation of all major ocular structures, including the cone-dense areas (visual streak/macula/fovea) of the retina for rabbits, dogs, minipigs and nonhuman primates, which is a regulatory expectation for ocular toxicity studies
Flow Chemistry Highlights column in Chimia (monthly contribution)
No full textThe content will consist of either short articles describing the Flow Chemistry Network activities (the network is part of the Swiss Chemical Society), or flow chemistry literature mini-reviews. An upload will be made to this OAK case each time a contribution has been co-authored by myself. My regular co-author will be Agnieszka Ladosz (Idorsia), as well as Claudio Battilocchio (Syngenta)