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Interferon regulatory factor 5 and nuclear factor kappa-B exhibit cooperating but also divergent roles in the regulation of pro-inflammatory cytokines important for the development of TH1 and TH17 responses
No full textA large body of data demonstrates that interferon regulatory factor 5 (IRF5) and nuclear factor kappa B (NF-κB) are the two major transcription factors in classically activated macrophages responsible for the transcriptional control of proinflammatory genes. Although recent evidence suggests that IRF5 interacts with certain members of the nuclear factor kappa B pathway, the extent of cooperation and its implications in disease are ambiguous. Since both pathways are known for their strong contributions in TLR8 signaling we used the human monocytic cell line THP-1.Dual, featuring gene reporters for NF-κB and IRFs, to simultaneously study the roles of IRF5 and the NF-κB subunit p65 in TLR8-mediated gene reporter activities. Furthermore, we profiled from these cells the proinflammatory cytokines involved in the differentiation of TH1 and TH17 cells. After ablation of IRF5 and/or p65 we activated the resultant cells with the TLR8 agonists R848 or the psoriasis-associated antimicrobial peptide LL-37 complexed with ssRNA and demonstrate that IRF5 deficiency drastically impairs the secretion of IL-1β, IL-6, IL-12, IL-23 and TNFα. In contrast, the lack of p65 impaired only IL-6, IL-12, and IL-23 secretion. Furthermore, we discovered that upon TLR8 stimulation, IRF5 but not NF-κB signaling is essential to provide a cytokine milieu supporting TH1 responses. Additionally, we demonstrate that IRF5 and NF-κB cooperate to provide a cytokine milieu supporting TH17 responses. Therefore, the distinct role of IRF5 in the intricate signaling network downstream of TLR8 may open new treatment options interfering with but not disrupting NF-κB signaling in human diseases
In Vitro Monitoring of the Mitochondrial Beta-Oxidation Flux of Palmitic Acid and Investigation of Its Pharmacological Alteration by Therapeutics
No full textBackground and Objective: The present study was designed to validate the functional assay that enables rapid screening of therapeutic candidates for their effect on mitochondrial fatty acid oxidation. Methods: The two whole-cell systems (tissue homogenates and hepatocytes) have been evaluated to monitor the total beta-oxidation flux of physiologically important 3H-palmitic acid by measurement of tritiated water enrichment in incubations using UPLC coupled on-line to radioactivity monitoring and mass spectrometry. Results: Our results with several known inhibitors of fatty acid oxidation showed that this simple assay could correctly predict a potential in alteration of mitochondrial function by drug candidates. Since the beta-oxidation of palmitic acid takes place almost exclusively in mitochondria of human hepatocytes, this model can be also utilized to distinguish between the mitochondrial and peroxisomal routes of this essential metabolic pathway in some cases. Conclusions: The present work offers a new in vitro screen of changes in mitochondrial beta-oxidation by xenobiotics as well as a model to study the mechanism of this pathway
A phase I/II study to evaluate the safety, tolerability and early efficacy of MGV354 in healthy subjects and in patients with ocular hypertension or glaucoma
No full textPurpose: To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma.
Design: Double-masked, prospective, randomized and vehicle-controlled three-part multicenter study with 98 subjects (ClinicalTrials.gov NCT02743780).
Methods: Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2). Part 3 evaluated IOP-lowering efficacy of the MTD administered nightly for one week in 50 patients with minimum IOP of 24mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at Day 8 compared to baseline.
Results: There was no difference in favor of MGV354 for IOP lowering; change from Baseline to Day 8 in mean diurnal IOP was -0.6 mmHg for MGV354-treated patients and -1.1 mmHg for Vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common AEs reported following MGV354 administration in all the three parts was conjunctival and ocular hyperemia.
Conclusions: Overall, MGV354 0.1% demonstrated no statistically significant effect compared to Vehicle in lowering IOP based upon the study’s main outcome measure. MGV354 produced ocular hyperemia consistent with target engagement in the conjunctiva, but human glaucomatous trabecular meshwork may have levels of oxidized sGC that are too low to benefit from MGV354
Postdoc position advertisement
No full textPostdoctoral position in Computational Sciences, Basel
We are currently seeking candidates for a postdoctoral position for a computational project. We are characterizing fibrotic processes in the liver in the context of non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) with the full range of discovery technologies available: both single cell and bulk transcriptomic sequencing of patient-derived material, genome-wide CRISPR/Cas9 genetic screening, low molecular weight compound screening, preclinical animal models and organoid systems. Concurrently, we characterize the biology of non-healing topical wounds (e.g. diabetic foot ulcer). Focusing on the biological processes involved, it is emerging that the biology at the core of fibrosis, wound healing, and some connective tissue disorders, is conserved.
The aim of the research project is to better understand the pathophysiology of extracellular matrix biology in the context of fibrotic disease. We will use all appropriate data of sufficient quality to generate a comprehensive understanding of conserved homeostatic and disease processes of relevance to fibrosis across different organs. These data will allow us to derive tissue-specific disease signatures that we then use to identify potential novel approaches for pharmacological modulation (e.g., computational repurposing) of the malfunctioning processes and pathways, and we will pursue experimental validation of these.
This research project is highly interdisciplinary, and combines data from current state-of-the-art technologies. Through the application of advanced computational analysis of extracellular matrix biology we aim to define new starting points for drug discovery
Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition
No full textThe role of KRAS, when activated through canonical mutations, has been well established in cancer. Here we explore a secondary means of KRAS activation in cancer, focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas. KRAS amplified gastric cancer models possess marked overexpression of KRAS protein and are insensitive to MAPK blockade due to their capacity to adaptively respond by rapidly increasing KRAS-GTP levels. We demonstrate that inhibition of guanine exchange factors SOS1/2 or protein tyrosine phosphatase, SHP2, can attenuate this adaptive process and that targeting of these factors, both genetically and pharmacologically, can enhance sensitivity of KRAS-amplified models to MEK inhibition both in in vitro and in vivo settings. These data demonstrate amplification to be an under-recognized means of KRAS activation, that tumors with these amplifications have unique adaptive physiology and that SOS or SHP2 blockade has potential to be a critical component of new therapies for these cancers.
SAMPL6: pKa prediction blind challenge submission
No full textIn a collaboration with Prof. Stefan Grimme (Bonn University, Germany) we will submit predicted pKa (dissociation constant) values for the pKa prediction part of the SAMPL6 challenge (https://drugdesigndata.org/about/sampl6/pka-prediction).
The SAMPL6 challenge is a public challenge where participants are invited to submit predictions of microscopic and macroscopic pKa values of 24 small molecules. The structures of the small molecules are in the public domain and can be found here: https://github.com/MobleyLab/SAMPL6/blob/pKa/images/pKa_challenge_small_molecules.jpg .
We will apply the conformational workflow ReSCoSS (previously externally presented as OAK IDs 32054 and 34000) to this challenge.
All molecules treated are in the public domain and the tools used have all been previously published. In line with the concept of the SAMPL6 challenge, we plan to submit the predicted values to the SAMPL6 challenge organizers, and for generating them we are collaborating with Prof. Grimme's group
Beta secretase BACE1 promotes surface expression and function of Kv3.4 potassium channels in hippocampal mossy fiber synapses
No full textIn the brain, expression of BACE1 is high during development and declines subsequently. One remarkable exception is the hippocampus, in parts of which BACE1 levels remain elevated into adulthood. The sites of elevated BACE1 in mature hippocampus show a striking overlap with those of the voltage-gated K+ channel 3.4 (Kv3.4), which has also been linked to AD. This presynaptically located K+ channel gives rise to fast activating and inactivating currents that serve to repolarize action potentials that invade the terminals, thereby shaping the kinetics of transmitter release. In view of the prominent parallel enrichment of BACE1 and Kv3.4 in the hippocampus, we investigated whether BACE1 plays a role in Kv3.4 expression and function. Our main findings were:
1) BACE1 and Kv3.4 strictly co-localize in the mossy fiber tract of the hippocampus.
2) Kv3.4-dependent synaptic transmission is altered in BACE1 knockout mice.
3) Hippocampal cell surface levels and synaptic levels (Figure 1) of Kv3.4 are reduced in BACE1 knockout mice.
4) In a cell line, co-expressed BACE1 drastically increases Kv3.4 channel density at the plasma membrane when proteolytically active and, most importantly, also when pharmacologically rendered inactive.
5) BACE1 was immunoprecipitated (IP) with Kv3.4 and vice versa in co-IP experiments in the expression system.
The last two points argue in favor of a non-proteolytic interaction between BACE1 and Kv3.4 as already demonstrated for other K+ channels (Agsten et al., 2015; Hessler et al., 2015). We therefore hypothesized that pharmacological inhibition of the enzymatic activity of BACE1 should not affect its interaction with Kv3.4. This hypothesis, however, was based on over- expression experiments in cell lines. To substantiate the notion that BACE1 interacts with Kv3.4 in a non-enzymatic manner, we planned to investigate synaptic Kv3.4 protein level in C57BL/6 mice which were either fed with BACE1 inhibitor NB-360 containing food pellets or with control pellets for four week
Discovery and design of first benzylamine-based ligands binding to an unlocked conformation of the Complement Factor D
No full textComplement Factor D, a serine protease of the S1 family and key component of the alternative pathway amplification loop, represents a promising target for the treatment of several prevalent and rare diseases linked to the innate immune system. Previously reported FD inhibitors have been shown to bind to the FD active site in its self-inhibited conformation characterized by the presence of a salt bridge at the bottom of the S1 pocket between Asp189 and Arg218. We report herein a new set of small-molecule FD ligands that harbor a basic S1 binding moiety directly binding to the carboxylate of Asp189, thereby displacing the Asp189-Arg218 ionic interaction and significantly changing the conformation of the self-inhibitory loop
In vitro activity of LYS228, a novel monobactam antibiotic, against multidrug-resistant enterobacteriaceae
No full textLYS228 is a novel monobactam with potent activity against Enterobacteriaceae. LYS228 is stable to metallo-β-lactamases (MBLs) and serine carbapenemases, including Klebsiella pneumoniae carbapenemases (KPCs), resulting in potency against the majority of extended-spectrum β-lactamase (ESBL)-producing and carbapenemresistant Enterobacteriaceae strains tested. Overall, LYS228 demonstrated potent activity against 271 Enterobacteriaceae strains, including multidrug-resistant isolates. Based on MIC90 values, LYS228 (MIC90, 1 μg/ml) was ≥32-fold more active against those strains than were aztreonam, ceftazidime, ceftazidime-avibactam, cefepime, and meropenem. The tigecycline MIC90 was 4 μg/ml against the strains tested. Against Enterobacteriaceae isolates expressing ESBLs (n = 37) or displaying carbapenem resistance (n = 77), LYS228 had MIC90 values of 1 and 4 μg/ml, respectively. LYS228 exhibited potent bactericidal activity, as indicated by low minimal bactericidal concentration (MBC) to MIC ratios (MBC/MIC ratios of ≤4) against 97.4% of the Enterobacteriaceae strains tested (264/271 strains). In time-kill studies, LYS228 consistently achieved reductions in CFU per milliliter of 3 log10 units (≥99.9% killing) at concentrations ≥4× MIC for Escherichia coli and K. pneumoniae reference strains, as well as isolates encoding TEM-1, SHV-1, CTX-M-14, CTX-M-15, KPC-2, KPC-3, and NDM-1 β-lactamases