7196 research outputs found
Sort by
Comparative study for the IMI2-NeuroDeRisk project on microelectrode arrays to derisk drug-induced seizure liability.
No full textIn the framework of the IMI2-NeuroDeRisk consortium, three in vitro electrophysiology assays were compared to improve preclinical prediction of seizure-inducing liabilities.Two cell models, primary rat cortical neurons and human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with hiPSC-derived astrocytes were tested on two different microelectrode array (MEA) platforms, Maestro Pro (Axion Biosystems) and Multiwell-MEA-System (Multi Channel Systems), in three separate laboratories. Pentylenetetrazole (PTZ) and/or picrotoxin (PTX) were included in each plate as positive (n = 3-6 wells) and ≤0.2% DMSO was used as negative controls (n = 3-12 wells). In general, concentrations in a range of 0.1-30 μM were tested, anchored, when possible, on clinically relevant exposures (unbound Cmax) were tested. Activity thresholds for drug-induced changes were set at 20%. To evaluate sensitivity, specificity and predictivity of the cell models, seizurogenic responses were defined as changes in 4 or more endpoints. Concentration dependence trends were also considered.Neuronal activity of 33 compounds categorized as positive tool drugs, seizure-positive or seizure-negative compounds was evaluated. Acute drug effects (<60 min) were compared to baseline recordings. Time points < 15 min exhibited stronger, less variable responses to many of the test agents. For many compounds a reduction and cessation of neuronal activity was detected at higher test concentrations. There was not a single pattern of seizurogenic activity detected, even among tool compounds, likely due to different mechanisms of actions and/or off-target profiles. A post-hoc analysis focusing on changes indicative of neuronal excitation is presented.All cell models showed good sensitivity, ranging from 70 to 86%. Specificity ranged from 40 to 70%. Compared to more conventional measurements of evoked activity in hippocampal slices, these plate-based models provide higher throughput and the potential to study subacute responses. Yet, they may be limited by the random, spontaneous nature of their network activity
Novartis overcomes space limitations to bring high-quality data analysis on-site
No full textThis article describes the setup and use of the cryo-EM facility in Cambridge
Quantitative and functional characterisation of extracellular vesicle loading with small molecules by passive adsorption versus cholesterol mediated membrane anchoring
No full textExtracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application
potential as biological drug delivery vehicles. Much progress has been made for loading or decorating
EVs with proteins, peptides or RNA by genetic engineering of the donor cells, but post-isolation loading
with synthetic drugs and/or using EVs from natural sources remains challenging. In particular,
quantitative and unambiguous data of whether and how small molecules associate with EVs versus
other components in the samples are still lacking. Here we describe the systematic and quantitative
characterisation of EV loading with small molecules by passive adsorption based on hydrophobic
interactions versus membrane anchoring of hydrophilic ligands via cholesterol tags. As revealed by
single vesicle imaging, both ligand types bind to CD63 positive EVs, however also unspecifically to other
vesicles, particles and serum proteins. With increasing concentrations, insoluble compounds such as
Curcumin and Terbinafine agglomerate on EVs with no apparent saturation up to 106-107 molecules
per vesicle as quantified by liquid chromatography – high resolution mass spectrometry (LC-HRMS),
forming a population of large, electron-dense vesicles detected by cryo-transmission electron
microscopy (TEM). High density EV loading resulted in reduced EV cell uptake for both compounds,
and toxic gain of function for Curcumin-EVs. In contrast, Cholesterol tagging of a hydrophilic mdm2-derived cyclic peptide saturated at densities of ca 104 – 105 molecules per vesicle, whereas lipidomics showed an addition rather than replacement of endogenous Cholesterol. Cholesterol anchored ligands did not change the EV size or morphology and retained their cell uptake activity without inducing cell toxicity, however were shed from the vesicles in presence of serum. Based on these data we conclude that (1) both methods allow loading of EVs with small molecules but are prone to unspecific compound binding to other components if present in the sample, (2) Cholesterol anchoring needs substantial
optimization of formulation stability for in vivo applications, whereas (3) careful titration of loading densities is warranted when relying on hydrophobic interactions of EVs with insoluble compounds to mitigate changes in physicochemical properties and loss of EV function and potential cell toxicity
Balancing the Objectives of Statistical Efficiency and Allocation Randomness in Randomized Controlled Trials
No full textVarious restricted randomization procedures are available to achieve equal (1:1) allocation in a randomized clinical trial. However, for some procedures, there is a nonnegligible probability of imbalance in the final numbers which may result in an underpowered study. It is important to assess such probability at the study planning stage and make adjustments in the design if needed. In this paper, we perform a quantitative assessment of the tradeoff between randomness, balance, and power of restricted randomization designs targeting equal allocation. First, we study the small-sample performance of biased coin designs with known asymptotic properties and identify a design with an excellent balance–randomness tradeoff. Second, we investigate the issue of randomization-induced treatment imbalance and the corresponding risk of an underpowered study. We propose two risk mitigation strategies: increasing the total sample size or fine-tuning the biased coin parameter to obtain the least restrictive randomization procedure that attains the target power with a high, user-defined probability for the given sample size. Our approach is simple and yet generalizable to more complex settings, including trials with stratified randomization and multi-arm trials with possibly unequal randomization ratios
Comprehensive Assessment of Pharmacokinetics, Pharmacodynamics, and Tolerability of Ligelizumab in Healthy Volunteers and Patients with Chronic Spontaneous Urticaria to Optimize Its Subcutaneous Delivery System.
No full textLigelizumab is a highly potent, humanized IgG1, anti-IgE monoclonal antibody. To explore its optimal subcutaneous delivery, the pharmacokinetics (PK), pharmacodynamics (PD), and tolerability of ligelizumab from two Phase 1 studies in healthy volunteers (HVs) and four Phase 2 and 3 studies in patients with chronic spontaneous urticaria (CSU) were assessed. Using different injection volumes or durations of a liquid-in-vial (LIVI) formulation or different formulations (LIVI vs. prefilled syringe (PFS)), single-dose ligelizumab showed comparable PK exposure in HVs. Steady-state exposure of ligelizumab was also comparable between LIVI and PFS following multiple dosing in CSU patients. The total IgE level (a PD marker) and tolerability were similar between the two formulations in both HVs and patients. Furthermore, the PK, total IgE, and tolerability were comparable for PFS administered either by patients or healthcare providers (HCPs). Collective evidence demonstrated that the injection duration or volume, formulation, or administrator had no apparent impact on the PK, PD, and tolerability of ligelizumab, supporting no clinically relevant difference between LIVI and PFS, and that PFS can be administered by patients or HCPs. This report provides a comprehensive assessment based on data of multiple clinical endpoints from both HVs and patients to inform formulation development and commercial use of a monoclonal antibody
A novel preclinical secondary pharmacology resource illuminates target-adverse drug reaction associations of marketed drugs
No full textIn vitro secondary pharmacology assays are an important tool for predicting clinical adverse drug reactions of investigational drugs. We tested 1958 marketed or withdrawn drugs using 200 safety pharmacology assays and compared our results to public and subscription resources to determine their on- and off-target effects. The aim of this study was the validation of (off)-target engagement and ADR associations. Approximately 95% of all results, and 36% of active results (AC50 < 1 µM) were unique to our database, with low quantitative agreement and bias towards higher activity on the subset of results appearing in public resources. By annotating drugs with their efficacious free maximal concentration in blood (free Cmax), we found 684 novel off-target activities at concentrations within 10 fold of free Cmax (potentially physiological). Comparing the known ADRs of drugs to assay activity revealed that 64% of putative ADRs linked to target activity in key literature reviews were not statistically significant in our dataset. Systematic analysis of all target vs. ADR pairs identified several novel associations with literature support, including α2C adrenergic receptor (ADRA2C) inhibition vs. hallucination and aggression, D3 dopamine receptor (DRD3) inhibition vs. tardive dyskinesia and progresterone receptor (PGR) agonism vs. hyperpigmentation disorders. Finally, the database was used to propose candidate mechanisms for known drug ADRs based on newly described off-target activities. Examples include M1/M2 muscarinic receptor (CHRM1/CHRM2) inhibition of trimipramine and zolpidem vs. accommodation disorder, and D1 dopamine receptor inhibition (DRD1) of citalopram vs. movement disorders. The Secondary Pharmacology Database (SPD) represents an important new resource for benchmarking ADR predictions, explaining novel phenotypic activity and investigating newly discovered clinical properties of marketed drugs
Estimating intrinsic cooperativities and concentrations of ternary complexes in biochemical or cellular environments from binary dissociation constants, apparent cooperativities and total or free ligand concentrations
No full textThere is an increasing interest to modulate difficult-to-tackle but disease-relevant target proteins by involving them with a chaperone protein into a compound-mediated ternary complex. In general, ternary complex-forming compounds can be classified by their direct affinity to chaperone and target protein and their degree of cooperativity which they exhibit upon ternary complex formation. As a trend, the smaller ternary complex-forming compounds are the stronger is the contribution of the intrinsic cooperativity to their thermodynamic stability relative to direct target (or chaperone) binding. This highlights the importance of the cooperativity as a desirable feature for the optimization of ternary complex-forming compounds – even more so as it provides higher and easier to achieve selectivity to target isoforms and allows the assessment of target occupancy via estimated concentrations of ternary complexes.
All of this emphasizes the need to quantify the natural constant of intrinsic cooperativity α which is defined as the gain (or loss) in affinity of a compound to its target in pre-bound vs. unbound state. In this publication, a workflow involving a mathematical model is presented that requires the input of the two relevant binary Kds and the two protein concentrations of target and chaperone for estimating the intrinsic cooperativity from experimentally observed apparent cooperativities. Intrinsic cooperativities are in the simplest version assessable through EC50 shifts in binary binding curve(s) of the ternary complex-forming compound with either target or chaperone relative to the same experiment but in the presence of also the counter protein.
This approach is then extended from closed systems (like biochemical assays) to the open system of a cellular assay by accounting for differences in total ligand vs. free ligand concentrations, which then allows calculating ternary complexes concentrations. Finally, this model is used to translate biochemical potency of ternary complex-forming compounds into expected cellular target occupancy, which could ultimately serve as way for validation or de-validation of hypothesized biological mechanisms of action
A high-throughput cigarette smoke-treated bronchosphere model for disease-relevant phenotypic compound screening.
No full textCigarette smoking (CS) is the leading cause of COPD, and identifying the pathways that are driving pathogenesis in the airway due to CS exposure can aid in the discovery of novel therapies for COPD. An additional barrier to the identification of key pathways that are involved in the CS-induced pathogenesis is the difficulty in building relevant and high throughput models that can recapitulate the phenotypic and transcriptomic changes associated with CS exposure. To identify these drivers, we have developed a cigarette smoke extract (CSE)-treated bronchosphere assay in 384-well plate format that exhibits CSE-induced decreases in size and increase in luminal secretion of MUC5AC. Transcriptomic changes in CSE-treated bronchospheres resemble changes that occur in human smokers both with and without COPD compared to healthy groups, indicating that this model can capture human smoking signature. To identify new targets, we ran a small molecule compound deck screening with diversity in target mechanisms of action and identified hit compounds that attenuated CSE induced changes, either decreasing spheroid size or increasing secreted mucus. This work provides insight into the utility of this bronchopshere model to examine human respiratory disease impacted by CSE exposure and the ability to screen for therapeutics to reverse the pathogenic changes caused by CSE
Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings
No full textAbstract to be written, for journal. Please find the synopsis for OAK reviewers only below:
In this publication we disclose the discovery of DCAF1 E3 ligase binding matter and the functionalization to PROTACs for TPD of BRD9, Tyrosine kinases (Dasatinib) and BTK. A lot of data has been initiated as part of the work by the ONC9LIG team (I was an integral part of). Project work on DCAF1 has been discontinued roughly 6-7 years ago (focus on BTK bifunctional degraders as ONC9BTK team). All compounds (IP inventory) have been shared with GDC in a previous email and the proposal to publish DCAF1 binders as well as the DCAF1-degraders was formally approved in August 2021 by the TPD steering committee including Jay and Karin.
We disclose 5 key messages:
• Discovery of DCAF1 as an essential ligase (DepMAP) and hypothesis to overcome CBRN mediated resistance. Leads into non-covalent DCAF1 binder discovery and characterisation, which can be functionalized for TPD for PROTACs (Figure 1)
• With a first degrader prototype we do show that endogenous mainly nuclear ligase DCAF1 can degrade BRD9 (BRD9-PROTAC, another nuclear protein. Key message is around in-depth chemical and genetic validation (Figure 2)
• We expand the degradation space with a second more promiscuous degrader prototype against tyrosine kinases (Dasatinib-DCAF1 PROTAC) and show that DCAF1 can even tackle cytoplasmic as well as membrane bound tyrosine kinases (Figure 3) – we touch here also on the tox observation
• We expand this further to an ONC target BTK with more specific PROTACs (linear and more complex linkers) that we characterize in-depth (Figure 4), here we describe a very systematic in vitro approach and show that unfortunately there is not a simple correlation between in vitro findings (complex, ubi etc.) and cellular degradation potency (as seen by many but not really worked up so systematically). Here we build up the path towards discovery of our most potent DCAF1-BTK degrader (JRF)
• JRF449 characterization and ultimate rescue experiment in CBRN-BTK PROTAC resistant cells where we do see BTK degradation and effect in proliferation with a DCAF1-BTK degrader even in CRBN resistant settings (in finalization) (draft Figure 5)
Key internal flags towards DCAF1 as an alternative ligase that led to termination were and still are:
• Discovery of only 1 chemical series (to be published in accompanying manuscript OAK 49705)
• This chemical series shows pan toxicity at >5uM (CLiP studies >300 cell lines tested revealed this)
o Rescue experiments to understand if this is on-target toxicity remained unsuccessful (Focused mutagenesis around donut-hole binding site, whole gene Variomics, endogenous gene editing around donut-hole locus)
o Until now tough we do not have a clear answer if this is off or on-target tox (DCAF1 is an essential ligase)
• We comment on these observations of tox briefly in the manuscript
• In addition, even tough we had extensive efforts on DCAF1-BTK degraders (50+ molecules) we never reached a potency range compared to CRBN-BTK (in-house) or (VHL, external). Our best degrader JRF449 (in publication has a DC50 of about 100nM)
o Literature also suggests that DCAF1 is somehow in a tetrameric self-inhibitory mode
We have had discussions with GDC colleagues concerning the chemical matter to be published in this manuscript and got verbal consent that the compounds can be disclosed. GDC decided not to patent the DCAF1 binders on their own and all the other elements of the PROTACs in this manuscript, thelinkers and war-heads are covered by own patents (BTK-binders and linkers) or in the public domain (BRD9 binder and Dasatinib)
Inhibiting WNT secretion reduces high bone mass caused by Sost loss-of-function or gain-of-function mutations in Lrp5.
No full textProper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5–6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models