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ON THE USE OF WEARABLE SENSORS AS MOBILITY BIOMARKERS IN THE MARKETING AUTHORISATION OF NEW DRUGS: A REGULATORY PERSPECTIVE
No full textThe loss of mobility is a common trait in multiple health conditions (e.g., Parkinson’s disease)
and is associated with reduced quality of life. In this context, being able to monitor mobility
in the real world is important. Until recently, the technology was not mature enough for this;
but today, miniaturised sensors and novel algorithms promise to monitor mobility accurately
and continuously in the real world, also in pathological populations.
However, before any such methodology can be employed to support the development and
testing of new drugs in clinical trials, they need to be qualified by the competent regulatory
agencies (e.g., European Medicines Agency). Nonetheless, to date, only very narrow scoped
requests for regulatory qualification were successful.
In this work, the Mobilise-D Consortium shares its positive experience with the European
regulator, summarising the two requests for Qualification Advice for the Mobilise-D
methodologies submitted in October 2019 and June 2020, as well as the feedback received,
which resulted in two Letters of Support publicly available for consultation on the website of
the European Medicines Agency. Leveraging on this experience, we hereby propose a refined
qualification strategy for the use of digital mobility outcome (DMO) measures as monitoring
biomarkers for mobility in drug trials
Cooperative assembly and structure of the RAS-SHOC2-PP1c holophosphatase provides mechanistic insights into disease-relevant mutations
No full textThe RAS-RAF-MAPK signaling pathway is fundamental for cell proliferation and altered in a
majority of human cancers. However, our mechanistic understanding of how RAS proteins
activate RAF kinases is still incomplete. Recent elegant studies revealed structural details on an
inhibitory RAF complex with 14-3-3, although steps leading to RAF
activation remain unclear. As a potential activator of RAF, MRAS-SHOC2-PP1c holophosphatase
activity has been postulated to de-phosphorylate RAF on a specific Serine residue resulting in 14-
3-3 release enabling RAF activation. Despite its importance, to date structural evidence for such a
holophosphatase complex and its organization is missing. Here we reveal a 1.95 Å high-resolution
X-ray crystal structure of the ternary MRAS-SHOC2-PP1c complex showing SHOC2 enclosing in
its concave side both PP1c and MRAS. Biophysical characterization indicates a cooperative
assembly requiring active GTP-loaded MRAS, an observation we could expand to other RAS
isoforms, measuring different affinities. Our findings support a RAS-driven and multimeric model
for RAF activation in which two neighboring RAS-GTP molecules can simultaneously recruit RAF-
14-3-3 and SHOC2-PP1c to produce downstream pathway activation. Finally, MRAS, SHOC2 and
PP1c are mutated in Rasopathies, a spectrum of developmental syndromes caused by aberrant
MAPK pathway activation and SHOC2 itself has recently emerged as potential target in RTK-RAS
driven tumors. We find that Rasopathy mutations reside at protein-protein interfaces within the
holophosphatase and result in affinity changes. Collectively our findings shed light on a
fundamental mechanism of RAS biology and on mechanisms for clinically observed enhanced
RAS-MAPK signaling, thus providing the structural basis for therapeutic interventions
Meeting report of the second European Biotransformation Workshop
No full textChallenges and opportunities in the field of biotransformation were presented and discussed at the 2nd European Biotransformation workshop which was conducted virtually in collaboration with the DMDG November 24/25, 2021. Here we summarise the presentations and discussions from this workshop.
The following topics were covered:
1) Regulatory requirements and biotransformation studies for ADCs and ASOs
2) Solutions for mass spectral data processing of peptides and oligonucleotides
3) Future outsourcing needs in biotransformation for new modalities
4) Established quantitative and qualitative workflows for metabolite identification
5) In vitro and microphysiological systems to study new chemical entities (NCEs) with low metabolic turnover
6) New strategies to frontload the human ADME study and to investigate the impact of human microbiome on drug development
Keywords: Biotransformation of new modalities, ADCs, ASOs, metabolite quantitation and qualification, microphysiological systems, human ADME, microbiom
A proteogenomic signature of age-related macular degeneration in blood.
No full textAge-related macular degeneration (AMD) is one of the most common causes of visual impairment in the elderly, with a complex and still poorly understood etiology. Whole-genome association studies have discovered 34 genomic regions associated with AMD. However, the genes and cognate proteins that mediate the risk, are largely unknown. In the current study, we integrate levels of 4782 human serum proteins with all genetic risk loci for AMD in a large population-based study of the elderly, revealing many proteins and pathways linked to the disease. Serum proteins are also found to reflect AMD severity independent of genetics and predict progression from early to advanced AMD after five years in this population. A two-sample Mendelian randomization study identifies several proteins that are causally related to the disease and are directionally consistent with the observational estimates. In this work, we present a robust and unique framework for elucidating the pathobiology of AMD
Unbiased screen for pathogens in human paraffin-embedded tissue samples by whole genome sequencing and metagenomics.
No full textIdentification of bacterial pathogens in formalin fixed, paraffin embedded (FFPE) tissue samples is limited to targeted and resource-intensive methods such as sequential PCR analyses. To enable unbiased screening for pathogens in FFPE tissue samples, we established a whole genome sequencing (WGS) method that combines shotgun sequencing and metagenomics for taxonomic identification of bacterial pathogens after subtraction of human genomic reads. To validate the assay, we analyzed more than 100 samples of known composition as well as FFPE lung autopsy tissues with and without histological signs of infections. Metagenomics analysis confirmed the pathogenic species that were previously identified by species-specific PCR in 62% of samples, showing that metagenomics is less sensitive than species-specific PCR. On the other hand, metagenomics analysis identified pathogens in samples, which had been tested negative for multiple common microorganisms and showed histological signs of infection. This highlights the ability of this assay to screen for unknown pathogens and detect multi-microbial infections which is not possible by histomorphology and species-specific PCR alone
Raman marker bands for secondary structure changes of frozen therapeutic monoclonal antibody formulations during thawing
No full textIn this work we use Raman spectroscopy for protein characterization in the frozen state. We investigate the behavior of frozen therapeutic monoclonal antibody IgG1 formulation upon thawing by Raman spectroscopy. Secondary and tertiary structure of the protein in three different mab formulations in the frozen state are followed through observation of marker bands for α-helix, β-sheet and random coil. We identify the tyrosine intensity ratio I856/I830 as a marker for mab aggregation. Upon fast cooling (40°C/min) to –80°C we observe a significant increase of random coil and α –helical structures, while this is not the case for slower cooling (20°C/min) to –80°C. Most changes in the protein's secondary structure are observed in the course of thawing in the range up to -20°C, when passing through the glass transitions and cold-crystallization of the two types of freeze-concentrated solutions formed through macro- and microcryoconcentration. An increase of protein concentration and the addition of mannitol suppress secondary structural changes but do no impact on aggregation
Controlled Delivery of Corticosteroids Using Tunable Tough Adhesives.
No full textHydrogel-based drug delivery systems typically aim to release drugs locally to tissue in an extended manner. Tissue adhesive alginate-polyacrylamide tough hydrogels are recently demonstrated to serve as an extended-release system for the corticosteroid triamcinolone acetonide. Here, the stimuli-responsive controlled release of triamcinolone acetonide from the alginate-polyacrylamide tough hydrogel drug delivery systems (TADDS) and evolving new approaches to combine alginate-polyacrylamide tough hydrogel with drug-loaded nano and microparticles, generating composite TADDS is described. Stimulation with ultrasound pulses or temperature changes is demonstrated to control the release of triamcinolone acetonide from the TADDS. The incorporation of laponite nanoparticles or PLGA microparticles into the tough hydrogel is shown to further enhance the versatility to control and modulate the release of triamcinolone acetonide. A first technical exploration of a TADDS shelf-life concept is performed using lyophilization, where lyophilized TADDS are physically stable and the bioactive integrity of released triamcinolone acetonide is demonstrated. Given the tunability of properties, the TADDS are a suggested technology platform for controlled drug delivery
Understanding the chronic kidney disease landscape using patient representation learning from electronic health records
No full textUnderstanding various subpopulations in chronic kidney disease can improve patient care and aid in developing treatments targeted to patients’ needs. Due to the general slow disease progression, electronic health records, which comprise a rich source of longitudinal real-world patient-level information, offer an approach for generating insights into disease. Here we apply the open-source ConvAE framework to train an unsupervised deep learning network using a real-world kidney disease cohort consisting of 2.2 million US patients from the OPTUM® EHR database. Numerical patient representations derived from ConvAE are used to derive disease subtypes, inform comorbidities and understand rare disease populations. To identify patients at high risk to develop end-stage kidney disease, we extend a validated algorithm classifying disease severity to hypothesize subpopulations of rapid chronic kidney disease progressors. We demonstrate that using a combination of data-driven methods offers a powerful exploratory approach to understand disease heterogeneity and identify high-risk patients who could be targeted for early therapeutic intervention to prevent end-stage kidney disease
Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms.
No full textWith the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance
