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A genome-wide microRNA screen identifies the microRNA-183/96/182 cluster as a modulator of circadian rhythms
No full textThe regulatory mechanisms of circadian rhythms have been studied primarily at the level of the transcription-translation feedback loops of protein-coding genes. Regulatory modules involving noncoding RNAs are less thoroughly understood. In particular, emerging evidence has revealed the important role of microRNAs (miRNAs) in maintaining the robustness of the circadian system. To identify miRNAs that have the potential to modulate circadian rhythms, we conducted a genome-wide miRNA screen using U2OS luciferase reporter cells. Among 989 miRNAs in the library, 120 changed the period length in a dose-dependent manner. We further validated the circadian regulatory function of an miRNA cluster, miR-183/96/182, both in vitro and in vivo. We found that all three members of this miRNA cluster can modulate circadian rhythms. Particularly, miR-96 directly targeted a core circadian clock gene, PER2. The knockout of the miR-183/96/182 cluster in mice showed tissue-specific effects on circadian parameters and altered circadian rhythms at the behavioral level. This study identified a large number of miRNAs, including the miR-183/96/182 cluster, as circadian modulators. We provide a resource for further understanding the role of miRNAs in the circadian network and highlight the importance of miRNAs as a genome-wide layer of circadian clock regulation
Population Pharmacokinetic Analysis of Fevipiprant in Healthy Subjects and Asthma Patients using a Tukey's g-and-h Distribution
No full textAim The objective of this analysis was to characterize the population pharmacokinetics (PK) of fevipiprant in asthma patients and to evaluate the effect of baseline covariates on the PK of fevipiprant. Methods PK data from 1281 healthy subjects or asthma patients were available after single or once daily dosing of fevipiprant. Population PK analysis was conducted to describe fevipiprant plasma concentration data using a non-linear mixed effect modeling approach. Results Fevipiprant PK was described by a two-compartment model with first-order absorption and first-order elimination. Exploration of fevipiprant PK in the population from the phase III studies revealed an over-dispersed and skewed distribution. This unusual distribution was described using Tukey's g-and-h distribution (TGH) on the between-subject variability of apparent clearance (CL/F). The model identified a significant impact of disease status on CL/F, with the value in healthy subjects being 62% higher than that in asthma patients. Bodyweight, age and renal function showed statistically significant impact on fevipiprant clearance; however, compared with a typical asthma patient, the simulated difference in steady-state exposure was at most 16%. Conclusion Fevipiprant PK was described by a two-compartment model with first-order absorption and first-order elimination. The TGH distribution was appropriate to describe the over-dispersed and skewed PK data as observed in the current studies. Asthma patients had approximately 37% higher exposure than healthy subjects did. Other covariates changed exposure by at most 16%
Safe, Scalable, Inexpensive, and Mild Nickel-Catalyzed Migita-like C–S Cross-Couplings in Recyclable Water
No full textA new approach to C–S couplings is reported that relies on nickel catalysis under mild conditions, enabled by micellar catalysis in recyclable water as reaction medium. The protocol tolerates a wide range of heteroaromatic halides and thiols, including alkyl and heteroaryl thiols, leading to a variety of thioethers in good isolated yields. The method is scalable, results in low residual metal in the products, and is applicable to syntheses of targets in the pharmaceutical area. The procedure also features an associated low E Factor, suggesting a far more attractive entry than is otherwise currently available, especially those based on unsustainable loadings of Pd catalysts
FZD6 triggers Wnt–signalling driven by WNT10BIVS1 expression and highlights new targets in T cell acute lymphoblastic leukemia
No full textWnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute
leukemia establishment. In our previous work we described a recurrent rearrangement involving the
WNT10B locus (WNT10BR), characterized by the expression of WNT10BIVS1 transcript variant, in
acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic
leukemia (T-ALL), we retrospectively analysed an Italian cohort of patients (n=20) and detected a
high incidence (13/20) of WNT10BIVS1 expression. To address genes involved in WNT10B
molecular response, we have designed a Wnt targeted RNA sequencing panel. Identifying Wnt
agonists and antagonists, it results that the expression of FZD6, LRP5, and PROM1 genes stands
out in WNT10BIVS1 positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as
leukemic cell models, which are characterized by the expression of WNT10BIVS1, we have observed
that WNT10B drives major Wnt activation to the FZD6 receptor complex through receipt of ligand.
Additionally, short hairpin RNAs (shRNAs)-mediated gene silencing and small molecules-mediated
inhibition of WNTs secretion, have been observed to interfere with the WNT10B/FZD6 interaction.
We have therefore identified that WNT10BIVS1 knockdown, or pharmacological interference by the
LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and
significantly impairs intracellular effectors and leukemic expansion. These results describe the
molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as a new target in the T-ALL
treatment strategy
Development of the late-phase manufacturing process to ZPL389: control of process impurities by enhanced process knowledge
No full textThe development of the late-phase manufacturing process to the drug candidate ZPL389 is described. The strategies for the control of impurities are outlined in detail. The development of the late-phase synthesis route allowed a thorough control of the process impurities leading to an improvement of the quality of the drug substance, as well as to an increase of the overall yield of the manufacturing process and a reduction of the PMI
Mutagenesis, Hydrogen–Deuterium Exchange, and Molecular Docking Investigations Establish the Dimeric Interface of Human Platelet-Type 12-Lipoxygenase
No full textIt was previously shown that human platelet 12S-lipoxygenase (h12-LOX) exists as a dimer; however, the specific structure is unknown. In this study, we create a model of the dimer through a combination of computational methods, experimental mutagenesis, and hydrogen–deuterium exchange (HDX) investigations. Initially, Leu183 and Leu187 were replaced by negatively charged glutamate residues and neighboring aromatic residues were replaced with alanine residues (F174A/W176A/L183E/L187E/Y191A). This quintuple mutant disrupted both the hydrophobic and π–π interactions, generating an h12-LOX monomer. To refine the determinants for dimer formation further, the L183E/L187E mutant was generated and the equilibrium shifted mostly toward the monomer. We then submitted the predicted monomeric structure to protein–protein docking to create a model of the dimeric complex. A total of nine of the top 10 most energetically favorable docking conformations predict a TOP-to-TOP dimeric arrangement of h12-LOX, with the α-helices containing a Leu-rich region (L172, L183, L187, and L194), corroborating our experimental results showing the importance of these hydrophobic interactions for dimerization. This model was supported by HDX investigations that demonstrated the stabilization of four, non-overlapping peptides within helix α2 of the TOP subdomain for wt-h12-LOX, consistent with the dimer interface. Most importantly, our data reveal that the dimer and monomer of h12-LOX have distinct biochemical properties, suggesting that the structural changes due to dimerization have allosteric effects on active site catalysis and inhibitor binding
Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.
No full textThe lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses
How Team Diversity Drives Innovation: Modeling in the Global Pharmaceutical Industry
No full textPurpose – The innovative capacity of an organization is typically realized through unit-level teams.
Previous studies correlate innovation performance with cultural diversity of teams, but note that team
dynamics need to be optimized to derive maximum benefit. Herein, this study offers an assessment of
available team building instruments through the lens of diverse innovation teams. In a demonstration
project in the pharmaceutical industry, this study then outlines specific tools and approaches which
can be successfully deployed through team coaching and mentoring.
Design/methodology/approach – A cluster of nine innovation teams with varying degrees of cultural
diversity was provided with assessment and management instruments which had been identified and field
tested by a mentoring team. Content included cultural awareness tools, innovation team profiling methods
and Team Science (SciTS) ideology. Teams were funded, coached and mentored through a six-month
performance period and assessed at regular intervals.
Findings – Team assessments provided correlations between performance (measured by project completion
and new intellectual property generated) and diversity together with wealth of information on intra-team
culture and dynamics. Concrete recommendations from the study include adoption of appropriate
communication standards to promote inclusivity, use of SciTS operational tracking metrics to enhance
engagement, use of the FourSight group profiling methodology and cultural quotient scale cultural awareness
instruments at team-forming stage to promote effective dynamics and enhance inclusivity.
Practical implications – Cultural diversity has a positive impact on innovation teams. This said, for
maximum benefit cultural awareness of team members should be optimized to avoid unintended conflicts
developing. Such issues can be exacerbated when teams are deployed remotely and preventative measures
should be established. These issues became of heightened significance as a result of telecommuting imposed
by the COVID-19 pandemic and have longer-term implications, as corporations consider global air travel
reduction through environmental concerns. A tracking tool is described to monitor team engagement and
promote inclusivity. It is expected that the learnings can influence how teams can best form, normalize and
operate within corporate innovation programs and form the basis of long-term impact studies
In vivo susceptibility to energy failure parkinsonism and LRRK2 kinase activity.
No full textThe G2019S mutation of LRRK2 represents a risk factor for idiopathic Parkinson's disease. Here, we investigate whether LRRK2 kinase activity regulates susceptibility to the environmental toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). G2019S knock-in mice (bearing enhanced kinase activity) showed greater nigro-striatal degeneration compared to LRRK2 knock-out, LRRK2 kinase-dead and wild-type mice following subacute MPTP treatment. LRRK2 kinase inhibitors PF-06447475 and MLi-2, tested under preventive or therapeutic treatments, protected against nigral dopamine cell loss in G2019S knock-in mice. MLi-2 also rescued striatal dopaminergic terminal degeneration in both G2019S knock-in and wild-type mice. Immunoblot analysis of LRRK2 Serine935 phosphorylation levels confirmed target engagement of LRRK2 inhibitors. However, MLi-2 abolished phosphoSerine935 levels in the striatum and midbrain of both wild-type and G2019S knock-in mice whereas PF-06447475 partly reduced phosphoSerine935 levels in the midbrain of both genotypes. In vivo and ex vivo uptake of the 18-kDa translocator protein (TSPO) ligand [18F]-VC701 revealed a similar TSPO binding in MPTP-treated wild-type and G2019S knock-in mice which was consistent with an increased GFAP striatal expression as revealed by Real Time PCR. We conclude that LRRK2 G2019S, likely through enhanced kinase activity, confers greater susceptibility to mitochondrial toxin-induced parkinsonism. LRRK2 kinase inhibitors are neuroprotective in this model
A cross-industry survey on photosafety evaluation of pharmaceuticals after implementation of ICH S10.
No full textA cross-industry survey was conducted by EFPIA/IQ DruSafe in 2018 to provide information on photosafety evaluation of pharmaceuticals after implementation of ICH S10. This survey focused on the strategy utilized for photosafety risk assessment, the design of nonclinical (in vitro and in vivo) and clinical evaluations, the use of exposure margins in risk assessment, and regulatory interactions. The survey results indicated that a staged approach for phototoxicity assessment has been widely accepted by regulatory authorities globally. The OECD-based 3T3 NRU Phototoxicity Test is the most frequently used in vitro approach. Modifications to this assay suggested by ICH S10 are commonly applied. For in-vitro-positives, substantial margins from in vitro IC50 values under irradiation to Cmax (clinical) have enabled further development without the need for additional photosafety data. In vivo phototoxicity studies typically involve dosing rodents and exposing skin and eyes to simulated sunlight, and subsequently evaluating at least the skin for erythema and edema. However, no formal guidelines exist and protocols are less standardized across companies. A margin-of-safety approach (based on Cmax at NOAEL) has been successfully applied to support clinical development. Experience with dedicated clinical phototoxicity studies was limited, perhaps due to effective de-risking approaches employed based on ICH S10