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The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A1/A2A receptor signalling through the activation of the Ca2+ -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2. We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice.
Anti-CD20 Disrupts Meningeal B-Cell Aggregates in a Model of Secondary Progressive Multiple Sclerosis.
OBJECTIVE: Therapies targeting B cells have been used in the clinic for multiple sclerosis (MS). In patients with relapsing MS, anti-CD20 therapy often suppresses relapse activity; yet, their effect on disease progression has been disappointing. Most anti-CD20 therapeutic antibodies are type I, but within the unique microenvironment of the brain, type II antibodies may be more beneficial, as type II antibodies exhibit reduced complement-dependent cytotoxicity and they have an increased capacity to induce direct cell death that is independent of the host immune response. METHODS: We compared the effect of type I with type II anti-CD20 therapy in a new rodent model of secondary progressive MS (SPMS), which recapitulates the principal histopathologic features of MS including meningeal B-cell aggregates. Focal MS-like lesions were induced by injecting heat-killed Mycobacterium tuberculosis into the piriform cortex of MOG-immunized mice. Groups of mice were treated with anti-CD20 antibodies (type I [rituxumab, 10 mg/kg] or type II [GA101, 10 mg/kg]) 4 weeks after lesion initiation, and outcomes were evaluated by immunohistochemistry. RESULTS: Anti-CD20 therapy decreased the extent of glial activation, significantly decreased the number of B and T lymphocytes in the lesion, and resulted in disruption of the meningeal aggregates. Moreover, at the given dose, the type II anti-CD20 therapy was more efficacious than the type I and also protected against neuronal death. CONCLUSIONS: These results indicate that anti-CD20 may be an effective therapy for SPMS with B-cell aggregates and that the elimination of CD20+ B cells alone is sufficient to cause disruption of aggregates in the brain.
Combining tissue engineering and optical imaging approaches to explore interactions along the neuro-cardiac axis: Neuro-cardiac research
© 2020 The Authors. Interactions along the neuro-cardiac axis are being explored with regard to their involvement in cardiac diseases, including catecholaminergic polymorphic ventricular tachycardia, hypertension, atrial fibrillation, long QT syndrome and sudden death in epilepsy. Interrogation of the pathophysiology and pathogenesis of neuro-cardiac diseases in animal models present challenges resulting from species differences, phenotypic variation, developmental effects and limited availability of data relevant at both the tissue and cellular level. By contrast, tissue-engineered models containing cardiomyocytes and peripheral sympathetic and parasympathetic neurons afford characterization of cellular- and tissue-level behaviours while maintaining precise control over developmental conditions, cellular genotype and phenotype. Such approaches are uniquely suited to long-term, high-throughput characterization using optical recording techniques with the potential for increased translational benefit compared to more established techniques. Furthermore, tissue-engineered constructs provide an intermediary between whole animal/tissue experiments and in silico models. This paper reviews the advantages of tissue engineering methods of multiple cell types and optical imaging techniques for the characterization of neuro-cardiac diseases.
Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.
Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 μM, solubility: 477 μM, mouse hepatocyte T1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.
Targeting the bacterial SOS response for new antimicrobial agents: drug targets, molecular mechanisms and inhibitors.
Antimicrobial resistance is a pressing threat to global health, with multidrug-resistant pathogens becoming increasingly prevalent. The bacterial SOS pathway functions in response to DNA damage that occurs during infection, initiating several pro-survival and resistance mechanisms, such as DNA repair and hypermutation. This makes SOS pathway components potential targets that may combat drug-resistant pathogens and decrease resistance emergence. This review discusses the mechanism of the SOS pathway; the structure and function of potential targets AddAB, RecBCD, RecA and LexA; and efforts to develop selective small-molecule inhibitors of these proteins. These inhibitors may serve as valuable tools for target validation and provide the foundations for desperately needed novel antibacterial therapeutics.
The mammalian neocortex is characterised by precise patterns of synaptic connections, which dictate how information flows through cortical circuits. The degree of convergence and divergence of excitatory information results from highly non-random, short-range and long-range glutamatergic synaptic connections (1-11). Previous seminal work has shown that connections across cortical layers can form preferentially between sister neurons born from the same individual progenitor cell (12). However, cortical neurons also form synaptic connections with many neurons within the same layer (1-6) and this intralaminar connectivity has been shown to reflect specific patterns of longer-range connectivity (1-3, 9-10). Given that neurons within the same cortical layer can be derived from a heterogeneous population of embryonic progenitor cells (13-18), we examined how defined progenitor pools influence local excitatory synaptic connectivity in mouse primary somatosensory cortex. We find that upper layer neurons derived from a subpopulation of progenitors, called short neural precursors, connect out-of-class, such that they preferentially form intralaminar connections with neurons derived from other types of progenitor. Similar local connectivity rules have been associated with differential long-range connectivity (1). Indeed, while we find no differences in basic intrinsic or morphological properties, optogenetic circuit mapping reveals that progenitor pool predicts the long-range thalamic input a neuron receives, as well as a neuron's output to deeper layers of cortex. Neurons derived from short neural precursors receive more input from a higher-order thalamic nucleus and provide stronger output to layer 5a, which is associated with cortico-cortical projections. In contrast, adjacent neurons derived from other progenitors receive more input from a first-order thalamic nucleus and provide stronger output to layer 5b, which is associated with subcortical projections. These data suggest that the emergence of progenitor pools has enabled the cortex to generate distinct local and long-range subnetworks for the differential routing of excitatory information.
The mammalian neocortex is characterized by a variety of neuronal cell types and precise arrangements of synaptic connections, but the processes that generate this diversity are poorly understood. Here we examine how a pool of embryonic progenitor cells consisting of apical intermediate progenitors (aIPs) contribute to diversity within the upper layers of mouse cortex. In utero labeling combined with single-cell RNA-sequencing reveals that aIPs can generate transcriptionally defined glutamatergic cell types, when compared to neighboring neurons born from other embryonic progenitor pools. Whilst sharing layer-associated morphological and functional properties, simultaneous patch clamp recordings and optogenetic studies reveal that aIP-derived neurons exhibit systematic biases in both their intralaminar monosynaptic connectivity and the post-synaptic partners that they target within deeper layers of cortex. Multiple cortical progenitor pools therefore represent an important factor in establishing diversity amongst local and long-range fine-scale glutamatergic connectivity, which generates subnetworks for routing excitatory synaptic information.
The posteromedial thalamus (POm) has extensive recurrent connectivity with the whisker-related primary somatosensory cortex (wS1) of rodents. However, its functional contribution to somatosensory processing in wS1 remains unclear. This article reviews several recent findings, which begin to elucidate the role of POm in sensory-evoked plasticity and discusses their implications for somatosensory processing.
Stomatin-like protein-3 (STOML3) is an integral membrane protein expressed in the cilia of olfactory sensory neurons, but its functional role in this cell type has never been addressed. STOML3 is also expressed in dorsal root ganglia neurons, where it has been shown to be required for normal touch sensation. Here, we extended previous results indicating that STOML3 is mainly expressed in the knob and proximal cilia of olfactory sensory neurons. We additionally showed that mice lacking STOML3 have a morphologically normal olfactory epithelium. Due to its presence in the cilia, together with known olfactory transduction components, we hypothesized that STOML3 could be involved in modulating odorant responses in olfactory sensory neurons. To investigate the functional role of STOML3, we performed loose patch recordings from wild type and Stoml3 KO olfactory sensory neurons. We found that spontaneous mean firing activity was lower with additional shift in interspike intervals distributions in Stoml3 KOs compared to wild type neurons. Moreover, the firing activity in response to stimuli was reduced both in spike number and duration in neurons lacking STOML3 compared to wildtype neurons. Control experiments suggested that the primary deficit in neurons lacking STOML3 was at the level of transduction and not at the level of action potential generation. We conclude that STOML3 has a physiological role in olfaction, being required for normal sensory encoding by olfactory sensory neurons.Significance Statement Olfactory transduction comprises a series of well-characterized molecular steps that take place in the cilia of olfactory sensory neurons (OSNs) terminating in action potential firing. Here, we introduce a possible new player: stomatin-like protein 3 (STOML3). Indeed, STOML3 is localized in olfactory cilia, and we show that STOML3 plays a role in OSN physiology. First, it allows OSNs to broaden the possible frequency range of their spontaneous activity. Second, STOML3 modulates odorant-evoked action potential firing by regulating both the number of spikes and response duration. These new findings call for a reconsideration of the patterns of the peripheral coding of sensory stimuli.
We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family of serine/threonine kinases. Here we described the optimisation of those compounds to improve their physicochemical and ADME properties as well as reducing their off-targets activities against other kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies, advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in a concentration dependent manner.
NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions-NMR waterLOGSY.
In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.
© 2020 American Chemical Society. Macromolecules such as antibodies and antibody fragments have been reported to interfere with intracellular targets, but their use is limited to delivery systems where expression is achieved from vectors such as plasmids or viruses. We have developed PEGylated nanoparticles of poly-lactic acid (PLA), including the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which are functionalized with monoclonal anti-CD7, anti-CD53, or anti-GPR56 antibodies for receptor-mediated endocytic delivery into T-cell leukemia cell lines. Incorporation of DOTAP as the lipid component of the PLA nanoparticles enhanced the release of the immuno-nanoparticles from the endosomes into the cytosol compared to nanoparticles made from PLA alone. Systemic delivery of these anti-CD7 immuno-nanoparticles into humanized CD7 transgenic mice resulted in localization in the spleen, enhanced uptake into CD7-expressing splenocytes, and release of low amounts of reporter mRNA for translation. These functionalized polymer lipid nanoparticles are the basis for elaboration and optimization for realizing their potential in therapeutic applications to carry specific macromolecules such as mRNAs for translation into therapeutic proteins that can target intracellular proteins which mediate disease.
2,4-Diaminothieno[3,2-d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm.
The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle.
Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.
The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein-protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein-protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein-protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein-protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.
Targeting the protein-protein interaction between p53 and MDM2/MDMX (MDM4) represents an attractive anticancer strategy for the treatment of p53-competent tumors. Several selective and potent MDM2 inhibitors have been developed and entered the clinic; however, the repertoire of MDMX antagonists is still limited. The arylmethylidenepyrazolinone SJ-172550 has been reported as a selective MDMX antagonist; yet, uncertainties about its mechanism of action have raised doubts about its use as a chemical probe. Here, we show that, in addition to its unclear mode of action, SJ-172550 is unstable in aqueous buffers, giving rise to side products of unknown biological activity. Using an SJ-172550-derived affinity probe, we observed promiscuous binding to cellular proteins whereas cellular thermal shift assays did not reveal a stabilizing effect on MDMX. Overall, our results raise further questions about the interpretation of data using SJ-172550 and related compounds to investigate cellular phenotypes.