E2814 is a humanized monoclonal antibody that recognizes the microtubule-binding region (MTBR) of tau, a region of the protein essential for filament formation and propagation in neurodegenerative diseases. Epitope mapping showed that E2814 binds to a specific sequence motif HVPGG in the MTBR. To elucidate the atomic interactions of E2814-tau binding, we performed X-ray crystallography studies with E2814 and various tau peptides containing the HVPGG motif. The F ab fragment of E2814 was incubated with 5 different tau peptides containing the 299-HVPGG-303 epitope at a 1:10 molar ratio prior to co-crystallization screening. Co-crystals of E2814/tau peptides were grown by sitting drop vapor diffusion, and the diffraction data were collected at synchrotron. Structures of E2814 bound to tau peptides were determined by molecular replacement. Crystal structures of E2814 F ab bound to five tau peptides containing 299-HVPGG-303 epitope were determined at 1.44 –1.64 Å resolution. The comparison of the five crystal structures revealed the sequence 299-HVPG-302 of tau presents the core region mediating recognition by E2814, which is also facilitated by I297 in the flanking region. The tau peptides are accommodated in the recognition groove formed by CDR regions from the VH and VL domains, and exhibited a U-shaped conformation with close-to quarter turns when bound to E2814. Both H299 and P301 at the turns mediate major interactions with E2814. Our results reveal the structural details of the key interactions between E2814 and the tau HVPGG motif, which adopts a U-shaped conformation upon binding to E2814. These findings provide atomic insights into the mechanism of E2814-mediated inhibition of tau aggregation and propagation.

Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of ∼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified in this work provide a novel starting point for further investigation and optimization.

Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene, loss of function variants of which cause an autosomal recessive lysosomal storage disorder, Gaucher disease (GD). Heterozygous variants of GBA1 are also known as the strongest common genetic risk factor for Parkinson's disease (PD). Restoration of GCase enzymatic function using a pharmacological chaperone strategy is considered a promising therapeutic approach for PD and GD. We identified compound 4 as a GCase pharmacological chaperone with sub-micromolar activity from a high-throughput screening (HTS) campaign. Compound 4 was further optimised to ER-001230194 (compound 25). ER-001230194 shows improved ADME and physicochemical properties and therefore represents a novel pharmacological chaperone with which to investigate GCase pharmacology further.

As Alzheimer’s disease progresses, pathological tau spreads through the brain via synaptically connected pathways. Tau proteins containing the microtubule binding region (MTBR) may be essential to form extracellular ‘seeds’ that initiate and propagate pathology. To simulate this process preclinically, we have further characterised an AD brain-derived seed-injection in vivo model using hTau mice that overexpress all six isoforms of human wild-type tau (Andorfer et al., 2003). In further follow up studies, mice were treated with the 7G6-IgG2a antibody, which is the murine counterpart of E2814, an anti-tau antibody currently in clinical trials that recognises the MTBR (Roberts et al., 2020). In a validation study, insoluble fraction or whole homogenate from human AD brain (‘seed’) was directly injected into the left hippocampus of 6-month-old hTau mice or their tau null littermates. At different times after seed injection, the generation of insoluble tau was quantified in selected brain regions (hippocampus and cortex) by western blotting following tissue fractionation with detergents. In a subsequent therapeutic intervention study, animals were treated peripherally with a control mouse IgG2a antibody or 7G6-IgG2a at different doses (3, 10, and 40 mg/kg). Levels of insoluble tau in the hippocampus and cortex were quantified as described above. Terminal CSF was also collected to measure target engagement of antibody. Additionally, immunohistochemistry using the AT8 antibody that detects pathological forms of phospho-tau proteins was performed on brains from a limited number of animals receiving the highest dose of control IgG2a or 7G6-IgG2a. AD brain insoluble fraction was superior to homogenate when used as a seed in the hTau mouse. Peripheral treatment of seeded hTau mice with the 7G6-IgG2a antibody (40 mg/kg) resulted in a significant reduction of insoluble tau in multiple brain regions. Antibody target engagement also increased in the CSF from treated animals in a dose-dependent manner. AD seed-injection in hTau mice has been further validated and can be used in therapeutic intervention studies. The murine version of E2814, 7G6-IgG2a, reduced pathological tau seeding and spreading in this model. The nonclinical data obtained here further supports E2814 being tested in clinical trials.

External innovation initiatives in the pharmaceutical industry have become an integral part of research and development. Collaborations have been built to enhance innovation, mitigate risk, and share cost, especially for neurodegenerative diseases, a therapeutic area that has suffered from high attrition rates. This article outlines the Eisai-University College London (UCL) Drug Discovery and Development Collaboration as a case study of how to implement a productive industry-academic partnership. In the first 10 years, seven projects have been established and the first project, a novel anti-tau antibody for Alzheimer's disease, has entered clinical trials, providing early validation of this collaboration model.

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

The R47H variant of the microglial membrane receptor TREM2 is linked to increased risk of late onset Alzheimer's disease. Human induced pluripotent stem cell derived microglia (iPS-Mg) from patient iPSC lines expressing the AD-linked R47Hhet TREM2 variant, common variant (Cv) or an R47Hhom CRISPR edited line and its isogeneic control, demonstrated that R47H-expressing iPS-Mg expressed a deficit in signal transduction in response to the TREM2 endogenous ligand phosphatidylserine with reduced pSYK-pERK1/2 signalling and a reduced NLRP3 inflammasome response, (including ASC speck formation, Caspase-1 activation and IL-1beta secretion). Apoptotic cell phagocytosis and soluble TREM2 shedding were unaltered, suggesting a disjoint between these pathways and the signalling cascades downstream of TREM2 in R47H-expressing iPS-Mg, whilst metabolic deficits in glycolytic capacity and maximum respiration were reversed when R47H expressing iPS-Mg were exposed to PS+ expressing cells. These findings suggest that R47H-expressing microglia are unable to respond fully to cell damage signals such as phosphatidylserine, which may contribute to the progression of neurodegeneration in late-onset AD.

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer’s disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species (“seeds”) containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG 1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro . They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in the glucocerebrosidase 1 ( GBA1 ) gene encoding the lysosomal enzyme glucocerebrosidase. Patients with type 1 GD present with accumulation of glucosylceramide in macrophages leading to a range of systemic manifestations,

Neurofibrillary tangles composed of aggregated tau protein are a pathological hallmark of Alzheimer's disease (AD). Extracellular tau can form ‘seeds’ capable of inducing intracellular tau aggregation and propagating pathology through the brain. The presence of the tau microtubule binding region (MTBR) is essential for the extracellular seeds to exert their effect, suggesting a therapeutic antibody targeting this region may attenuate the spread of pathology in AD. E2814 is a novel anti-tau therapeutic antibody that binds to the MTBR. To develop a clinically useful target engagement assay for E2814, it was necessary to confirm the presence of MTBR-containing tau in cerebrospinal fluid (CSF). This work describes a focused LC/MS method quantifying MTBR-containing tau species in CSF. We analyzed MTBR-tau in CSF from several patients with different tauopathies using unique methodology. Briefly, multi-step fractionation techniques were applied to enrich MTBR-tau from CSF, followed then by tryptic digestion to obtain a measurable proxy peptide. Quantification of the peptide was performed by mass spectrometry equipped with nano-flow capillary liquid chromatography interfaced with nano-electrospray ionization. To demonstrate target engagement of E2814, antibody-bound and -unbound proteins in CSF were collected from E2814-treated non-human primates and passed through a Protein A column. MTBR-tau levels were then quantified in each fraction. The method revealed: (1) Existence of MTBR-tau in CSF; (2) significant increase of MTBR-tau in AD CSF over control; (3) significant increase of E2814-bound MTBR-tau in AD CSF over control and other tauopathies; (4) both antibody-bound and -unbound concentrations of CSF MTBR-tau changed in a dose-dependent manner in non-human primates treated with E2814. These results suggest that secretion of MTBR-tau into the extracellular space is measurable in human CSF and is increased specifically in AD. Notably, the majority of MTBR-tau could also be bound by E2814 spiked into AD CSF. It was also demonstrated in non-human primate CSF that antibody-bound and -unbound levels changed with antibody dose levels, eventually saturating at a specific concentration of E2814. Overall the data suggest this assay method may be used to evaluate E2814 target engagement in human clinical trials.

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders that include Alzheimer's disease (AD). As disease progresses, Braak staging of post-mortem tissue suggest that pathological tau, spreads through the brain via synaptically connected pathways. Tau proteins (full length or fragments) containing the microtubule binding region (MTBR) may be essential to form extracellular ‘seeds’ that initiate and propagate pathology. Indeed, the MTBR forms the core of tau filaments observed in AD brain. Tau immunotherapy has emerged as a promising approach to slow or limit AD progression. Therefore, targeting the tau MTBR-containing seeds with a therapeutic antibody may be a useful strategy to provide disease-modifying treatment for AD. E2814 is a novel humanised IgG1 antibody that binds to the tau MTBR. The preclinical characterisation of this antibody and its murine counterpart 7G6, will be presented. Human and murine antibodies were fine epitope mapped using PEPperPRINT technology. Their effect on inhibiting tau aggregation and ability to immunodeplete seeds was assessed in vitro. The in vivo efficacy of 7G6 was evaluated in an intrahippocampal seed injection model. Finally, the ability of E2814 to bind pathological human tau was tested by immunogold labelling of AD fibrils and by immunohistochemical staining of tissue sections. E2814 and 7G6 are high affinity antibodies bi-epitopic for 4R tau isoforms. Both antibodies effectively inhibited tau aggregation in vitro and immunodepleted different MTBR-containing tau seeds. An attenuation of brain sarkosyl-insoluble tau could also be observed in an in vivo model of tau seeding and transmission. In analysis of AD brain, E2814 was also shown to bind both different types of isolated tau filaments and pathological tau structures in tissue sections. Collectively, these data suggest that E2814 should be investigated further in human clinical trials.

Mitochondrial Ca 2+ uptake has a key role in cellular Ca 2+ homeostasis. Excessive matrix Ca 2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH 2 -linked substrates. The functional consequences of Ca 2+ -induced mPTP opening were assessed by Ca 2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca 2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca 2+ overload.

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.

From Braak staging in Alzheimer's disease, it is now recognized that tau pathology spreads along anatomically connected pathways with progression of disease. The agent of this “prion-like” aggregation and trans-synaptic spread are pathological conformers of tau that are released by cells, endocytosed by neighbouring healthy neurons and in turn convert normal tau in a cascade-like fashion. Studies have shown the potential therapeutic benefit of targeting this extracellular tau with specific antibodies.RD3 and RD4 antibodies that target regions in the microtubule-binding repeat domain of tau that are crucial for aggregation were investigated to demonstrate their efficacy in preventing pathological tau uptake and aggregation in a cell-based model. Sequences encoding the wild-type, ΔK280 and P301L/V337M double mutant tau repeat region were cloned into pEGFP-N1 and pDsRed-monomer-N1. SH-SY5Y neuroblastoma cells were co-transfected with both mutant and WT tau constructs. After 48 hours, fixed cells were analysed by fluorescence resonance energy transfer (FRET) using both acceptor photo bleaching microscopy and a fluorescence plate reader with fixed excitation/emission windows. Strongest FRET signals in cells co-expressing the ΔK280 and P301L/V337M mutants were observed. Preincubation of the cells with Alzheimer's disease and Progressive Supranuclear Palsy patient brain homogenates further increased the signal. This was progressively abolished with preincubation of the brain homogenate with increasing concentrations of RD3 antibody with a more modest efficacy of the RD4 antibody. The well-characterized RD3 antibody appears to be effective in preventing the cellular uptake and seeding of Tau aggregation. These findings suggest that RD3 could be developed for passive immunotherapy.

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinsons disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD.