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Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT- active compounds hold promise as antitrypanosomal agents. MT-targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,β-tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against culturedTrypanosoma bruceienabled a robust quantitative structure-activity relationship (QSAR) model and the prioritization of two congeners forin vivopharmacokinetics (PK), tolerability and efficacy studies. Treatment ofT. brucei-infected mice with tolerable doses of TPDs3and4significantly decreased blood parasitemia within 24 h. Further, two once-weekly doses of4at 10 mg/kg significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS-active TPDs may provide alternative treatments for human African trypanosomiasis.

Microtubule (MT)-stabilizing 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) hold promise as candidate therapeutics for Alzheimer's disease (AD) and other neurodegenerative conditions. However, depending on the choice of substituents around the TPD core, these compounds can elicit markedly different cellular phenotypes that likely arise from the interaction of TPD congeners with either one or two spatially distinct binding sites within tubulin heterodimers (i.e., the seventh site and the vinca site). In the present study, we report the design, synthesis, and evaluation of a series of new TPD congeners, as well as matched molecular pair analyses and computational studies, that further elucidate the structure-activity relationships of MT-active TPDs. These studies led to the identification of novel MT-normalizing TPD candidates that exhibit favorable ADME-PK, including brain penetration and oral bioavailability, as well as brain pharmacodynamic activity.

Microtubule (MT)-stabilizing 1,2,4-triazolo[1,5

The microtubule-associated protein tau (tau) forms hyperphosphorylated aggregates in the brains of tauopathy patients that can be pathologically and biochemically defined as distinct tau strains. Recent studies show that these tau strains exhibit strain-specific biological activities, also referred to as pathogenicities, in the tau spreading models. Currently, the specific pathogenicity of human-derived tau strains cannot be fully recapitulated by synthetic tau preformed fibrils (pffs), which are generated from recombinant tau protein. Reproducing disease-relevant tau pathology in cell and animal models necessitates the use of human brain-derived tau seeds. However, the availability of human-derived tau is extremely limited. Generation of tau variants that can mimic the pathogenicity of human-derived tau seeds would significantly extend the scale of experimental design within the field of tauopathy research. Previous studies have demonstrated that in vitro seeding reactions can amplify the beta-sheet structure of tau protein from a minute quantity of human-derived tau. However, whether the strain-specific pathogenicities of the original, human-derived tau seeds are conserved in the amplified tau strains has yet to be experimentally validated. Here, we used biochemically enriched brain-derived tau seeds from Alzheimer’s disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) patient brains with a modified seeding protocol to template the recruitment of recombinant 2N4R (T40) tau in vitro. We quantitatively interrogated efficacy of the amplification reactions and the pathogenic fidelity of the amplified material to the original tau seeds using recently developed sporadic tau spreading models. Our data suggest that different tau strains can be faithfully amplified in vitro from tau isolated from different tauopathy brains and that the amplified tau variants retain their strain-dependent pathogenic characteristics. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-020-02253-4.

The hallmark pathological features of Alzheimer's disease (AD) brains are senile plaques, comprising β-amyloid (Aβ) peptides, and neuronal inclusions formed from tau protein. These plaques form 10–20 years before AD symptom onset, whereas robust tau pathology is more closely associated with symptoms and correlates with cognitive status. This temporal sequence of AD pathology development, coupled with repeated clinical failures of Aβ-directed drugs, suggests that molecules that reduce tau inclusions have therapeutic potential. Few tau-directed drugs are presently in clinical testing, in part because of the difficulty in identifying molecules that reduce tau inclusions. We describe here two cell-based assays of tau inclusion formation that we employed to screen for compounds that inhibit tau pathology: a HEK293 cell-based tau overexpression assay, and a primary rat cortical neuron assay with physiological tau expression. Screening a collection of ∼3500 pharmaceutical compounds with the HEK293 cell tau aggregation assay, we obtained only a low number of hit compounds. Moreover, these compounds generally failed to inhibit tau inclusion formation in the cortical neuron assay. We then screened the Prestwick library of mostly approved drugs in the cortical neuron assay, leading to the identification of a greater number of tau inclusion inhibitors. These included four dopamine D2 receptor antagonists, with D2 receptors having previously been suggested to regulate tau inclusions in a Caenorhabditis elegans model. These results suggest that neurons, the cells most affected by tau pathology in AD, are very suitable for screening for tau inclusion inhibitors.

Limbic-predominant age-related TDP-43 encephalopathy (LATE) is characterized by the accumulation of TAR-DNA-binding protein 43 (TDP-43) aggregates in older adults. LATE coexists with Lewy body disease (LBD) as well as other neuropathological changes including Alzheimer’s disease (AD). We aimed to identify the pathological, clinical, and genetic characteristics of LATE in LBD (LATE-LBD) by comparing it with LATE in AD (LATE-AD), LATE with mixed pathology of LBD and AD (LATE-LBD + AD), and LATE alone (Pure LATE). We analyzed four cohorts of autopsy-confirmed LBD (n = 313), AD (n = 282), LBD + AD (n = 355), and aging (n = 111). We assessed the association of LATE with patient profiles including LBD subtype and AD neuropathologic change (ADNC). We studied the morphological and distributional differences between LATE-LBD and LATE-AD. By frequency analysis, we staged LATE-LBD and examined the association with cognitive impairment and genetic risk factors. Demographic analysis showed LATE associated with age in all four cohorts and the frequency of LATE was the highest in LBD + AD followed by AD, LBD, and Aging. LBD subtype and ADNC associated with LATE in LBD or AD but not in LBD + AD. Pathological analysis revealed that the hippocampal distribution of LATE was different between LATE-LBD and LATE-AD: neuronal cytoplasmic inclusions were more frequent in cornu ammonis 3 (CA3) in LATE-LBD compared to LATE-AD and abundant fine neurites composed of C-terminal truncated TDP-43 were found mainly in CA2 to subiculum in LATE-LBD, which were not as numerous in LATE-AD. Some of these fine neurites colocalized with phosphorylated α-synuclein. LATE-LBD staging showed LATE neuropathological changes spread in the dentate gyrus and brainstem earlier than in LATE-AD. The presence and prevalence of LATE in LBD associated with cognitive impairment independent of either LBD subtype or ADNC; LATE-LBD stage also associated with the genetic risk variants of TMEM106B rs1990622 and GRN rs5848. These data highlight clinicopathological and genetic features of LATE-LBD.

Dystrophic neuronal processes harboring neuritic plaque (NP) tau pathology are found in association with Aβ plaques in Alzheimer’s disease (AD) brain. Microglia are also in proximity to these plaques and microglial gene variants are known risk factors in AD, including loss-of-function variants of TREM2. We have further investigated the role of Aβ plaque-associated microglia in 5XFAD mice in which NP tau pathology forms after intracerebral injection of AD brain-derived pathologic tau (AD-tau), focusing on the consequences of reduced TREM2 expression and microglial depletion after treatment with the colony-stimulating factor 1 (CSFR1) inhibitor, PLX3397. Young 5XFAD mice treated with PLX3397 had a large reduction of brain microglia, including cortical plaque-associated microglia, with a significant reduction of Aβ plaque burden in the cortex. A corresponding decrease in cortical APP-positive dystrophic processes and NP tau pathology were observed after intracerebral AD-tau injection in the PLX3397-treated 5XFAD mice. Consistent with prior reports, 5XFAD × TREM2−/− mice showed a significant reduction of plaque-associated microglial, whereas 5XFAD × TREM2+/− mice had significantly more plaque-associated microglia than 5XFAD × TREM2−/− mice. Nonetheless, AD-tau injected 5XFAD × TREM2+/− mice showed greatly increased AT8-positive NP tau relative to 5XFAD × TREM2+/+ mice. Expression profiling revealed that 5XFAD × TREM2+/− mice had a disease-associated microglial (DAM) gene expression profile in the brain that was generally intermediate between 5XFAD × TREM2+/+ and 5XFAD × TREM2−/− mice. Microarray analysis revealed significant differences in cortical and hippocampal gene expression between AD-tau injected 5XFAD × TREM2+/− and 5XFAD × TREM2−/− mice, including pathways linked to microglial function. These data suggest there is not a simple correlation between the extent of microglia plaque interaction and plaque-associated neuritic damage. Moreover, the differences in gene expression and microglial phenotype between TREM2+/− and TREM2−/− mice suggest that the former may better model the single copy TREM2 variants associated with AD risk.

Schistosomiasis is a parasitic disease that affects approximately 200 million people in developing countries. Current treatment relies on just one partially effective drug and new drugs are needed. Tubulin and microtubules (MTs) are essential constituents of the cytoskeleton in all eukaryotic cells and considered potential drug targets to treat parasitic infections. The α- and β-tubulin of Schistosoma mansoni have ~96% and ~91% sequence identity to their respective human tubulins suggesting that compounds which bind mammalian tubulin may interfere with MT-mediated functions in the parasite. To explore the potential of different classes of tubulin-binding molecules as anti-schistosomal leads, we completed a series of in vitro whole-organism screens of a target-based compound library against S. mansoni adults and somules (post-infective larvae), and identified multiple biologically active compounds, among which phenylpyrimidines were the most promising. Further structure-activity relationship studies of these hits identified a series of novel thiophen-2-yl-pyrimidine congeners, which induce a potent and long-lasting paralysis of the parasite. Moreover, compared to the originating compounds, which showed cytotoxicity values in the low nanomolar range, these new derivatives were 1 – 4 orders of magnitude less cytotoxic and exhibited weak or undetectable activity against mammalian MTs in a cell-based assay of MT stabilization. Given their selective anti-schistosomal activity and relatively simple drug-like structures, these molecules hold promise as candidates for the development of new treatments for schistosomiasis.

Studies in tau and Aβ plaque transgenic mouse models demonstrated that brain-penetrant microtubule (MT)-stabilizing compounds, including the 1,2,4-triazolo[1,5-a]pyrimidines, hold promise as candidate treatments for Alzheimer’s disease (AD) and related neurodegenerative tauopathies. Triazolopyrimidines have already been investigated as anti-cancer agents; however, the anti-mitotic activity of these compounds does not always correlate with stabilization of MTs in cells. Indeed, previous studies from our laboratories identified a critical role for the fragment linked at C6 in determining whether triazolopyrimidines promote MT stabilization or, conversely, disrupt MT integrity in cells. To further elucidate the structure-activity relationship (SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68 triazolopyrimidine congeners bearing structural modifications at C6 and/or C7 was designed, synthesized and evaluated. These studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identification of novel analogues that, relative to the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity and pharmacokinetics.

BackgroundThe spread of tau pathology in Alzheimer’s disease (AD) is mediated by cell-to-cell transmission of pathological tau seeds released from neurons that, upon internalization by recipient neurons, template the misfolding of naïve cellular tau, thereby propagating fibrillization. We hypothesize that anti-tau monoclonal antibodies (mAbs) that selectively bind to pathological tau seeds will inhibit propagation of tau aggregates and reduce the spread of tau pathology in vivo.MethodsWe inoculated mice with human AD brain-derived extracts containing tau paired helical filaments (AD-tau) and identified two novel mAbs, DMR7 and SKT82, that selectively bind to a misfolded pathological conformation of tau relative to recombinant tau monomer. To evaluate the effects of these mAbs on the spread of pathological tau in vivo,5xFAD mice harboring significant brain Aβ plaque burden were unilaterally injected with AD-tau in the hippocampus, to initiate the formation of neuritic plaque (NP) tau pathology, and were treated weekly with intraperitoneal (i.p.) injections of DMR7, SKT82, or IgG isotype control mAbs.ResultsDMR7 and SKT82 bind epitopes comprised of the proline-rich domain and c-terminal region of tau and binding is reduced upon disruption of the pathological conformation of AD-tau by chemical and thermal denaturation. We found that both DMR7 and SKT82 immunoprecipitate pathological tau and significantly reduce the seeding of cellular tau aggregates induced by AD-tau in primary neurons by 60.5 + 13.8% and 82.2 + 8.3%, respectively, compared to IgG control. To investigate the mechanism of mAb inhibition, we generated pH-sensitive fluorophore-labeled recombinant tau fibrils seeded by AD-tau to track internalization of tau seeds and demonstrate that the conformation-selective tau mAbs inhibit the internalization of tau seeds. DMR7 and SKT82 treatment reduced hyperphosphorylated NP tau as measured with AT8 immunohistochemistry (IHC) staining, but did not achieve statistical significance in the contralateral cortex and SKT82 significantly reduced tau pathology in the ipsilateral hippocampus by 24.2%;p = 0.044.ConclusionsThese findings demonstrate that conformation-selective tau mAbs, DMR7 and SKT82, inhibit tau pathology in primary neurons by preventing the uptake of tau seeds and reduce tau pathology in vivo,providing potential novel therapeutic candidates for the treatment of AD.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are progressive neurodegenerative diseases for which there is no disease-modifying treatment. PD and DLB are characterized by aggregation of the synaptic protein α-synuclein, and there is compelling evidence to suggest that progression of these diseases is associated with the trans-cellular spread of pathogenic α-synuclein through the brains of afflicted individuals. Therapies targeting extracellular, pathogenic α-synuclein may therefore hold promise for slowing or halting disease progression. In this regard, it has been suggested that highly-selective antibodies can be administered as therapeutic agents targeting pathogenic proteins. In the current study, we screened a series of antibodies using multiple selection criterion to identify those that selectively bind pathogenic α-synuclein and show potent inhibition of pathology seeding in a neuronal model of α-synucleinopathy. A lead antibody was tested in a mouse model of PD, and it was able to reduce the spread of α-synuclein pathology in the brain and attenuate dopamine reductions in the striatum. This study highlights the therapeutic potential of α-synuclein immunotherapy for the treatment of PD and DLB, and provides a framework for screening of α-synuclein antibodies to identify those with preferred properties.

Background Alzheimer’s disease (AD) and related tauopathies are neurodegenerative diseases that are characterized by the presence of insoluble inclusions of the protein tau within brain neurons and often glia. Tau is normally found associated with axonal microtubules (MTs) in the brain, and in tauopathies this MT binding is diminished due to tau hyperphosphorylation. As MTs play a critical role in the movement of cellular constituents within neurons via axonal transport, it is likely that the dissociation of tau from MTs alters MT structure and axonal transport, and there is evidence of this in tauopathy mouse models as well as in AD brain. We previously demonstrated that different natural products which stabilize MTs by interacting with β-tubulin at the taxane binding site provide significant benefit in transgenic mouse models of tauopathy. More recently, we have reported on a series of MT-stabilizing triazolopyrimidines (TPDs), which interact with β-tubulin at the vinblastine binding site, that exhibit favorable properties including brain penetration and oral bioavailability. Here, we have examined a prototype TPD example, CNDR-51657, in a secondary prevention study utilizing aged tau transgenic mice. Methods 9-Month old female PS19 mice with a low amount of existing tau pathology received twice-weekly administration of vehicle, or 3 or 10 mg/kg of CNDR-51657, for 3 months. Mice were examined in the Barnes maze at the end of the dosing period, and brain tissue and optic nerves were examined immunohistochemically or biochemically for changes in MT density, axonal dystrophy, and tau pathology. Mice were also assessed for changes in organ weights and blood cell numbers. Results CNDR-51657 caused a significant amelioration of the MT deficit and axonal dystrophy observed in vehicle-treated aged PS19 mice. Moreover, PS19 mice receiving CNDR-51657 had significantly lower tau pathology, with a trend toward improved Barnes maze performance. Importantly, no adverse effects were observed in the compound-treated mice, including no change in white blood cell counts as is often observed in cancer patients receiving high doses of MT-stabilizing drugs. Conclusions A brain-penetrant MT-stabilizing TPD can safely correct MT and axonal deficits in an established mouse model of tauopathy, resulting in reduced tau pathology. Electronic supplementary material The online version of this article (10.1186/s13024-018-0291-3) contains supplementary material, which is available to authorized users.

Alzheimer’s disease (AD) is characterized by extracellular amyloid-β (Aβ) plaques and intracellular tau inclusions. However, the exact mechanistic link between these two AD lesions remains enigmatic. Through injection of human AD-brain-derived pathological tau (AD-tau) into Aβ plaque–bearing mouse models that do not overexpress tau, we recapitulated the formation of three major types of AD-relevant tau pathologies: tau aggregates in dystrophic neurites surrounding Aβ plaques (NP tau), AD-like neurofibrillary tangles (NFTs) and neuropil threads (NTs). These distinct tau pathologies have different temporal onsets and functional consequences on neural activity and behavior. Notably, we found that Aβ plaques created a unique environment that facilitated the rapid amplification of proteopathic AD-tau seeds into large tau aggregates, initially appearing as NP tau, which was followed by the formation and spread of NFTs and NTs, likely through secondary seeding events. Our study provides insights into a new multistep mechanism underlying Aβ plaque–associated tau pathogenesis.

Many neurodegenerative diseases are characterized by deficiencies in neuronal axonal transport, a process in which cellular cargo is shuttled with the aid of molecular motors from the cell body to axonal termini and back along microtubules (MTs). Proper axonal transport is critical to the normal functioning of neurons, and impairments in this process could contribute to the neuronal damage and death that is characteristic of neurodegenerative disease. Although the causes of axonal transport abnormalities may vary among the various neurodegenerative conditions, in many cases it appears that the transport deficiencies result from a diminution of axonal MT stability. Here we review the evidence of MT abnormalities in a number of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and traumatic brain injury, and highlight the potential benefit of MT-stabilizing agents in improving axonal transport and nerve function in these diseases. Moreover, we discuss the challenges associated with the utilization of MT-stabilizing drugs as therapeutic candidates for neurodegenerative conditions.

The 1,2,4-triazolo[1,5-a]pyrimidine (TP) heterocycle, in spite of its relatively simple structure, has proved to be remarkably versatile as evidenced by its use in many different applications reported over the years in different areas of drug design. For example, as the ring system of TPs is isoelectronic with that of purines, this heterocycle has been proposed as a possible surrogate of the purine ring. However, depending on the choice of substituents, the TP ring has also been described as a potentially viable bio-isostere of the carboxylic acid functional group and of the N-acetyl fragment of ε-N-acetylated lysine. In addition, the metal-chelating properties of the TP ring have also been exploited to generate candidate treatments for cancer and parasitic diseases. In the present review article, we discuss recent applications of the TP scaffold in medicinal chemistry, and provide an overview of its properties and methods of synthesis.

Deposition of tau aggregates in the brain is a pathological hallmark of several neurodegenerative diseases, termed tauopathies, such as Alzheimer's disease (AD), corticobasal degeneration, and progressive supranuclear palsy (PSP). As transcellular spread of pathological tau aggregates has been implicated in disease progression, immunotherapy is being considered as a treatment for tauopathies. Here we report a detailed biochemical and biophysical characterization of the tau-binding properties of gosuranemab, a humanized monoclonal antibody directed against N-terminal tau that is currently being investigated as a treatment for AD. Binding experiments showed that gosuranemab exhibited high affinity for tau monomer, tau fibrils, and insoluble tau from different tauopathies. Epitope mapping studies conducted using X-ray crystallography and mutagenesis showed that gosuranemab bound to human tau residues 15–22. Immunodepletion of pathological human brain homogenates and transgenic mouse interstitial fluid (ISF) with gosuranemab resulted in reduced tau aggregation in tau biosensor cells. Preincubation of seed-competent AD-tau with gosuranemab significantly inhibited tau aggregation in mouse primary cortical neurons. Gosuranemab also significantly reduced unbound N-terminal tau in cerebrospinal fluid (CSF) from individuals with PSP and AD, and in ISF and CSF of treated transgenic mice. These results are consistent with the >90% target engagement observed in the CSF of some clinical trial dosing cohorts and support the evaluation of gosuranemab as a potential treatment for AD.

A group of neurodegenerative diseases referred to as tauopathies are characterized by the presence of brain cells harboring inclusions of pathological species of the tau protein. These disorders include Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) due to tau pathology, including progressive supranuclear palsy, corticobasal degeneration and Pick’s disease. Tau is normally a microtubule-associated protein that appears to play an important role in ensuring proper axonal transport, but in tauopathies tau becomes hyperphosphorylated and disengages from microtubules, with consequent misfolding and deposition into inclusions that mainly affect neurons, but also glia. A body of experimental evidence suggests that the development of tau inclusions leads to the neurodegeneration observed in tauopathies, and there is a growing interest in developing tau-directed therapeutic agents. The following review provides a summary of strategies under investigation for the potential treatment of tauopathies, highlighting both the promises and challenges associated with these various therapeutic approaches.

Filamentous tau aggregates, the hallmark lesions of Alzheimer disease (AD), play key roles in neurodegeneration. Activation of protein degradation systems has been proposed to be a potential strategy for removing pathological tau, but it remains unclear how effectively tau aggregates can be degraded by these systems. By applying our previously established cellular model system of AD-like tau aggregate induction using preformed tau fibrils, we demonstrate that tau aggregates induced in cells with regulated expression of full-length mutant tau can be gradually cleared when soluble tau expression is suppressed. This clearance is at least partially mediated by the autophagy-lysosome pathway, although both the ubiquitin-proteasome system and the autophagy-lysosome pathway are deficient in handling large tau aggregates. Importantly, residual tau aggregates left after the clearance phase leads to a rapid reinstatement of robust tau pathology once soluble tau expression is turned on again. Moreover, we succeeded in generating monoclonal cells persistently carrying tau aggregates without obvious cytotoxicity. Live imaging of GFP-tagged tau aggregates showed that tau inclusions are dynamic structures constantly undergoing "fission" and "fusion," which facilitate stable propagation of tau pathology in dividing cells. These findings provide a greater understanding of cell-to-cell transmission of tau aggregates in dividing cells and possibly neurons.

The microtubule (MT)–stabilizing protein tau disengages from MTs and forms intracellular inclusions known as neurofibrillary tangles in Alzheimer’s disease and related tauopathies. Reduced tau binding to MTs in tauopathies may contribute to neuronal dysfunction through decreased MT stabilization and disrupted axonal transport. Thus, the introduction of brain-penetrant MT-stabilizing compounds might normalize MT dynamics and axonal deficits in these disorders. We previously described a number of phenylpyrimidines and triazolopyrimidines (TPDs) that induce tubulin post-translational modifications indicative of MT stabilization. We now further characterize the biologic properties of these small molecules, and our results reveal that these compounds can be divided into two general classes based on the cellular response they evoke. One group composed of the phenylpyrimidines and several TPD examples showed a bell-shaped concentration-response effect on markers of MT stabilization in cellular assays. Moreover, these compounds induced proteasome-dependent degradation of α- and β-tubulin and caused altered MT morphology in both dividing cells and neuron cultures. In contrast, a second group comprising a subset of TPD molecules (TPD+) increased markers of stable MTs in a concentration-dependent manner in dividing cells and in neurons without affecting total tubulin levels or disrupting MT architecture. Moreover, an example TPD+ compound was shown to increase MTs in a neuron culture model with induced tau hyperphosphorylation and associated MT deficits. Several TPD+ compounds were shown to be both brain penetrant and orally bioavailable, and a TPD+ example increased MT stabilization in the mouse brain, making these compounds potential candidate therapeutics for neurodegenerative tauopathies such as Alzheimer’s disease.

In vitro whole-organism screens of Trypanosoma brucei with representative examples of brain-penetrant microtubule (MT)-stabilizing agents identified lethal triazolopyrimidines and phenylpyrimidines with sub-micromolar potency. In mammalian cells, these antiproliferative compounds disrupt MT integrity and decrease total tubulin levels. Their parasiticidal potency, combined with their generally favorable pharmacokinetic properties, which include oral bioavailability and brain penetration, suggest that these compounds are potential leads against human African trypanosomiasis.

The [1,2,4]triazolo[1,5-a]pyrimidines comprise a promising class of non-naturally occurring microtubule (MT)-active compounds. Prior studies revealed that different triazolopyrimidine substitutions can yield molecules that either promote MT stabilization or disrupt MT integrity. These differences can have important ramifications in the therapeutic applications of triazolopyrimidines and suggest that different analogues may exhibit different binding modes within the same site or possibly interact with tubulin/MTs at alternative binding sites. To help discern these possibilities, a series of photoactivatable triazolopyrimidine congeners was designed, synthesized and evaluated in cellular assays with the goal of identifying candidate probes for photoaffinity labeling experiments. These studies led to the identification of different derivatives that incorporate a diazirine ring in the amine substituent at position 7 of the triazolopyrimidine heterocycle, resulting in molecules that either promote stabilization of MTs or disrupt MT integrity. These photoactivatable candidate probes hold promise to investigate the mode of action of MT-active triazolopyrimidines.

Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a time-dependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.

Several independent studies indicate that microtubule (MT)-stabilizing agents hold considerable promise as candidate therapeutics for a wide spectrum of conditions of the central nervous system (CNS), from brain tumors to spinal cord injury, as well as a number of neurodegenerative diseases, including Alzheimer’s disease, frontotemporal lobar degeneration, Parkinson’s disease, and amyotrophic lateral sclerosis. Although the identification and development of candidate compounds for CNS-directed MT-stabilizing therapies has been a challenge in drug discovery for many years, a growing number of molecules have now been identified that exhibit both MT-stabilizing activity and brain penetration. In this Viewpoint, we will highlight the potential utility of MT-active triazolopyrimidines, phenylpyrimidines, and related classes of non-naturally occurring small molecules that exhibit favorable druglike properties, including brain penetration and oral bioavailability. The mode of action of these small molecules has not as yet been fully elucidated at the molecular level. However, based on all available data, compounds from these classes appear to act on MTs in a potentially unique manner. Further characterization of these molecules may have important ramifications for drug discovery, especially in the area of CNS diseases.

The oxetane ring serves as an isostere of the carbonyl moiety, suggesting that oxetan-3-ol may be considered as a potential surrogate of the carboxylic acid functional group. To investigate this structural unit, as well as thietan-3-ol and the corresponding sulfoxide and sulfone derivatives, as potential carboxylic acid bioisosteres, a set of model compounds has been designed, synthesized, and evaluated for physicochemical properties. Similar derivatives of the cyclooxygenase inhibitor, ibuprofen, were also synthesized and evaluated for inhibition of eicosanoid biosynthesis in vitro. Collectively, the data suggest that oxetan-3-ol, thietan-3-ol, and related structures hold promise as isosteric replacements of the carboxylic acid moiety.

Previous studies revealed that examples of the non-naturally occurring microtubule (MT)-stabilizing triazolopyrimidines are both brain penetrant and orally bioavailable, indicating that this class of compounds may be potentially attractive in the development of MT-stabilizing therapies for the central nervous system (CNS). We now report on the pharmacokinetics (PK), pharmacodynamics (PD), and metabolism of a selected triazolopyrimidine congener, (S)-3-(4-(5-Chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy)-propan-1-ol (4). These studies revealed that 4 exhibits longer brain than plasma half-life that may be exploited to achieve a selective accumulation of the compound within the CNS. Furthermore, compound metabolism studies suggest that in plasma 4 is rapidly oxidized at the terminal hydroxyl group to form a comparatively inactive carboxylic acid metabolite. Peripheral administration of relatively low doses of 4 to normal mice was found to produce a significant elevation in acetylated α-tubulin, a marker of stable MTs, in the brain. Collectively, these results indicate that 4 may effectively target brain MTs at doses that produce minimal peripheral exposure.

Inclusions comprising the microtubule (MT)-stabilizing protein, tau, are found within neurons in the brains of patients with Alzheimer's disease and related neurodegenerative disorders that are broadly referred to as tauopathies. The sequestration of tau into inclusions is believed to cause a loss of tau function, such that MT structure and function are compromised, leading to neuronal damage. Recent data reveal that the brain-penetrant MT-stabilizing agent, epothilone D (EpoD), improves cognitive function and decreases both neuron loss and tau pathology in transgenic mouse models of tauopathy. There is thus a need to identify additional MT-stabilizing compounds with blood-brain barrier (BBB) permeability and slow brain clearance, as observed with EpoD. We report here that the MT-stabilizing natural product, dictyostatin, crosses the BBB in mice and has extended brain retention. Moreover, a single administration of dictyostatin to mice causes prolonged stabilization of MTs in the brain. In contrast, the structurally related MT-stabilizer, discodermolide, shows significantly less brain exposure. Thus, dictyostatin merits further investigation as a potential tauopathy therapeutic.

Alzheimer disease and several other neurodegenerative disorders are characterized by the accumulation of intraneuronal fibrils comprised of the protein Tau. Tau is normally a soluble protein that stabilizes microtubules, with splice isoforms that contain either three (3-R) or four (4-R) microtubule binding repeats. The formation of Tau fibrils is thought to result in neuronal damage, and inhibitors of Tau fibrillization may hold promise as therapeutic agents. The process of Tau fibrillization can be replicated in vitro, and a number of small molecules have been identified that inhibit Tau fibril formation. However, little is known about how these molecules affect Tau fibrillization. Here, we examined the mechanism by which the previously described aminothieno pyridazine (ATPZ) series of compounds inhibit Tau fibrillization. Active ATPZs were found to promote the oxidation of the two cysteine residues within 4-R Tau by a redox cycling mechanism, resulting in the formation of a disulfide-containing compact monomer that was refractory to fibrillization. Moreover, the ATPZs facilitated intermolecular disulfide formation between 3-R Tau monomers, leading to dimers that were capable of fibrillization. The ATPZs also caused cysteine oxidation in molecules unrelated to Tau. Interestingly, methylene blue, an inhibitor of Tau fibrillization under evaluation in Alzheimer disease clinical trials, caused a similar oxidation of cysteines in Tau and other molecules. These findings reveal that the ATPZs and methylene blue act by a mechanism that may affect their viability as potential therapeutic agents.

Neurodegenerative disorders referred to as tauopathies, which includes Alzheimer’s disease (AD), are characterized by insoluble deposits of the tau protein within neuron cell bodies and dendritic processes in the brain. Tau is normally associated with microtubules (MTs) in axons, where it provides MT stabilization and may modulate axonal transport. However, tau becomes hyperphosphorylated and dissociates from MTs in tauopathies, with evidence of reduced MT stability and defective axonal transport. This has led to the hypothesis that MT-stabilizing drugs may have potential for the treatment of tauopathies. Prior studies demonstrated that the brain-penetrant MT-stabilizing drug, epothilone D, had salutary effects in transgenic (Tg) mouse models of tauopathy, improving MT density and axonal transport, while reducing axonal dystrophy. Moreover, epothilone D enhanced cognitive performance and decreased hippocampal neuron loss, with evidence of reduced tau pathology. To date, epothilone D has been the only non-peptide small molecule MT-stabilizing agent to be evaluated in Tg tau mice. Herein, we demonstrate the efficacy of another small molecule brain-penetrant MT-stabilizing agent, dictyostatin, in the PS19 tau Tg mouse model. Although dictyostatin was poorly tolerated at once-weekly doses of 1 mg/kg or 0.3 mg/kg, likely due to gastrointestinal (GI) complications, a dictyostatin dose of 0.1 mg/kg was better tolerated, such that the majority of 6-month old PS19 mice, which harbor a moderate level of brain tau pathology, completed a 3-month dosing study without evidence of significant body weight loss. Importantly, as previously observed with epothilone D, the dictyostatin-treated PS19 mice displayed improved MT density and reduced axonal dystrophy, with a reduction of tau pathology and a trend toward increased hippocampal neuron survival relative to vehicle-treated PS19 mice. Thus, despite evidence of dose-limiting peripheral side effects, the observed positive brain outcomes in dictyostatin-treated aged PS19 mice reinforces the concept that MT-stabilizing compounds have significant potential for the treatment of tauopathies.

The replacement of a carboxylic acid with a surrogate structure, or (bio)-isostere, is a classical strategy in medicinal chemistry. The general underlying principle is that by maintaining the features of the carboxylic acid critical for biological activity, but appropriately modifying the physicochemical properties, improved analogs may result. In this context, a systematic assessment of the physicochemical properties of carboxylic acid isosteres would be desirable to enable more informed decisions of potential replacements to be used for analog design. Herein we report the structure–property relationships (SPR) of 35 phenylpropionic acid derivatives, in which the carboxylic acid moiety is replaced with a series of known isosteres. The data set generated provides an assessment of the relative impact on the physicochemical properties that these replacements may have compared to the carboxylic acid analog. As such, this study presents a framework for how to rationally apply isosteric replacements of the carboxylic acid functional group.

In neurodegenerative tauopathies, of which the most prevalent example is Alzheimer’s disease (AD), the aggregation of the microtubule (MT)-stabilizing protein tau is believed to have neuropathological consequences. Multiple studies indicate that deficits in axonal MTs and axonal transport may contribute to the neurodegenerative processes of these diseases. MT-stabilizing molecules have shown promise in restoring axonal MTs and transport, as well as cognitive performance, in animal models of human tauopathies. As a result, such compounds may be considered as potential candidates for the treatment of AD and related tauopathies. Many examples of MT-stabilizing natural products and derivatives thereof have been approved for cancer treatment; however, the use of these compounds for central nervous system (CNS) diseases may be challenging due to limited brain penetration and oral bioavailability, as well as potential systemic side effects. In this chapter, we review the progress made toward the identification and development of CNS-active MT-stabilizing candidate compounds, with an emphasis on nonnaturally occurring small molecules that exhibit favorable drug-like properties.

The emerging global epidemic of Alzheimer's disease (AD) demands novel paradigms to address the two unmet needs of the field: (a) cost-effective health care delivery programs/services, and (b) clinical and basic research to accelerate therapy discovery/development. This report outlines a model demonstration project, the Marian S. Ware Alzheimer Program at the University of Pennsylvania, which was designed to achieve four specific aims: (1) improve the integration and continuity of AD care; (2) identify biomarkers that detect the earliest presence of AD and related neurodegenerative cognitive disorders; (3) enhance both the design and conduct of clinical trials as well as review their results to more effectively test new AD therapies and translate valuable therapies into clinical practice; and (4) discover and develop novel disease-modifying small molecule treatments for AD. The "Ware-UPenn" program has been presented in this report as a useful prototype for partnerships between private philanthropy and academia in planning and developing programs to address a major national public health problem.

Neurons within the brains of those with AD (Alzheimer's disease) and related neurodegenerative disorders, collectively termed ‘tauopathies’, contain fibrillar inclusions composed of hyperphosphorylated tau protein. Tau is normally enriched in axons, where it binds and stabilizes MTs (microtubules). Tau hyperphosphorylation and aggregation probably result in reduced MT binding that could affect axonal transport and neuronal function. A possible therapeutic strategy to overcome a loss of tau function in tauopathies is administration of MT-stabilizing agents, such as those used in the treatment of cancer. However, these drugs elicit severe side effects, and most existing MT-stabilizing compounds have poor BBB (blood–brain barrier) permeability, which renders them unsuitable for tauopathy treatment. We identified EpoD (epothilone D) as a brain-penetrant MT-stabilizing agent with preferred pharmacokinetic and pharmacodynamic properties. EpoD was evaluated for its ability to compensate for tau loss-of-function in an established Tg (transgenic) mouse model, using both preventative and interventional dosing paradigms. EpoD at doses much lower than previously used in human cancer patients caused improved axonal MT density and decreased axonal dystrophy in the tau Tg mice, leading to an alleviation of cognitive deficits. Moreover, EpoD reduced the extent of tau pathology in aged tau Tg mice. Importantly, no adverse side effects were observed in the EpoD-treated mice. These results suggest that EpoD might be a viable drug candidate for the treatment of AD and related tauopathies.

Previous studies demonstrated that members of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors exhibit a promising combination of in vitro activity as well as favorable pharmacokinetic properties (i.e., brain-penetration and oral bioavailability). Here we report the synthesis and evaluation of several new analogues. These studies indicate that the thienopyridazine core is essential for inhibition of tau fibrillization in vitro, while the choice of the appropriate scaffold decoration is critical to impart desirable ADME-PK properties. Among the active, brain-penetrant ATPZ inhibitors evaluated, 5-amino-N-cyclopropyl-3-(4-fluorophenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide (43) was selected to undergo maximum tolerated dose and one-month tolerability testing in mice. The latter studies revealed that this compound is well-tolerated with no notable side-effects at an oral dose of 50 mg/kg/day.

Filamentous tau aggregates, the hallmark lesions of Alzheimer disease (AD), play key roles in neurodegeneration. Activation of protein degradation systems has been proposed to be a potential strategy for removing pathological tau, but it remains unclear how effectively tau aggregates can be degraded by these systems. By applying our previously established cellular model system of AD-like tau aggregate induction using preformed tau fibrils, we demonstrate that tau aggregates induced in cells with regulated expression of full-length mutant tau can be gradually cleared when soluble tau expression is suppressed. This clearance is at least partially mediated by the autophagy-lysosome pathway, although both the ubiquitin-proteasome system and the autophagy-lysosome pathway are deficient in handling large tau aggregates. Importantly, residual tau aggregates left after the clearance phase leads to a rapid reinstatement of robust tau pathology once soluble tau expression is turned on again. Moreover, we succeeded in generating monoclonal cells persistently carrying tau aggregates without obvious cytotoxicity. Live imaging of GFP-tagged tau aggregates showed that tau inclusions are dynamic structures constantly undergoing “fission” and “fusion,” which facilitate stable propagation of tau pathology in dividing cells. These findings provide a greater understanding of cell-to-cell transmission of tau aggregates in dividing cells and possibly neurons.

Tau, a protein that is enriched in neurons of the central nervous system (CNS), is thought to play a critical role in the stabilization of microtubules (MTs). Several neurodegenerative disorders referred to as tauopathies, including Alzheimer's disease and certain types of frontotemporal lobar degeneration, are characterized by the intracellular accumulation of hyperphosphorylated tau fibrils. Tau deposition into insoluble aggregates is believed to result in a loss of tau function that leads to MT destabilization, and this could cause neurodegeneration as intact MTs are required for axonal transport and normal neuron function. This tau loss-of-function hypothesis has been validated in a tau transgenic mouse model with spinal cord tau inclusions, where the MT-stabilizing agent, paclitaxel, increased spinal nerve MT density and improved motor function after drug absorption at neuromuscular junctions. Unfortunately, paclitaxel is a P-glycoprotein substrate and has poor blood–brain barrier permeability, making it unsuitable for the treatment of human tauopathies. We therefore examined several MT-stabilizing compounds from the taxane and epothilone natural product families to assess their membrane permeability and to determine whether they act as substrates or inhibitors of P-glycoprotein. Moreover, we compared brain and plasma levels of the compounds after administration to mice. Finally, we assessed whether brain-penetrant compounds could stabilize mouse CNS MTs. We found that several epothilones have significantly greater brain penetration than the taxanes. Furthermore, certain epothilones cause an increase in CNS MT stabilization, with epothilone D demonstrating a favorable pharmacokinetic and pharmacodynamic profile which suggests this agent merits further study as a potential tauopathy drug candidate.

Neurons in the brains of those with Alzheimer's disease (AD) and many frontotemporal dementias (FTDs) contain neurofibrillary tangles comprised of hyperphosphorylated tau protein. Tau normally stabilizes microtubules (MTs), and tau misfolding could lead to a loss of this function with consequent MT destabilization and neuronal dysfunction. Accordingly, a possible therapeutic strategy for AD and related “tauopathies” is treatment with a MT-stabilizing anti-cancer drug such as paclitaxel. However, paclitaxel and related taxanes have poor blood–brain barrier permeability and thus are unsuitable for diseases of the brain. We demonstrate here that the MT-stabilizing agent, epothilone D (EpoD), is brain-penetrant and we subsequently evaluated whether EpoD can compensate for tau loss-of-function in PS19 tau transgenic mice that develop forebrain tau inclusions, axonal degeneration and MT deficits. Treatment of 3-month-old male PS19 mice with low doses of EpoD once weekly for a 3 month period significantly improved CNS MT density and axonal integrity without inducing notable side-effects. Moreover, EpoD treatment reduced cognitive deficits that were observed in the PS19 mice. These results suggest that certain brain-penetrant MT-stabilizing agents might provide a viable therapeutic strategy for the treatment of AD and FTDs.

CRTh2 (DP2) is a prostaglandin D2 receptor implicated in the recruitment of eosinophils and basophils within the asthmatic lung. Here we report the discovery of a novel series of 3-indolyl sultam antagonists with low nM affinity for CRTh2. These compounds proved to be selective over the other primary prostaglandin D2 receptor (DP1) as well as the thromboxane A2 receptor (TP).

The microtubule-associated protein tau forms insoluble filaments that deposit as neurofibrillary tangles (NFTs) in the brains of those with Alzheimer's disease (AD) and other related neurodegenerative disorders. The presence of both NFTs and amyloid beta (Abeta)-containing senile plaques within the brain is required to confirm the diagnosis of AD. However, the demonstration that familial AD can be caused by mutations that result in increased Abeta production has resulted in AD drug discovery strategies that are largely focused on reducing brain Abeta levels, with substantially less emphasis on tau-directed approaches. This trend may be changing, as there are an increasing number of research programs that are exploring ways to reduce NFTs in AD and related tauopathies. We briefly review recent advances in tau-based drug discovery, with an emphasis on the identification of compounds that inhibit the assembly of tau into multimers and fibrils.

Cytoplasmic inclusions containing alpha-synuclein (alpha-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although alpha-Syn fibrils can be generated from recombinant alpha-Syn protein in vitro, the production of fibrillar alpha-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular alpha-Syn aggregation can be triggered by the introduction of exogenously produced recombinant alpha-Syn fibrils into cultured cells engineered to overexpress alpha-Syn. Unlike unassembled alpha-Syn, these alpha-Syn fibrils "seeded" recruitment of endogenous soluble alpha-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular alpha-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of alpha-Syn fibrils can alter this balance by acting as seeds for aggregation.

The discovery that mutations within the tau gene lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) provided direct evidence that tau alterations can lead to neurodegenerative disease. While the presence of tau fibrils and tangles is a common feature of all tauopathies, including Alzheimer’s disease (AD), data are emerging from biochemical, cell-based and transgenic mouse studies which suggest that a pre-fibrillar form of pathological tau may play a key role in eliciting central nervous system (CNS) neurodegeneration and behavioral impairments. Herein we review recent findings that implicate diffusible tau pathology in the onset of neurodegeneration, and discuss the implications of these findings as they relate to tau tangles and possible therapeutic strategies for the treatment of AD and related tauopathies.

Senile plaques comprised of Aβ peptides are a hallmark of Alzheimer’s disease (AD) brain, as are activated glia that release inflammatory molecules, including eicosanoids. Previous studies have demonstrated that amyloid precursor protein (APP) and Aβ levels can be increased through activation of thromboxane A2-prostanoid (TP) receptors on neurons. We demonstrate that TP receptor regulation of APP expression depends on Gαq-signaling and conventional protein kinase C isoforms. Importantly, we discovered that Gαq-linked prostaglandin E2 and leukotriene D4 receptors also regulate APP expression. Prostaglandin E2 and thromboxane A2, as well as total APP levels, were found to be elevated in the brains of aged 5XFAD transgenic mice harboring Aβ plaques and activated glia, suggesting that increased APP expression resulted from eicosanoid binding to Gαq-linked neuronal receptors. Notably, inhibition of eicosanoid synthesis significantly lowered brain APP protein levels in aged 5XFAD mice. These results provide new insights into potential AD therapeutic strategies.