Discovery and characterization of novel TRPML1 agonists.

Screening a library of >100,000 compounds identified the substituted tetrazole compound 1 as a selective TRPML1 agonist. Both enantiomers of compound 1 were separated and profiled in vitro and in vivo. Their selectivity, ready availability and CNS penetration should enable them to serve as the tool compounds of choice in future TRPML1 channel activation studies. SAR studies on conformationally locked macrocyclic analogs further improved the TRPML1 agonist potency while retaining the selectivity.

Discovery of oxazoline enhancers of cellular progranulin release.

Phenotypic screening of an annotated small molecule library and initial SAR studies identified compound 2 as a robust enhancer of progranulin secretion. Detailed SAR development on conformationally restricted carbamate isosteres led to the identification of compound 60 with a 3-fold improvement in BV-2 potency and a 9-fold decrease in hERG inhibition over compound 2, substantially improving this important margin of safety relative to compound 2.

Discovery of quinuclidine modulators of cellular progranulin.

Phenotypic screening of an annotated small molecule library identified the quinuclidine tetrahydroisoquinoline solifenacin (1) as a robust enhancer of progranulin secretion with single digit micromolar potency in a murine microglial (BV-2) cell line. Subsequent SAR development led to the identification of 29 with a 38-fold decrease in muscarinic receptor antagonist activity and a 10-fold improvement in BV-2 potency.

Granulins rescue inflammation, lysosome dysfunction, and neuropathology in a mouse model of progranulin deficiency.

Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.

Discovery of the Oxadiazine FRM-024: A Potent CNS-Penetrant Gamma Secretase Modulator.

The recent approval of aducanumab for Alzheimer's disease has heightened the interest in therapies targeting the amyloid hypothesis. Our research has focused on identification of novel compounds to improve amyloid processing by modulating gamma secretase activity, thereby addressing a significant biological deficit known to plague the familial form of the disease. Herein, we describe the design, synthesis, and optimization of new gamma secretase modulators (GSMs) based on previously reported oxadiazine 1. Potency improvements with a focus on predicted and measured properties afforded high-quality compounds further differentiated via robust Aß42 reductions in both rodents and nonhuman primates. Extensive preclinical profiling, efficacy studies, and safety studies resulted in the nomination of FRM-024, (+)-cis-5-(4-chlorophenyl)-6-cyclopropyl-3-(6-methoxy-5-(4-methyl-1H-imidazole-1-yl)pyridin-2-yl)-5,6-dihydro-4H-1,2,4-oxadiazine, as a GSM preclinical candidate for familial Alzheimer's disease.

Pro-inflammatory cytokines modulate glial apolipoprotein E secretion.

Alzheimer's disease (AD) is a neurological disorder characterized by plaques and an elevated immune response. Specifically, increased expression of interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha, has been observed in AD cerebrospinal fluid and temporal brain tissue. Both of these immunomodulators were shown to carry genetic variants that increase the risk of developing AD. Studies have also established the apolipoprotein E (apoE) gene to be a risk factor for AD with epsilon4 carriers having been found to show lower levels of brain apoE. In the present study, treatment of primary rat mixed glial cell cultures with IL-1beta induced a significant increase in extracellular apoE protein. In contrast, treatment primary rat astrocyte and mixed glial cell cultures with TNF-alpha significantly reduced extracellular apoE protein levels. These results are consistent with the notion that elevated cytokine expression directly modulates immunosuppression and indirectly apoE-mediated neuronal remodeling.

BACE1 Inhibitor MK-8931 Alters Formation but Not Stability of Dendritic Spines.

Beta-site amyloid-precursor-protein cleaving enzyme 1 (BACE1) is the rate limiting protease in the production of the amyloid-beta peptide (Aß), which is considered to be the causative agent in the pathogenesis of Alzheimer's Disease (AD). Therefore, the therapeutic potential of pharmacological BACE1 inhibitors is currently tested in clinical trials for AD treatment. To ensure a positive clinical outcome it is crucial to identify and evaluate adverse effects associated with BACE1 inhibition. Preclinical studies show that chronic blockade of BACE1 activity alters synaptic functions and leads to loss of dendritic spines. To assess the mechanism of synapse loss, dendritic spine dynamics of pyramidal layer V cells were monitored by in vivo two-photon microscopy in the somatosensory cortex of mice, treated with the BACE1 inhibitor MK-8931. MK-8931 treatment significantly reduced levels of Aß40 and density of dendritic spines in the brain. However, the steady decline in dendritic spine density specifically resulted from a diminished formation of new spines and not from a loss of stable spines. Furthermore, the described effects on spine formation were transient and recovered after inhibitor withdrawal. Since MK-8931 inhibition did not completely abolish spine formation, our findings suggest that carefully dosed inhibitors might be therapeutically effective without affecting the structural integrity of excitatory synapses if given at an early disease stage.

Generation and Partial Characterization of Rabbit Monoclonal Antibody to Amyloid-ß Peptide 1-37 (Aß37).

Secreted soluble amyloid-ß 1-37 (Aß37) peptide is one of the prominent Aß forms next to Aß40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aß37 levels in combination with Aß38, Aß40, and Aß42 to support the diagnosis of patients with probable Alzheimer's disease (AD), and the value of antibody to Aß37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aß37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aß37, and 2) to determine whether the antibody detects changes in Aß37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aß37 was found to be specific to Aß37, since it did not react with Aß36, Aß38, Aß39, Aß40, and Aß42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aß37. The antibody was sensitive enough to measure CSF and plasma Aß37 levels in ELISA. Immunohistological studies showed the presence of Aß37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aß37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.

Design, Synthesis, and Evaluation of a Novel Series of Oxadiazine Gamma Secretase Modulators for Familial Alzheimer's Disease.

Herein we describe the design, synthesis, and evaluation of a novel series of oxadiazine-based gamma secretase modulators obtained via isosteric amide replacement and critical consideration of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent predicted CNS drug-like properties and desirable ADME/PK profile. This lead compound demonstrated robust Aß42 reductions and subsequent Aß37 increases in both rodent brain and CSF at 30 mg/kg dosed orally.

Gamma Secretase Modulators: New Alzheimer's Drugs on the Horizon?

The rapidly aging population desperately requires new therapies for Alzheimer's disease. Despite years of pharmaceutical research, limited clinical success has been realized, with several failed disease modification therapies in recent years. On the basis of compelling genetic evidence, the pharmaceutical industry has put a large emphasis on brain beta amyloid (Aß) either through its removal via antibodies or by targeting the proteases responsible for its production. In this Perspective, we focus on the development of small molecules that improve the activity of one such protease, gamma secretase, through an allosteric binding site to preferentially increase the concentration of the shorter non-amyloidogenic Aß species. After a few early failures due to poor drug-like properties, the industry is now on the cusp of delivering gamma secretase modulators for clinical proof-of-mechanism studies that combine potency and efficacy with improved drug-like properties such as lower cLogP, high central nervous system multiparameter optimization scores, and high sp(3) character.

Characterization of FRM-36143 as a new γ-secretase modulator for the potential treatment of familial Alzheimer's disease.

BACKGROUND: Familial Alzheimer's disease (FAD) is caused by mutations in the amyloid precursor protein (APP) or presenilin (PS). Most PS mutations, which account for the majority of FAD cases, lead to an increased ratio of longer to shorter forms of the amyloid beta (Aß) peptide. The therapeutic rationale of γ-secretase modulators (GSMs) for Alzheimer's disease is based on this genetic evidence as well as on enzyme kinetics measurements showing changes in the processivity of the γ-secretase complex. This analysis suggests that GSMs could potentially offset some of the effects of PS mutations on APP processing, thereby addressing the root cause of early onset FAD. Unfortunately, the field has generated few, if any, molecules with good central nervous system (CNS) drug-like properties to enable proof-of-mechanism studies. METHOD: We characterized the novel GSM FRM-36143 using multiple cellular assays to determine its in vitro potency and off-target activity as well as its potential to reverse the effect of PS mutations. We also tested its efficacy in vivo in wild-type mice and rats. RESULTS: FRM-36143 has much improved CNS drug-like properties compared to published GSMs. It has an in vitro EC50 for Aß42 of 35 nM in H4 cells, can reduce Aß42 to 58 % of the baseline in rat cerebrospinal fluid, and also increases the non-amyloidogenic peptides Aß37 and Aß38. It does not inhibit Notch processing, nor does it inhibit 24-dehydrocholesterol reductase (DHCR24) activity. Most interestingly, it can reverse the effects of presenilin mutations on APP processing in vitro. CONCLUSIONS: FRM-36143 possesses all the characteristics of a GSM in terms of Aß modulation Because FRM-36143 was able to reverse the effect of PS mutations, we suggest that targeting patients with this genetic defect would be the best approach at testing the efficacy of a GSM in the clinic. While the amyloid hypothesis is still being tested with ß-site APP-cleaving enzyme inhibitors and monoclonal antibodies in sporadic AD, we believe it is not a hypothesis for FAD. Since GSMs can correct the molecular defect caused by PS mutations, they have the promise to provide benefits to the patients when treated early enough in the course of the disease.

Could lipoprotein lipase play a role in Alzheimer's disease?

This paper reviews recent literature on the role of lipoprotein lipase in the central nervous system with a focus on its recently described role in synaptic remodeling. This novel role could have implication for Alzheimer's disease treatment.

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice.

BACKGROUND: A hallmark of Alzheimer's disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aß42 and Aß40. Many drug discovery efforts have focused on decreasing the production of Aß42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aß production to favor shorter, less amyloidogenic peptides than Aß42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer's disease. RESULTS: Here we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aß42 in H4 cells (IC50 = 67 nM) and increased the shorter Aß38 by 1.7 fold at the IC50 for lowering of Aß42. AßTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aß42 and did not alter AßTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aß deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aß aggregates, amyloid plaques, inflammatory markers, and cognitive deficits. CONCLUSIONS: EVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aß42, attenuated memory deficits, and reduced Aß plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer's disease.

A deficit in astroglial organization causes the impaired reactive sprouting in human apolipoprotein E4 targeted replacement mice.

The epsilon4 allele of apolipoprotein (apo)E associates with an increased risk of developing Alzheimer's disease (AD) as well as an earlier age of onset. However, the exact mechanisms by which apoE4 confers such susceptibility is currently unknown. We used a human apoE targeted replacement (hE-TR) mouse model to investigate the allele-specific response to entorhinal cortex lesion (ECL). We observed a marked impairment in reactive sprouting in hE4 mice compared to hE3 mice. ApoE expression was similar between genotypes at days post-lesion (DPL) 2 and 14. Thirty days post-lesion, hE4 mice had more reactive astrocytes as well as a defective outward migration pattern of the astrocytes in the dentate gyrus. The expression of the anti-inflammatory cytokine IL-1ra was delayed in hE4 mice compared to hE3 mice. ApoE and beta-amyloid (Abeta) 1-40 accumulated at 30 DPL in hE4 mice. These results suggest that the presence of apoE4 delays the astroglial repair process and indirectly compromises synaptic remodeling.

A polymorphism in lipoprotein lipase affects the severity of Alzheimer's disease pathophysiology.

Emerging evidences indicate a role for lipoprotein lipase (LPL) in degenerative states. Genetic variations in the LPL gene were previously associated to lipid imbalance and coronary artery disease (CAD) risk and severity, a condition that shares pathological features with common Alzheimer's disease (AD). To evaluate whether these genetic variations associate with the risk and pathophysiology of common AD, autopsy-confirmed patients (242 controls, 153 AD) were genotyped for a PvuII single nucleotide polymorphism (SNP; rs285; referred to as the P+ allele) of LPL. Brain LPL mRNA levels, cholesterol levels, amyloid concentration, senile plaques and neurofibrillary tangles density counts were measured and contrasted with specific LPL genotypes. When adjusted for age and sex, homozygosity for the P+ allele resulted in an odds ratio of 2.3 for the risk of developing AD. More importantly, we report that the presence of the P+ allele of LPL significantly affects its mRNA expression level (n = 51; P = 0.026), brain tissue cholesterol levels (n = 55; P = 0.0013), neurofibrillary tangles (n = 52; P = 0.025) and senile plaque (n = 52; P = 0.022) densities. These results indicate that a common polymorphism in the lipoprotein lipase gene modulates the risk level for sporadic AD in the eastern Canadian population but more importantly, indirectly modulates the pathophysiology of the brain in autopsy-confirmed cases.

A role for caveolin-1 in post-injury reactive neuronal plasticity.

Remodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin-1 is a cholesterol-binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up-regulation of caveolin-1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin-1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin-1-overexpressing cells revealed increases in synaptophysin and GAP-43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin-1-overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin-1-overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin-1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin-1 plays an active role in the regulation of injury-induced synaptic and terminal remodeling in the adult CNS.

The absence of ABCA1 decreases soluble ApoE levels but does not diminish amyloid deposition in two murine models of Alzheimer disease.

ABCA1, a cholesterol transporter expressed in the brain, has been shown recently to be required to maintain normal apoE levels and lipidation in the central nervous system. In addition, ABCA1 has been reported to modulate beta-amyloid (Abeta) production in vitro. These observations raise the possibility that ABCA1 may play a role in the pathogenesis of Alzheimer disease. Here we report that the deficiency of ABCA1 does not affect soluble or guanidine-extractable Abeta levels in Tg-SwDI/B or amyloid precursor protein/presenilin 1 (APP/PS1) mice, but rather is associated with a dramatic reduction in soluble apoE levels in brain. Although this reduction in apoE was expected to reduce the amyloid burden in vivo, we observed that the parenchymal and vascular amyloid load was increased in Tg-SwDI/B animals and was not diminished in APP/PS1 mice. Furthermore, we observed an increase in the proportion of apoE retained in the insoluble fraction, particularly in the APP/PS1 model. These data suggested that ABCA1-mediated effects on apoE levels and lipidation influenced amyloidogenesis in vivo.

Cholesterol homeostasis and the pathophysiology of Alzheimer's disease.

The past 4 years have seen a growing interest in cholesterol metabolism and its relationship to Alzheimer's disease. From the first report linking cholesterol and beta-amyloid metabolisms to the recent positive report on the use of atorvastatin (Lipitor, Pfizer Inc.), a cholesterol-lowering drug, in mild-to-moderate Alzheimer's disease, this review examines the scientific progress pertaining to etiopathology of Alzheimer's disease over the past 15 years and the central role of lipids in this field of research. The role of key proteins involved in this metabolic pathway such as apolipoprotein E, lipoprotein lipase, caveolin, hydroxy-methylglutaryl Coenzyme A reductase, low-density lipoprotein receptors, cholesterol 24-hydroxylase, acyl-coenzyme A:cholesterol acyltransferase and beta-amyloid are discussed.

A role for lipoprotein lipase during synaptic remodeling in the adult mouse brain.

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules found in lipoprotein particles. This particular lipase also has a role in the binding of lipoprotein particles to different cell-surface receptors. LPL has been identified in the brain but has no specific function yet. This study aimed at elucidating the role of LPL in the brain in response to injury. Mice were subjected to hippocampal deafferentation using the entorhinal cortex lesion and mRNA and protein expression were assessed over a time-course of degeneration/reinnervation. Hippocampal LPL levels peaked at 2 days post-lesion (DPL) both at the mRNA and protein levels. No change was observed for receptors of the LDL-receptor family or RAP at DPL 2 in the hippocampus but the glia-specific syndecan-4 was found to be significantly upregulated at DPL 2. These results suggest that LPL is involved in the recycling of cholesterol and lipids released from degenerating terminals after a lesion through a syndecan-4-dependent pathway.