γ-Secretase Modulator BPN15606 Reduced Aß42 and Aß40 and Countered Alzheimer-Related Pathologies in a Mouse Model of Down Syndrome.

OBJECTIVES: Due to increased gene dose for the amyloid precursor protein (APP), elderly adults with Down syndrome (DS) are at a markedly increased risk of Alzheimer's disease (AD), known as DS-AD. How the increased APP gene dose acts and which APP products are responsible for DS-AD is not well understood, thus limiting strategies to target pathogenesis. As one approach to address this question, we used a novel class of γ-secretase modulators that promote γ-site cleavages by the γ-secretase complex, resulting in lower levels of the Aß42 and Aß40 peptides. METHODS: Ts65Dn mice, which serve as a model of DS, were treated via oral gavage with 10 mg/kg/weekday of BPN15606 (a potent and novel pyridazine-containing γ-secretase modulators). Treatment started at 3 months-of-age and lasted for 4 months. RESULTS: Demonstrating successful target engagement, treatment with BPN15606 significantly decreased levels of Aß40 and Aß42 in the cortex and hippocampus; it had no effect on full-length APP or its C-terminal fragments in either 2 N or Ts65Dn mice. Importantly, the levels of total amyloid-ß were not impacted, pointing to BPN15606-mediated enhancement of processivity of γ-secretase. Additionally, BPN15606 rescued hyperactivation of Rab5, a protein responsible for regulating endosome function, and normalized neurotrophin signaling deficits. BPN15606 treatment also normalized the levels of synaptic proteins and tau phosphorylation, while reducing astrocytosis and microgliosis, and countering cognitive deficits. INTERPRETATION: Our findings point to the involvement of increased levels of Aß42 and/or Aß40 in contributing to several molecular and cognitive traits associated with DS-AD. They speak to increased dosage of the APP gene acting through heightened levels of Aß42 and/or Aß40 as supporting pathogenesis. These findings further the interest in the potential use of γ-secretase modulators for treating and possibly preventing AD in individuals with DS. ANN NEUROL 2024.

Retromer Proteins Reduced in Down Syndrome and the Dp16 Model: Impact of APP Dose and Preclinical Studies of a γ-Secretase Modulator.

OBJECTIVE: The retromer complex plays an essential role in intracellular endosomal sorting. Deficits in the retromer complex are linked to enhanced Aß production. The levels of the components of the retromer complex are reported to be downregulated in Alzheimer disease (AD). Down syndrome (DS) shares neuropathological features with AD. Recent evidence points to dysregulation of the retromer complex in DS. The mechanisms underlying retromer deficits in DS and AD are poorly understood. METHODS: We measured the levels of retromer components in the frontal cortex of cases of DS-AD (AD in DS) as well as DS; the frontal cortex of a person partially trisomic (PT-DS) for human chromosome 21 (HSA21), whose genome had only the normal 2 copies of the APP gene, was also examined. We also analyzed these proteins in the Dp16 mouse model of DS. To further explore the molecular mechanism for changes in the retromer complex, we treated Dp16 mice with a γ-secretase modulator (GSM; 776890), a treatment that reduces the levels of Aß42 and Aß40. RESULTS: We found VPS26A, VPS26B, and VPS29, but not VPS35, were significantly reduced in both DS and DS-AD, but not in PT-DS. Downregulation of VPS26A, VPS26B, and VPS29 was recapitulated in the brains of old Dp16 mice (at 16 months of age) and required increased App gene dose. Significantly, GSM treatment completely prevented reductions of the retromer complex. INTERPRETATION: Our studies point to increased APP gene dose as a compromising retromer function in DS and suggest a causal role for Aß42 and Aß40. ANN NEUROL 2023;94:245-258.

Endotype reversal as a novel strategy for screening drugs targeting familial Alzheimer's disease.

While amyloid-ß (Aß) plaques are considered a hallmark of Alzheimer's disease, clinical trials focused on targeting gamma secretase, an enzyme involved in aberrant Aß peptide production, have not led to amelioration of AD symptoms or synaptic dysregulation. Screening strategies based on mechanistic, multi-omics approaches that go beyond pathological readouts can aid in the evaluation of therapeutics. Using early-onset Alzheimer's (EOFAD) disease patient lineage PSEN1A246E iPSC-derived neurons, we performed RNA-seq to characterize AD-associated endotypes, which are in turn used as a screening evaluation metric for two gamma secretase drugs, the inhibitor Semagacestat and the modulator BPN-15606. We demonstrate that drug treatment partially restores the neuronal state while concomitantly inhibiting cell cycle re-entry and dedifferentiation endotypes to different degrees depending on the mechanism of gamma secretase engagement. Our endotype-centric screening approach offers a new paradigm by which candidate AD therapeutics can be evaluated for their overall ability to reverse disease endotypes.

Preclinical validation of a potent γ-secretase modulator for Alzheimer's disease prevention.

A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer's disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aß42 and to a lesser extent Aß40, while concomitantly augmenting production of Aß38 and Aß37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUCeffective, the systemic exposure required for reducing levels of Aß42 in rat brain by 50%.

Design and synthesis of novel methoxypyridine-derived gamma-secretase modulators.

The evolution of gamma-secretase modulators (GSMs) through the introduction of novel heterocycles with the goal of aligning activity for reducing the levels of Aß42 and properties consistent with a drug-like molecule are described. The insertion of a methoxypyridine motif within the tetracyclic scaffold provided compounds with improved activity for arresting Aß42 production as well as improved properties, including solubility. In vivo pharmacokinetic analysis demonstrated that several compounds within the novel series were capable of crossing the BBB and accessing the therapeutic target. Treatment with methoxypyridine-derived compound 64 reduced Aß42 levels in the plasma of J20 mice, in addition to reducing Aß42 levels in the plasma and brain of Tg2576 mice.

Molecular imaging of Alzheimer's disease-related gamma-secretase in mice and nonhuman primates.

The pathogenesis of Alzheimer's disease (AD) is primarily driven by brain accumulation of the amyloid-ß-42 (Aß42) peptide generated from the amyloid-ß precursor protein (APP) via cleavages by ß- and γ-secretase. γ-Secretase is a prime drug target for AD; however, its brain regional expression and distribution remain largely unknown. Here, we are aimed at developing molecular imaging tools for visualizing γ-secretase. We used our recently developed γ-secretase modulators (GSMs) and synthesized our GSM-based imaging agent, [11C]SGSM-15606. We subsequently performed molecular imaging in rodents, including AD transgenic animals, and macaques, which revealed that our probe displayed good brain uptake and selectivity, stable metabolism, and appropriate kinetics and distribution for imaging γ-secretase in the brain. Interestingly, rodents and macaques shared certain brain areas with high γ-secretase expression, suggesting a functional conservation of γ-secretase. Collectively, we have provided the first molecular brain imaging of γ-secretase, which may not only accelerate our drug discovery for AD but also advance our understanding of AD.

γ-Secretase Partitioning into Lipid Bilayers Remodels Membrane Microdomains after Direct Insertion.

γ-Secretase is a multisubunit complex that catalyzes intramembranous cleavage of transmembrane proteins. The lipid environment forms membrane microdomains that serve as spatio-temporal platforms for proteins to function properly. Despite substantial advances in the regulation of γ-secretase, the effect of the local membrane lipid microenvironment on the regulation of γ-secretase is poorly understood. Here, we characterized and quantified the partitioning of γ-secretase and its substrates, the amyloid precursor protein (APP) and Notch, into lipid bilayers using solid-supported model membranes. Notch substrate is preferentially localized in the liquid-disordered (Ld) lipid domains, whereas APP and γ-secretase partition as single or higher complex in both phases but highly favor the ordered phase, especially after recruiting lipids from the ordered phase, indicating that the activity and specificity of γ-secretase against these two substrates are modulated by membrane lateral organization. Moreover, time-elapse measurements reveal that γ-secretase can recruit specific membrane components from the cholesterol-rich Lo phase and thus creates a favorable lipid environment for substrate recognition and therefore activity. This work offers insight into how γ-secretase and lipid modulate each other and control its activity and specificity.

The GSM BPN-15606 as a Potential Candidate for Preventative Therapy in Alzheimer's Disease.

BACKGROUND: In the amyloid hypothesis of Alzheimer's disease (AD), the dysregulation of amyloid-ß protein (Aß) production and clearance leads to amyloid deposits, tau tangles, neuronal loss, and cognitive dysfunction. Thus far, therapies targeting the enzymes responsible for Aß production have been found ineffective or having significant side effects. OBJECTIVE: To test whether a γ-secretase modulator, BPN-15606, is an effective disease-modifying or preventative treatment in the PSAPP mouse model of AD. METHODS: We treated pre-plaque (3-month-old) and post-plaque (6-month-old) PSAPP AD transgenic mice for 3 months and examined behavioral, biochemical, and pathological end points. RESULTS: BPN-15606 attenuated cognitive impairment and reduced amyloid plaque load, microgliosis, and astrogliosis associated with the AD phenotype of PSAPP mice when administered to pre-plaque (3-month-old) but was ineffective when administered to post-plaque (6-month-old) mice. No treatment-related toxicity was observed. CONCLUSION: BPN-15606 appears efficacious when administered prior to significant pathology.

Cholesterol Metabolism Is a Druggable Axis that Independently Regulates Tau and Amyloid-ß in iPSC-Derived Alzheimer's Disease Neurons.

Genetic, epidemiologic, and biochemical evidence suggests that predisposition to Alzheimer's disease (AD) may arise from altered cholesterol metabolism, although the molecular pathways that may link cholesterol to AD phenotypes are only partially understood. Here, we perform a phenotypic screen for pTau accumulation in AD-patient iPSC-derived neurons and identify cholesteryl esters (CE), the storage product of excess cholesterol, as upstream regulators of Tau early during AD development. Using isogenic induced pluripotent stem cell (iPSC) lines carrying mutations in the cholesterol-binding domain of APP or APP null alleles, we found that while CE also regulate Aß secretion, the effects of CE on Tau and Aß are mediated by independent pathways. Efficacy and toxicity screening in iPSC-derived astrocytes and neurons showed that allosteric activation of CYP46A1 lowers CE specifically in neurons and is well tolerated by astrocytes. These data reveal that CE independently regulate Tau and Aß and identify a druggable CYP46A1-CE-Tau axis in AD.

Soluble Gamma-secretase Modulators Attenuate Alzheimer's ß-amyloid Pathology and Induce Conformational Changes in Presenilin 1.

A central pathogenic event of Alzheimer's disease (AD) is the accumulation of the Aß42 peptide, which is generated from amyloid-ß precursor protein (APP) via cleavages by ß- and γ-secretase. We have developed a class of soluble 2-aminothiazole γ-secretase modulators (SGSMs) that preferentially decreases Aß42 levels. However, the effects of SGSMs in AD animals and cells expressing familial AD mutations, as well as the mechanism of γ-secretase modulation remain largely unknown. Here, a representative of this SGSM scaffold, SGSM-36, was investigated using animals and cells expressing FAD mutations. SGSM-36 preferentially reduced Aß42 levels without affecting either α- and ß-secretase processing of APP nor Notch processing. Furthermore, an allosteric site was identified within the γ-secretase complex that allowed access of SGSM-36 using cell-based, fluorescence lifetime imaging microscopy analysis. Collectively, these studies provide mechanistic insights regarding SGSMs of this class and reinforce their therapeutic potential in AD.

Pharmacological and Toxicological Properties of the Potent Oral γ-Secretase Modulator BPN-15606.

Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42-amino-acid amyloid-ß peptide (Aß), as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the Aß42 peptide in the brain, as a result of either increased production or decreased clearance, are postulated to initiate and drive the AD pathologic process. We initially introduced a novel class of bridged aromatics referred tγ-secretase modulatoro as γ-secretase modulators that inhibited the production of the Aß42 peptide and to a lesser degree the Aß40 peptide while concomitantly increasing the production of the carboxyl-truncated Aß38 and Aß37 peptides. These modulators potently lower Aß42 levels without inhibiting the γ-secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. In this study, we report a large number of pharmacological studies and early assessment of toxicology characterizing a highly potent γ-secretase modulator (GSM), (S)-N-(1-(4-fluorophenyl)ethyl)-6-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)-4-methylpyridazin-3-amine (BPN-15606). BPN-15606 displayed the ability to significantly lower Aß42 levels in the central nervous system of rats and mice at doses as low as 5-10 mg/kg, significantly reduce Aß neuritic plaque load in an AD transgenic mouse model, and significantly reduce levels of insoluble Aß42 and pThr181 tau in a three-dimensional human neural cell culture model. Results from repeat-dose toxicity studies in rats and dose escalation/repeat-dose toxicity studies in nonhuman primates have designated this GSM for 28-day Investigational New Drug-enabling good laboratory practice studies and positioned it as a candidate for human clinical trials.

Design and synthesis of aminothiazole modulators of the gamma-secretase enzyme.

The design and construction of a series of novel aminothiazole-derived γ-secretase modulators is described. The incorporation of heterocyclic replacements of the terminal phenyl D-ring of lead compound 1 was conducted in order to align potency with favorable drug-like properties. γ-Secretase modulator 28 displayed good activity for in vitro inhibition of Aß42, as well as substantial improvement in ADME and physicochemical properties, including aqueous solubility. Pharmacokinetic evaluation of compound 28 in mice revealed good brain penetration, as well as good clearance, half-life, and volume of distribution which collectively support the continued development of this class of compounds.

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site.

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of substituted benzoxazole ligands were investigated.

Nanopore-based conformational analysis of a viral RNA drug target.

Nanopores are single-molecule sensors that show exceptional promise as a biomolecular analysis tool by enabling label-free detection of small amounts of sample. In this paper, we demonstrate that nanopores are capable of detecting the conformation of an antiviral RNA drug target. The hepatitis C virus uses an internal ribosome entry site (IRES) motif in order to initiate translation by docking to ribosomes in its host cell. The IRES is therefore a viable and important drug target. Drug-induced changes to the conformation of the HCV IRES motif, from a bent to a straight conformation, have been shown to inhibit HCV replication. However, there is presently no straightforward method to analyze the effect of candidate small-molecule drugs on the RNA conformation. In this paper, we show that RNA translocation dynamics through a 3 nm diameter nanopore is conformation-sensitive by demonstrating a difference in transport times between bent and straight conformations of a short viral RNA motif. Detection is possible because bent RNA is stalled in the 3 nm pore, resulting in longer molecular dwell times than straight RNA. Control experiments show that binding of a weaker drug does not produce a conformational change, as consistent with independent fluorescence measurements. Nanopore measurements of RNA conformation can thus be useful for probing the structure of various RNA motifs, as well as structural changes to the RNA upon small-molecule binding.

Soluble γ-secretase modulators selectively inhibit the production of the 42-amino acid amyloid ß peptide variant and augment the production of multiple carboxy-truncated amyloid ß species.

Alzheimer's disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid ß peptide variant (Aß42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aß42 compared to the levels of Aß40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769-780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255-1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aß42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aß42 levels. The effects of one potent SGSM on Aß peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aß species. This SGSM also inhibited Aß42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.

Hepatitis C virus translation inhibitors targeting the internal ribosomal entry site.

The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5' UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide, and small molecule ligands. Emphasis will be given to the IRES subdomain IIa, which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA.

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.

1,3-Diazepanes of natural product-like complexity from cyanamide-induced rearrangement of epoxy-delta-lactams.

A synthetic procedure toward 1,3-diazepane scaffolds of natural product-like complexity was developed for the construction of RNA-directed ligand libraries. A molecular building block was designed that combines the characteristics of RNA-binding natural products, including a high density of hydrogen bond donors and acceptors around a rigid, nonplanar scaffold with straightforward total-synthetic accessibility that permits extensive control over the chemical space. The synthesis of the 1,3-diazepane scaffold was achieved via an unprecedented cyanamide-induced rearrangement of epoxy-delta-lactams.