Γ-secretase Modulators Research Articles

Iminoheterocycles as γ-secretase modulators

The synthesis of a novel series of iminoheterocycles and their structure–activity relationship (SAR) as modulators of γ-secretase activity will be detailed. Encouraging SAR generated from a monocyclic core led to a structurally unique bicyclic core. Selected compounds exhibit good potency as γ-secretase modulators, excellent rat pharmacokinetics, and lowering of Aβ 42 levels in various in vivo models.

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

Following ectodomain shedding by beta-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by gamma-secretase result in the release of amyloid-beta (Abeta) peptides of variable length. Abeta peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent gamma-secretase modulator (GSM), selectively reduces Abeta42 production in favor of shorter Abeta species, such as Abeta38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Abeta42 levels and concomitantly increased Abeta38 levels. However, the precise molecular mechanism by which GSMs modulate Abeta production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related Abeta42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Abeta sequence. Strikingly, the attenuated APP-TMS interaction by GSMs correlated strongly with Abeta42-lowering activity and binding strength to the Abeta sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Abeta42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease.

Pharmacokinetic and pharmacodynamic analysis of the gamma-secretase modulator (GSM) EVP-0015962

Gamma (γ)-secretase has been characterized as the enzyme responsible for the Aβ C-terminal cleavage, which results in the production of Aβ peptides of various lengths including Aβx-40 (> 80%) and Aβx-42 (˜10%). The latter amyloidogenic Aβ42 is deposited early in the course of Alzheimer's disease (AD) and also readily forms soluble and potent neurotoxic oligomers. In early onset AD, a greater proportion of Aβ42 is formed, strongly implicating it as central to the disease etiology. Selective inhibition of this form of Aβ without the affecting other γ-secretase substrates may represent a disease modifying approach for the treatment of AD, as represented by the novel γ-secretase modulator EVP-0015962. - In-vitro characterization of EVP-0015962, carried out in transfected human cells and in normal rat primary cortical neurons, has shown potent reduction of Aβ42 with a concomitant increase in Aβ38 while total levels of Aβ remain essentially unchanged. These modulatory effects have also been observed in cell extracts enriched for γ-secretase activity and do not inhibit signaling via, e.g., the Notch pathway. In-vivo proof of principle in normal rodents (C57/Bl6 mice and Sprague-Dawley rats) demonstrates that a single orally administered dose of EVP-0015962 will exert the modulatory effect on γ-secretase activity, significantly reducing Aβ42 with a concomitant increase in Aβ38 while maintaining total Aβ at a constant level. These overall effects are qualitatively consistent between brain, CSF, and plasma although the exact magnitude of the effect may differ between the compartments. A full pharmacokinetic- pharmacodynamic (PK-PD) time-course profile has been generated for brain, CSF, and plasma, demonstrating a simple relationship between drug exposure and the Aβ effects. However, analysis suggests a difference in the timing of the appearance of Aβ38 and the reduction of Aβ42 in CSF which may ultimately have significant implications for target engagement/biomarker measurements in clinical trials. Overall, our characterization of the GSM, EVP-0015962, continues to support it's clinical development as a potentially disease modifying agent for the treatment of AD, aimed at the selective reduction of the neurotoxic Aβ42.

Exploring the chemical space of γ-secretase modulators

gamma-Secretase is a key enzyme in the pathophysiology of Alzheimer's disease (AD) and is responsible for the production of potentially toxic amyloid-beta (Abeta) 42 peptides. gamma-Secretase modulators (GSMs) are small molecules (<600 Da) causing a product shift from Abeta42 toward shorter and less toxic Abeta fragments. Classical non-steroidal anti-inflammatory drugs (NSAIDs) constituted the first class of GSMs, and therefore many of today's GSMs exhibit NSAID-like overall structure combining an acidic head group with a lipophilic backbone. Recent developments include structurally different non-acidic GSMs. Here we summarize common structural features of GSMs, pick up the controversial discussion regarding their mechanism of action, and show how computational analysis of pharmacophoric features can help reveal their pharmacological profile.

Tarenflurbil

Alzheimer disease (AD), a serious neurodegenerative illness characterized by cognitive impairment and functional in-capacity, is thought to affect more than 26 million people worldwide, a number projected to expand to 106 million over the next several decades. The costs associated with this disease are estimated to be $25 billion, costs largely associated with the loss of independence and the need for care of these patients. It is no wonder that search for treatments is vigorous, with more than 700 trials registered on clinicaltrials.gov since 1997. One of the most common targets for treatment for AD is the manipulation of amyloid, which accumulates in the brain of those with the disease. One mechanism for this manipulation is the modulation of the enzyme γ-secretase, which may favor the production of a less toxic form of β-amyloid (Aβ). Tarenflurbil has demonstrated such activity in vitro and in in vivo animal studies, and it has shown relative safety in human trials. Hence, it was considered a viable candidate for phase 3 clinical trials in AD. The randomized placebo-controlled trial of tarenflurbil in patients with mild AD reported by Green et al1 is a well-conducted study of more than 1600 subjects with AD recruited from 133 sites across the United States. These investigators used a rigorous design and a long exposure period of 18 months. During the trial protocol, modifications were made (based on reanalysis of phase 2 data) consisting of shifting from 2 doses to a single high dose and modifying entry criteria to include only very mildly impaired subjects (with Mini-Mental State Examination [MMSE] scores of 20-26 inclusive). In the face of these modifications and the relatively long exposure period, most subjects (97%) were available for the primary analysis and the mean medication compliance rate was higher than 90%. It is therefore quite disappointing to see the dramatic lack of effect in the primary and secondary outcomes, with the exception of the Clinical Dementia Rating sum of boxes scores, which favored placebo. While so many things could go wrong in one of the few long trials that have been published to date, it appears that this cohort was typical of all that we know about patients with AD, with standard use of prescriptive cognitive enhancers, predictable decline in the placebo group in cognition, function, and behavior, no unusual adverse events, and no unreasonable loss to follow-up. We can therefore conclude with conviction that there was no benefit of tarenflurbil in this very typical group of patients with very mild AD. So why did this fail? Were there any data that should have limited enthusiasm to move to a phase 3 study? Was there false enthusiasm that moved this trial beyond the apparent data? There are several things to consider. The intent-to-treat analysis of the phase 2 study in mild to moderate AD demonstrated no significant benefit on the primary outcomes.2 Additional analyses that examined subgroups defined by MMSE scores identified a benefit in the group with the highest MMSE scores and a significant negative effect in several outcomes in those with lower MMSE scores. The decision to focus on this subgroup (mild) of a subgroup (mild to moderate) was solidified based on an analysis that demonstrated a differential treatment effect for the subgroup defined by a specific cut point on the MMSE. This approach to defining disease severity ex post facto identified a mild subgroup that demonstrated a benefit and a moderate subgroup that actually worsened. Dramatic differences in the direction of effect in subgroups seem biologically implausible. Should one suspect that at the shift of 1 point in an MMSE score, a patient was at risk for significant adverse effects with the agent that was otherwise beneficial? Could the results of the current trial suggest mathematical regression to the mean? The literature in clinical trials for the treatment of AD has many similar cases of failures in large trials that have based subject selection on a result in a subgroup of previous trials including acetylcarnitine, propentofylline, and most recently bapineuzumab. It would seem that the preliminary studies provided important information that forewarned of questionable effect, and further phase 2 testing might have been advisable before embarking on the “largest phase 3 trial to date.”1

Design, Synthesis, and Biological Evaluation of a Novel Class of γ-Secretase Modulators with PPARγ Activity

We present a novel class of dual modulators of gamma-secretase and peroxisome proliferator-activated receptor gamma (PPARgamma) based on the structure of 2-(bis(phenethoxy)pyrimidine-2-ylthio)hexanoic acid 8 (IC(50)(Abeta42) = 22.8 microM, EC(50)(PPARgamma) = 8.3 microM). The modulation of both targets with approved drugs (i.e., amyloid-beta 42 (Abeta42)-lowering NSAIDs for gamma-secretase and glitazones for PPARgamma) has demonstrated beneficial effects in in vitro and in vivo models of Alzheimer's disease (AD). However, although NSAIDs and PPARgamma agonists share similar structural features, no druglike compounds with dual activities as gamma-secretase modulators (GSMs) and PPARgamma agonists have been designed so far. On the basis of our initial lead structure 8, we present the structure-activity relationships (SARs) of broad structural variations. A significant improvement was reached by the introduction of p-trifluoromethyl substituents at the phenyl residues yielding compound 16 (IC(50)(Abeta42) = 6.0 microM, EC(50)(PPARgamma) = 11.0 microM) and the replacement of the two phenyl residues of 8 by cyclohexyl yielding compound 22 (IC(50)(Abeta42) = 5.1 microM, EC(50)(PPARgamma) = 6.6 microM).

The Discovery of Pyridone and Pyridazone Heterocycles as γ-Secretase Modulators.

A series of novel pyridazone and pyridone compounds as γ-secretase modulators were discovered. Starting from the initial lead, structure-activity relationship studies were carried out in which an internal hydrogen bond was introduced to conformationally fix the side chain, and compounds with improved in vitro Aβ42 inhibition activity and good Aβtotal/Aβ42 selectivity were quickly discovered. Compound 35 displayed very good in vitro activity and excellent selectivity with good in vivo efficacy in both CRND8 mouse and nontransgenic rat models. This compound displayed a good overall profile in terms of rat pharmacokinetics and ancillary profile. No abnormal behavior and side effects were observed in all of the studies.

&amp;#947;-Secretase as a Therapeutic Target in Alzheimers Disease

gamma-Secretase is an intramembranous multi-protein complex that cleaves many type-I proteins with critical roles in neuronal function. In Alzheimer's disease (AD) interest in gamma-secretase comes, in part, from the fact that this complex is responsible for the last cleavage step of the amyloid precursor protein (APP) that generates the amyloid-beta peptide (Abeta). Abeta represents the primary component of the amyloid plaque, one of the main pathological hallmarks of AD. Over the last years, considerable efforts have been made to develop drugs to reduce Abeta production with the aim to slow AD progression. Many inhibitors of this protease have been identified, although the clinical use has been limited by concerns about the possible toxicity of these compounds. gamma-secretase inhibitors have been shown to reduce Abeta in vitro and in vivo, but interference with Notch proteolysis causes immunological and gastrointestinal toxicity in animal models. The observation that some nonsteroidal anti-inflammatory drug (NSAID) derivatives are able to specifically lower Abeta42 and the development of inhibitors with Notch-sparing selectivity has revived the interest in gamma-secretase as an attractive target for drug intervention in AD. Despite the fact that all clinical trials with NSAIDs or gamma-secretase modulators in AD have failed to show clinical benefit thus far, the main concern is that the Abeta-lowering potency of the tested compounds may be too low. Active efforts are being made to develop compounds able to penetrate into the brain to lower Abeta at physiological doses without interfering with the cleavage and function of other critical gamma-secretase substrates. These novel inhibitors and modulators may soon offer hope in the Alzheimer's fight.

Purine derivatives as potent γ-secretase modulators

The development of a novel series of purines as γ-secretase modulators for potential use in the treatment of Alzheimer’s disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based γ-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Αβ42 in an APP-YAC transgenic mouse model.

Late-Life Dementias: Does This Unyielding Global Challenge Require a Broader View?

Late-Life Dementias: Does This Unyielding Global Challenge Require a Broader View?

Nonspecificity of Binding of γ-Secretase Modulators to the Amyloid Precursor Protein

Evidence that certain gamma-secretase modulators (GSMs) target the 99-residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of gamma-secretase, but not the protease complex itself has been presented [Kukar, T. L., et al. (2008) Nature 453, 925-929]. Here, NMR results demonstrate a lack of specific binding of these GSMs to monodisperse C99 in LMPG micelles. In addition, results indicate that C99 was likely to have been aggregated in some of the key experiments of the previous work and that binding of GSMs to these C99 aggregates is also of a nonspecific nature.

Piperazinyl pyrimidine derivatives as potent γ-secretase modulators

The development of a novel series of piperazinyl pyrimidines as γ-secretase modulators for potential use in the treatment of Alzheimer’s disease is disclosed herein. Optimization of a screening hit provided a series of potent γ-secretase modulators with >180-fold in vitro selectivity over inhibition of Notch cleavage.

Fluorinated piperidine acetic acids as γ-secretase modulators

We report herein a novel series of difluoropiperidine acetic acids as modulators of γ-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective β-difluorination with Selectfluor®. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Aβ42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250 mg/kg per day, AUC 0–24 = 2100 μM h) did not exhibit Notch-related effects.

NSAID-derived γ-secretase modulators. Part III: Membrane anchoring

Selective lowering of Aβ42 levels with small-molecule substrate targeting γ-secretase modulators (sGSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. Here we present N-substituted carbazole- and O-substituted fenofibrate-derived sGSMs and their activity data. Seven out of 19 screened compounds exhibited promising activity against Aβ42 secretion at a low micromolar level. We presume that the sGSMs interact with lys624 at the membrane interface and that the lipophilic substituents anchor the compound orientation in the membrane.

TMP21 Transmembrane Domain Regulates γ-Secretase Cleavage

TMP21 has been shown to be associated with the γ-secretase complex and can specifically regulate γ-cleavage without affecting ϵ-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of γ-secretase activity was investigated independent of ϵ-cleavage using an amyloid-β precursor proteinϵ (APPϵ) construct which lacks the amyloid intracellular domain domain. The APPϵ construct behaves similarly to the full-length precursor protein with respect to α- and β-cleavages and is able to undergo normal γ-processing. Co-expression of APPϵ and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Aβ-positive fragments, consistent with an inhibition of γ-secretase cleavage. The APPϵ system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the γ-secretase complex and could decrease γ-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Aβ production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased γ-cleavage activity.

Piperidine-derived γ-secretase modulators

This Letter details the SAR of a novel series of piperidine-derived γ-secretase modulators. Compound 10h was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aβ42, increase Aβ38 and have no effect on Aβ40 levels. Furthermore, 10h demonstrated excellent pharmacokinetic parameters in the mouse, rat and dog in addition to good CNS penetration in the mouse.

A surrogate marker for Aβ42 production in the CNS

Alzheimer's disease (AD) is the most common cause of dementia. There are currently no effective treatments that may delay the onset, slow the progression or prevent the disease. Unless such treatments are developed, the number of AD cases is expected to double in the next 30 years. There is overwhelming genetic and biochemical evidence that the aggregation and buildup of the amyloid-β (Aβ) peptide plays a critical role in AD pathogenesis.

Presenilin-1 (PS1) and amyloid precursor protein (APP) mutations present in mouse models of Alzheimer's disease in their response to γ-secretase inhibitors and modulators

Presenilin-1 (PS1) and amyloid precursor protein (APP) mutations present in mouse models of Alzheimer's disease in their response to γ-secretase inhibitors and modulators

Purification, Pharmacological Modulation, and Biochemical Characterization of Interactors of Endogenous Human γ-Secretase

Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.

Distinct Pharmacological Effects of Inhibitors of Signal Peptide Peptidase and γ-Secretase

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.