Current Topics in Medicinal Chemistry - Volume 8, Issue 1, 2008

Alzheimer's disease (AD) is the most common cause of dementia. According to the so called “amyloid hypothesis”, the oligomeric forms of a 40-42 aminoacid peptide known as β-amyloid (Aβ), is the main cause of neuronal death in AD. Aβ is the metabolite of a large transmembrane protein called amyloid precursor protein (APP). The last metabolic step that generates Aβ involves the enzymatic intramembrane cleavage of APP by a high-molecular weight complex called γ-secretase. γ-Secretase is formed by at least four proteins: presenilin (PS), nicastrin, anterior pharynx (Aph-1) and presenilin enhancer 2 (Pen-2). Presenilins are of exceptional pathophysiological importance since more than 150 autosomal dominant point mutations are known in these proteins, all of which cause aggressive early-onset AD. These mutations result in increased production of Aβ42, the highly self-aggregating and neurotoxic form of Aβ. Thus, inhibition or modulation of γ-secretase appears to be a logical strategy to decrease Aβ accumulation in AD patients. To fully explore recent advances in the γ-secretase field, Current Topics in Medicinal Chemistry has devoted this entire issue to a review of the structure and function of the γ-secretase complex and summarizes SARs of the main peptidic and nonpeptidic inhibitors of the enzyme. In addition, allosteric modulators of γ-secretase are described since they may represent a safer approach to inhibit Aβ secretion in AD patients. Finally, I have reviewed the γ-secretase inhibitors and modulators that have been selected for development and describe data obtained so far from clinical trials. With a number of companies conducting clinical trials with γ-secretase inhibitors and modulators, it should become clear in the near future whether this attractive target for decreasing Aβ can lead to effective and safe drugs for AD. I would like to express my gratitude to all the Authors for their clear and comprehensive contributions to this special issue of Current Topics in Medicinal Chemistry. Our hope is that this issue will be an informative contribution to the field and will represent a key reference work for those involved in the discovery of γ-secretase inhibitors and modulators.

γ-Secretase proteolyzes a variety of membrane-associated fragments derived from type I integral membrane proteins, including the amyloid β-protein precursor, involved in Alzheimer's disease, and the Notch receptor, critical for cellular differentiation. This protease is composed of four integral membrane proteins: presenilin, nicastrin, Aph-1 and Pen-2. Assembly of these four components leads to presenilin autoproteolysis into two subunits, each of which contributes one aspartate to the active site of an aspartyl protease. The protease contains an initial docking site for substrate, where it binds prior to passing between the two presenilin subunits to the internal water-containing active site. The extracellular region of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Modulation of APP processing without interfering with Notch signaling is an important therapeutic goal, and allosteric sites on the protease allow such selective modulation. A better structural and mechanistic understanding of γ-secretase should ultimately allow structure-based design of more potent and selective modulators.

γ-secretase is an intramembranous protein complex that cleaves many type-I membrane proteins, including the Notch receptor and the β-amyloid precursor protein (APP). Interest in γ-secretase comes, in part, from the fact that this multiprotein complex is responsible for the cleavage of APP that generates the amyloid-β peptide (Aβ), one of the primary components of amyloid plaques in Alzheimer's disease (AD). Over the last years, molecular identification of the complex has shown that γ-secretase is an aspartyl protease composed of four different members that are essential for the enzymatic activity: presenilin 1, aph1, pen-2 and nicastrin. In recent years, an increasing number of type-I membrane proteins have been shown to be cleaved by γ-secretase. How the enzyme cleaves such a set of substrates with diverse functions and subcellular localizations is not well understood. In overexpression assays, the γ-secretase cleavage of some substrates releases intracellular domains with signaling properties. On the other hand, the loose specificity required for intramembrane cleavage has raised the possibility of γ-secretase as the membrane proteasome. The impact of γ-secretase on other substrates has clear implications for the development of new therapies for AD, and in particular for the search of γ-secretase inhibitors or modulators. Interference with the cleavage of some of the γ-secretase substrates has been shown to be associated with serious adverse effects in animal models. The understanding of the mechanism by which γ-secretase recognizes and cleaves all these proteins is of great importance to clarify the function of γ-secretase and its role as a therapeutic target in AD, and possibly in other diseases in which γ- secretase is involved.

Aβ is implicated in the initiation and progression of Alzheimer's disease (AD) by the phenotypic analysis of mutations in three human genes that lead to inherited, early forms of AD and data from preclinical studies. Based on this evidence, γ-secretase inhibitors are being actively pursued as potential AD therapeutics to reduce Aβ formation. This manuscript reviews recent progress in the medicinal chemistry of three major classes of γ-secretase inhibitors: peptide isosteres, azepines, and sulfonamides. Peptide isosteres have been useful for demonstrating that presenilin is the catalytic subunit of γ-secretase and probing the active site. The peptidic nature of these inhibitors has, however, interfered with their utility for in vivo studies. Instead, the pharmaceutical industry has focused on optimizing azepines and sulfonamides. Both azepines and sulfonamides bind to a common, allosteric site on presenilin that differs from the active site identified by the peptide isosteres. Significant progress in the optimization of both azepines and sulfonamides has led to compounds that inhibit brain Aβ synthesis in preclinical models and has culminated in the identification of γ-secretase inhibitors, including LY- 450139 and MK-0752, for human trials.

The genetics of Alzheimer's disease (AD) implies that restoring non-pathological levels or ratios of different amyloid-β (Aβ) peptide species in the brain could prevent the onset or delay the progression of this neurodegenerative disease. In particular, a selective reduction of the longer Aβ(1-42) peptide which is widely believed to be causative of AD is currently seen as an attractive approach for a disease-modifying therapy. Based on the knowledge that Aβ(1-42) and various shorter Aβ peptides are generated by the same γ-secretase enzyme, the concept of allosteric modulation of the cleavage specificity of this aspartic protease has been introduced to the field of protease drug discovery and fuelled novel medicinal chemistry efforts. γ-Secretase modulation holds the promise that chemical entities can be synthesized which restore non-pathological enzyme activity by shifting the actual substrate cleavage towards the generation of shorter Aβ peptides. It can be assumed that this approach has gained considerable attraction for pharmaceutical drug discovery since the development of non-selective protease inhibitors for γ-secretase has been proven to be difficult due to inherent mechanism-based liabilities.

γ-Secretase modulation holds the promise for the development of a disease-modifying therapy for Alzheimer's disease (AD). This novel concept of manipulating the cleavage specificity of the γ-secretase enzyme by pharmacological means implies that steady state levels of the potentially disease-causing amyloid-β(1-42) peptide can be lowered without the undesired side effects associated with full inhibition of this aspartyl-type protease. Following on from the initial discovery that certain non-steroidal anti-inflammatory drugs (NSAIDs) exhibit properties characteristic of γ-secretase modulators, this class of compounds has been extensively studied and exploited, leading to the discovery of NSAIDs derivatives endowed with improved potency for the reduction of amyloid-β(1-42) peptide production. In addition, a very limited number of non-NSAID derived γ-secretase modulators has also been recently claimed in the patent literature, suggesting that only a restricted number of pharmacophores might be involved in the modulation of γ-secretase.

Genetic and biochemical evidence continues to implicate the production and accumulation of the Aβ42 peptide as the causative factor in Alzheimer's disease (AD). Thus, a variety of strategies have been developed to decrease the production and/or aggregation of this peptide, which may be clinically useful for the treatment of this devastating disorder. Recently, the discovery that some non-steroidal anti-inflammatory drugs (NSAIDs) appear to selectively decrease the production of Aβ42 has opened a novel therapeutic avenue for AD treatment that may circumvent potential toxicity associated with long-term global inhibition of γ-secretase activity. One drug from this class of compounds, R-flurbiprofen, has advanced to phase 3 clinical trials and may soon provide insight into the viability of this strategy for the prevention or treatment of AD. Delineating the target and mechanism of these compounds is essential for developing new agents with increased potency and optimized pharmacologic properties. The evidence indicating that these chemicals modulate the production of Aβ peptides by directly interacting with the γ-secretase complex is summarized.

According to the β-amyloid (Aβ) hypothesis, compounds that inhibit γ-secretase, the pivotal enzyme that generates Aβ, are potential therapeutics for Alzheimer's disease (AD). Studies in both transgenic and non-transgenic animal models of AD have indicated that γ-secretase inhibitors, administered by the oral route, are able to lower brain Aβ concentrations. However, scanty data are available on the effects of these compounds on brain Aβ deposition after prolonged administration. Behavioral studies are also scarce with only one study indicating positive cognitive effects of a peptidomimetic compound (DAPT). γ-Secretase inhibitors may cause abnormalities in the gastrointestinal tract, thymus and spleen in rodents. These toxic effects are likely due to inhibition of Notch cleavage, a transmembrane receptor involved in regulating cell-fate decisions. Interestingly, some non-steroidal anti-inflammatory drugs (NSAIDs) and other small organic molecules have been found to modulate γ-secretase and to selectively reduce β-amyloid1-42 (Aβ42) levels without affecting Notch cleavage. Long-term histopathological and behavioral animal studies are available with these NSAIDs (mainly ibuprofen) but it is unclear if the observed in vivo effects on Aβ brain pathology and learning depend on their activity on γ-secretase or on other biological targets. The first published clinical studies in healthy subjects and in AD patients with a γ-secretase inhibitor, LY- 450139, confirmed the dose-dependent inhibition of plasma Aβ but evidenced a later rebound in Aβ plasma levels and absence of a significant effect on cerebrospinal fluid Aβ concentrations. Some observed gastrointestinal adverse events have raised concerns. Clinical studies with other potent γ-secretase inhibitors will tell us if these pharmacodynamic and tolerability profiles observed in humans are typical of the pharmacological class or are compound-specific. Given the uncertain Aβ reduction target and the potential for mechanismbased toxicity, it has been suggested that biomarkers for efficacy (cerebrospinal fluid Aβ42 levels) and toxicity (plasma adipsin levels) would be helpful in initial clinical trials with γ-secretase inhibitors. A large ongoing Phase 3 study with (R)-flurbiprofen, a claimed selective Aβ42 lowering agent, will tell us if allosteric modulation of γ-secretase is clinically effective.

Overcoming Resistance. Over four years ago, on May 2, 2003, the FDA approved Iressa™ (gefitinib) by AstraZeneca for the treatment of non-small cell lung cancer (NSCLC) in patients that failed to respond to two other types of chemotherapy. The potential therapeutic benefit of Iressa™, a once-daily tablet, was obvious, as the drug elicited an objective response and was well-tolerated in Phase II trials. The subsequent Phase III clinical trial, however, failed to prove that patients taking Iressa™ survived longer than those taking placebo. Due to these results, on June 17, 2005, the FDA limited the use of Iressa™ to patients who were currently benefiting or had previously benefited from the drug and patients that had formerly been enrolled in clinical trials. Importantly, although clinical trials of Iressa™ did not demonstrate a survival benefit for the overall patient population, the study showed a statistically significant increase in survival for patients of Asian origin and patients who had never smoked [1]. The epidermal growth factor receptor (EGFR) performs a critical role in cellular proliferation, differentiation and survival. Aberrant EGF-EGFR signaling, as in the case of mutations, leads to overexpression of wild-type EGFR - a hallmark of a broad range of cancers, including lung, breast and colon carcinomas [2]. Iressa™, a quinazoline derivative, is a tyrosine kinase inhibitor (TKI) that selectively targets EGFR. Despite the initial response to the drug in the majority of NSCLCs, most of the tumors eventually develop resistance to Iressa™. A secondary mutation in the EGFR gene is responsible for resistance in approximately half of these cases, but the cause of resistance in the remaining cancer cells is still being investigated. A recent study (Br. J. Cancer 2007, 97, 1560-1566) determined that classical mutations in the EGFR TK domain (exons 18, 19 and 21), but not other mutations, are associated with the clinical outcome in -treated patients with NSCLC [3]. A recent study by Dr. Jeffrey A. Engelman et al. (Science 2007, 316, 1039-1043) suggests that amplification of the MET oncogene may be another mechanism that leads to resistance, indicating combination therapy with MET kinase inhibitors as a possible solution [4]. Even if Iressa™ does not prove to be the cancer therapeutic that was hoped for, the molecule may serve as the precursor to a new effective anticancer agent. Tarceva™ (erlotinib), a joint product of Genentech, Inc. and OSI Pharmaceuticals, Inc., is a derivative of Iressa™ and another EGFR tyrosine kinase inhibitor that is currently in Phase III clinical trials[5]. Research is also being conducted to synthesize more analogues of Iressa™; Professor Jean-Pierre Henichart et al. synthesized 23 Iressa™ derivatives, many of which decreased proliferation and induced apoptosis in human prostate cancer cells[6]. The future of Iressa™ will be determined by further evaluation of the drug's effectiveness in subgroups, understanding of the cancer cells' resistance mechanisms, and novel therapies to overcome that resistance. REFERENCES [1] For detailed information and press releases describing Iressa™, see www.astrazeneca.com. [2] Cragg, M.S.; Kuroda, J.; Puthalakath, H.; Huang, D.C.S.; Strasser, A. PloS Medicine 2007, 4, 1681-1690. [3] Pallis, A.G.; Kalikaki, A.; Souglakos,