Alzheimer's Disease: A Brief History of Immunotherapies Targeting Amyloid ß.

Alzheimer's disease (AD) is the most common form of dementia and may contribute to 60-70% of cases. Worldwide, around 50 million people suffer from dementia and the prediction is that the number will more than triple by 2050, as the population ages. Extracellular protein aggregation and plaque deposition as well as accumulation of intracellular neurofibrillary tangles, all leading to neurodegeneration, are the hallmarks of brains with Alzheimer's disease. Therapeutic strategies including active and passive immunizations have been widely explored in the last two decades. Several compounds have shown promising results in many AD animal models. To date, only symptomatic treatments are available and because of the alarming epidemiological data, novel therapeutic strategies to prevent, mitigate, or delay the onset of AD are required. In this mini-review, we focus on our understanding of AD pathobiology and discuss current active and passive immunomodulating therapies targeting amyloid-ß protein.

A scalable and highly immunogenic virus-like particle-based vaccine against SARS-CoV-2.

BACKGROUND: SARS-CoV-2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock-downs, wearing masks, and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or adenoviruses, has greatly added to our ability to keep the virus at bay; again, however, in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long-term control of SARS-CoV-2 would be inexpensive production at large scale, ability to make multiple booster injections, and long-term stability at 4℃. METHODS: Here, we describe such a vaccine candidate, consisting of the SARS-CoV-2 receptor-binding motif (RBM) grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMVTT -RBM. RESULTS: Using bacterial fermentation and continuous flow centrifugation for purification, the yield of the production process is estimated to be >2.5 million doses per 1000-litre fermenter run. We demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera. The induced antibodies are more cross-reactive to mutant RBDs of variants of concern (VoC). Furthermore, antibody responses are neutralizing and long-lived. In addition, the vaccine candidate was stable for at least 14 months at 4℃. CONCLUSION: Thus, the here presented VLP-based vaccine may be a good candidate for use as conventional vaccine in the long term.

Structural binding site comparisons reveal Crizotinib as a novel LRRK2 inhibitor.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of autosomal dominant Parkinson's disease (PD) and have been associated with familial and sporadic PD. Reducing the kinase activity of LRRK2 is a promising therapeutic strategy since pathogenic mutations increase the kinase activity. Several small-molecule LRRK2 inhibitors are currently under investigation for the treatment of PD. However, drug discovery and development are always accompanied by high costs and a risk of late failure. The use of already approved drugs for a new indication, which is known as drug repositioning, can reduce the cost and risk. In this study, we applied a structure-based drug repositioning approach to identify new LRRK2 inhibitors that are already approved for a different indication. In a large-scale structure-based screening, we compared the protein-ligand interaction patterns of known LRRK2 inhibitors with protein-ligand complexes in the PDB. The screening yielded 6 drug repositioning candidates. Two of these candidates, Sunitinib and Crizotinib, demonstrated an inhibition potency (IC50) and binding affinity (Kd) in the nanomolar to micromolar range. While Sunitinib has already been known to inhibit LRRK2, Crizotinib is a novel LRRK2 binder. Our results underscore the potential of structure-based methods for drug discovery and development. In light of the recent breakthroughs in cryo-electron microscopy and structure prediction, we believe that structure-based approaches like ours will grow in importance.

Structure-based drug repositioning explains ibrutinib as VEGFR2 inhibitor.

Many drugs are promiscuous and bind to multiple targets. On the one hand, these targets may be linked to unwanted side effects, but on the other, they may achieve a combined desired effect (polypharmacology) or represent multiple diseases (drug repositioning). With the growth of 3D structures of drug-target complexes, it is today possible to study drug promiscuity at the structural level and to screen vast amounts of drug-target interactions to predict side effects, polypharmacological potential, and repositioning opportunities. Here, we pursue such an approach to identify drugs inactivating B-cells, whose dysregulation can function as a driver of autoimmune diseases. Screening over 500 kinases, we identified 22 candidate targets, whose knock out impeded the activation of B-cells. Among these 22 is the gene KDR, whose gene product VEGFR2 is a prominent cancer target with anti-VEGFR2 drugs on the market for over a decade. The main result of this paper is that structure-based drug repositioning for the identified kinase targets identified the cancer drug ibrutinib as micromolar VEGFR2 inhibitor with a very high therapeutic index in B-cell inactivation. These findings prove that ibrutinib is not only acting on the Bruton's tyrosine kinase BTK, against which it was designed. Instead, it may be a polypharmacological drug, which additionally targets angiogenesis via inhibition of VEGFR2. Therefore ibrutinib carries potential to treat other VEGFR2 associated disease. Structure-based drug repositioning explains ibrutinib's anti VEGFR2 action through the conservation of a specific pattern of interactions of the drug with BTK and VEGFR2. Overall, structure-based drug repositioning was able to predict these findings at a fraction of the time and cost of a conventional screen.

Immunization of Cats against Fel d 1 Results in Reduced Allergic Symptoms of Owners.

An innovative approach was tested to treat cat allergy in humans by vaccinating cats with Fel-CuMV (HypoCatTM), a vaccine against the major cat allergen Fel d 1 based on virus-like particles derived from cucumber mosaic virus (CuMV-VLPs). Upon vaccination, cats develop neutralizing antibodies against the allergen Fel d 1, which reduces the level of reactive allergen, thus lowering the symptoms or even preventing allergic reactions in humans. The combined methodological field study included ten cat-allergic participants who lived together with their cats (n = 13), that were immunized with Fel-CuMV. The aim was to determine methods for measuring a change in allergic symptoms. A home-based provocation test (petting time and organ specific symptom score (OSSS)) and a general weekly (or monthly) symptom score (G(W)SS) were used to assess changes in allergic symptoms. The petting time until a pre-defined level of allergic symptoms was reached increased already early after vaccination of the cats and was apparent over the course of the study. In addition, the OSSS after provocation and G(W)SS recorded a persistent reduction in symptoms over the study period and could serve for long-term assessment. Hence, the immunization of cats with HypoCatTM (Fel-CuMV) may have a positive impact on the cat allergy of the owner, and changes could be assessed by the provocation test as well as G(W)SS.

Miltefosine treatment reduces visceral hypersensitivity in a rat model for irritable bowel syndrome via multiple mechanisms.

Irritable bowel syndrome (IBS) is a heterogenic, functional gastrointestinal disorder of the gut-brain axis characterized by altered bowel habit and abdominal pain. Preclinical and clinical results suggested that, in part of these patients, pain may result from fungal induced release of mast cell derived histamine, subsequent activation of sensory afferent expressed histamine-1 receptors and related sensitization of the nociceptive transient reporter potential channel V1 (TRPV1)-ion channel. TRPV1 gating properties are regulated in lipid rafts. Miltefosine, an approved drug for the treatment of visceral Leishmaniasis, has fungicidal effects and is a known lipid raft modulator. We anticipated that miltefosine may act on different mechanistic levels of fungal-induced abdominal pain and may be repurposed to IBS. In the IBS-like rat model of maternal separation we assessed the visceromotor response to colonic distension as indirect readout for abdominal pain. Miltefosine reversed post-stress hypersensitivity to distension (i.e. visceral hypersensitivity) and this was associated with differences in the fungal microbiome (i.e. mycobiome). In vitro investigations confirmed fungicidal effects of miltefosine. In addition, miltefosine reduced the effect of TRPV1 activation in TRPV1-transfected cells and prevented TRPV1-dependent visceral hypersensitivity induced by intracolonic-capsaicin in rat. Miltefosine may be an attractive drug to treat abdominal pain in IBS.

Immunization of cats to induce neutralizing antibodies against Fel d 1, the major feline allergen in human subjects.

BACKGROUND: Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms. OBJECTIVE: We developed a new strategy to treat Fel d 1-induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1. METHODS: A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin-derived universal T-cell epitope tt830-843 (CuMVTT) was used to immunize cats. A first tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMVTT vaccine alone or in combination with an adjuvant. RESULTS: The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti-Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both in vitro and in vivo. A reduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed. CONCLUSION: Vaccination of cats with Fel-CuMVTT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters.

A vaccine against Alzheimer`s disease: anything left but faith?

INTRODUCTION: Alzheimer's disease looms as a profound and growing threat to future human health. The disease is thought to be primarily driven by aberrant proteolysis of the amyloid precursor protein (APP) and amyloid beta (Aß) plaque deposition. AREAS COVERED: We provide an overview of the molecular pathology that leads to an increase in Aß peptide accumulation, of the mechanism of action for antibody mediated therapies and of the therapeutic vaccines that target Aß under development. We also discuss the rationale for using vaccines in the early stages of the disease. EXPERT OPINION: The major components of ß-amyloid plaques are Aß1-42 and Aß1-40 peptides derived from the APP. Reducing these plaques by means of passive or active vaccination against Aß-peptides has been a long-running endeavor but with disappointing results as the impact on disease progression has been minimal. The data gathered to date could suggest that antibodies do not work, mainly because the studies have not been performed in an optimal fashion. The emerging views are that patients should be treated earlier, ideally in the prodromal or symptom free stage, antibody levels have to be high and the correct epitope must be targeted. More clinical trials to fully explore the potential of vaccines are therefore warranted.

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure.

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development.

Incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, Alzheimer's and cat allergy.

Monoclonal antibodies are widely used to treat non-infectious conditions but are costly. Vaccines could offer a cost-effective alternative but have been limited by sub-optimal T-cell stimulation and/or weak vaccine responses in recipients, for example, in elderly patients. We have previously shown that the repetitive structure of virus-like-particles (VLPs) can effectively bypass self-tolerance in therapeutic vaccines. Their efficacy could be increased even further by the incorporation of an epitope stimulating T cell help. However, the self-assembly and stability of VLPs from envelope monomer proteins is sensitive to geometry, rendering the incorporation of foreign epitopes difficult. We here show that it is possible to engineer VLPs derived from a non human-pathogenic plant virus to incorporate a powerful T-cell-stimulatory epitope derived from Tetanus toxoid. These VLPs (termed CMVTT) retain self-assembly as well as long-term stability. Since Th cell memory to Tetanus is near universal in humans, CMVTT-based vaccines can deliver robust antibody-responses even under limiting conditions. By way of proof of concept, we tested a range of such vaccines against chronic inflammatory conditions (model: psoriasis, antigen: interleukin-17), neurodegenerative (Alzheimer's, ß-amyloid), and allergic disease (cat allergy, Fel-d1), respectively. Vaccine responses were uniformly strong, selective, efficient in vivo, observed even in old mice, and employing low vaccine doses. In addition, randomly ascertained human blood cells were reactive to CMVTT-VLPs, confirming recognition of the incorporated Tetanus epitope. The CMVTT-VLP platform is adaptable to almost any antigen and its features and performance are ideally suited for the design of vaccines delivering enhanced responsiveness in aging populations.

Development of an Interleukin-1ß Vaccine in Patients with Type 2 Diabetes.

Interleukin-1ß (IL-1ß) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1ß activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1ß. The vaccine hIL1bQb consisting of full-length, recombinant IL-1ß coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1ß-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1ß-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1ß and represents a new treatment option for IL-1ß-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).

Particle-based platforms for malaria vaccines.

Recombinant subunit vaccines in general are poor immunogens likely due to the small size of peptides and proteins, combined with the lack or reduced presentation of repetitive motifs and missing complementary signal(s) for optimal triggering of the immune response. Therefore, recombinant subunit vaccines require enhancement by vaccine delivery vehicles in order to attain adequate protective immunity. Particle-based delivery platforms, including particulate antigens and particulate adjuvants, are promising delivery vehicles for modifying the way in which immunogens are presented to both the innate and adaptive immune systems. These particle delivery platforms can also co-deliver non-specific immunostimodulators as additional adjuvants. This paper reviews efforts and advances of the Particle-based delivery platforms in development of vaccines against malaria, a disease that claims over 600,000 lives per year, most of them are children under 5 years of age in sub-Sahara Africa.

Preclinical efficacy and safety of an anti-IL-1ß vaccine for the treatment of type 2 diabetes.

Neutralization of the inflammatory cytokine interleukin-1ß (IL-1ß) is a promising new strategy to prevent the ß-cell destruction, which leads to type 2 diabetes. Here, we describe the preclinical development of a therapeutic vaccine against IL-1ß consisting of a detoxified version of IL-1ß chemically cross-linked to virus-like particles of the bacteriophage Qß. The vaccine was well tolerated and induced robust antibody responses in mice, which neutralized the biological activity of IL-1ß, as shown both in cellular assays and in challenge experiments in vivo. Antibody titers were long lasting but reversible over time and not associated with the development of potentially harmful T cell responses against IL-1ß. Neutralization of IL-1ß by vaccine-induced antibodies had no influence on the immune responses of mice to Listeria monocytogenes and Mycobacterium tuberculosis. In a diet-induced model of type 2 diabetes, immunized mice showed improved glucose tolerance, which was mediated by improved insulin secretion by pancreatic ß-cells. Hence, immunization with IL-1ß conjugated to virus-like particles has the potential to become a safe, efficacious, and cost-effective therapy for the prevention and long-term treatment of type 2 diabetes.

Bacterially produced recombinant influenza vaccines based on virus-like particles.

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

Evaluation of steroidal amines as lipid raft modulators and potential anti-influenza agents.

The influenza A virus (IFV) possesses a highly ordered cholesterol-rich lipid envelope. A specific composition and structure of this membrane raft envelope are essential for viral entry into cells and virus budding. Several steroidal amines were investigated for antiviral activity against IFV. Both, a positively charged amino function and the highly hydrophobic (ClogP≥5.9) ring system are required for IC50 values in the low µM range. An amino substituent is preferential to an azacyclic A-ring. We showed that these compounds either disrupt or augment membrane rafts and in some cases inactivate the free virus. Some of the compounds also interfere with virus budding. The antiviral selectivity improved in the series 3-amino, 3-aminomethyl, 3-aminoethyl, or by introducing an OH function in the A-ring. Steroidal amines show a new mode of antiviral action in directly targeting the virus envelope and its biological functions.

A VLP-based vaccine against interleukin-1α protects mice from atherosclerosis.

Interleukin (IL)-1α is a potent proinflammatory cytokine that has been implicated in the development of atherosclerosis. We investigated whether a vaccine inducing IL-1α neutralizing antibodies could interfere with disease progression in a murine model of atherosclerosis. We immunized Apolipoprothin E (ApoE)-deficient mice with a vaccine (IL-1α-C-Qß) consisting of full-length, native IL-1α chemically conjugated to virus-like particles derived from the bacteriophage Qß. ApoE(-/-) mice were administered six injections of IL-1α-C-Qß or nonconjugated Qß over a period of 160 days while being maintained on a western diet. Atherosclerosis was measured in the descending aorta and in cross-sections at the aortic root. Macrophage infiltration in the aorta was measured using CD68. Expression levels of VCAM-1, ICAM-1, and MCP-1 were quantified by RT-PCR. Immunization against IL-1α reduced plaque progression in the descending aorta by 50% and at the aortic root by 37%. Macrophage infiltration in the aorta was reduced by 22%. Inflammation was also reduced in the adventitia, with a decrease of 54% in peri-aortic infiltrate score and reduced expression levels of VCAM-1 and ICAM-1. Active immunization targeting IL-1α reduced both the inflammatory reaction in the plaque as well as plaque progression. In summary, vaccination against IL-1α protected ApoE(-/-) mice against disease, suggesting that this may be a potential treatment option for atherosclerosis.

Therapeutic vaccines for chronic diseases: successes and technical challenges.

Chronic, non-communicable diseases are the major cause of death and disability worldwide and have replaced infectious diseases as the major burden of society in large parts of the world. Despite the complexity of chronic diseases, relatively few predisposing risk factors have been identified by the World Health Organization. Those include smoking, alcohol abuse, obesity, high cholesterol and high blood pressure as the cause of many of these chronic conditions. Here, we discuss several examples of vaccines that target these risk factors with the aim of preventing the associated diseases and some of the challenges they face.

The second-generation active Aß immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects.

Immunization against amyloid-ß (Aß) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer's disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aß1-6 coupled to the virus-like particle Qß. Immunization with this vaccine did not activate Aß-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aß antibody titers of different IgG subclasses mainly recognizing the Aß3-6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aß42 and Aß40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aß, which atypically was nonfibrillar. The efficacy of Aß immunotherapy depended on the Aß levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimer's disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aß monomers and oligomers and blocked Aß toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aß aggregation and its downstream detrimental effects.

A virus-like particle-based anti-nerve growth factor vaccine reduces inflammatory hyperalgesia: potential long-term therapy for chronic pain.

Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. For a substantial proportion of patients, conventional drug treatments do not provide adequate pain relief. Consequently, novel approaches to pain management, involving alternative targets and new therapeutic modalities compatible with chronic use, are being sought. Nerve growth factor (NGF) is a major mediator of chronic pain. Clinical testing of NGF antagonists is ongoing, and clinical proof of concept has been established with a neutralizing mAb. Active immunization, with the goal of inducing therapeutically effective neutralizing autoreactive Abs, is recognized as a potential treatment option for chronic diseases. We have sought to determine if such a strategy could be applied to chronic pain by targeting NGF with a virus-like particle (VLP)-based vaccine. A vaccine comprising recombinant murine NGF conjugated to VLPs from the bacteriophage Qß (NGFQß) was produced. Immunization of mice with NGFQß induced anti-NGF-specific IgG Abs capable of neutralizing NGF. Titers could be sustained over 1 y by periodic immunization but declined in the absence of boosting. Vaccination with NGFQß substantially reduced hyperalgesia in collagen-induced arthritis or postinjection of zymosan A, two models of inflammatory pain. Long-term NGFQß immunization did not change sensory or sympathetic innervation patterns or induce cholinergic deficits in the forebrain, nor did it interfere with blood-brain barrier integrity. Thus, autovaccination targeting NGF using a VLP-based approach may represent a novel modality for the treatment of chronic pain.

Structural design, solid-phase synthesis and activity of membrane-anchored ß-secretase inhibitors on Aß generation from wild-type and Swedish-mutant APP.

Covalent coupling of ß-secretase inhibitors to a raftophilic lipid anchor via a suitable spacer by using solid-phase peptide synthesis leads to tripartite structures displaying substantially improved inhibition of cellular secretion of the ß-amyloid peptide (Aß). Herein, we describe a series of novel tripartite structures, their full characterization by NMR spectroscopy and mass spectrometry, and the analysis of their biological activity in cell-based assays. The tripartite structure concept is applicable to different pharmacophores, and the potency in terms of ß-secretase inhibition can be optimized by adjusting the spacer length to achieve an optimal distance of the inhibitor from the lipid bilayer. A tripartite structure containing a transition-state mimic inhibitor was found to be less potent on Aß generation from Swedish-mutant amyloid precursor protein (APP) than from the wild-type protein. Moreover, our observations suggest that specific variants of Aß are generated from wild-type APP but not from Swedish-mutant APP and are resistant to ß-secretase inhibition. Efficient inhibition of Aß secretion by tripartite structures in the absence of appreciable neurotoxicity was confirmed in a primary neuronal cell culture, thus further supporting the concept.