WO2017095250A1 - Peptide inhibitors of bace1 for the treatment of neurological disorders - Google Patents

Abstract

The present application presents novel peptide inhibitors of beta amyloid cleavage enzyme, capable to permeate the blood-brain barrier and with no cytotoxic effects. Additionally, this application relates to a new methodology to inhibit BACE1 with peptidergic compounds. The peptides are incorporated in pharmaceutical compositions and applied in the treatment of Alzheimer's disease and in other neurological disorders such as Parkinson's disease, Vascular Dementia, Dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, Down's Syndrome, head trauma, and stroke.

Description

DESCRIPTION

"PEPTIDE INHIBITORS OF BACE1 FOR THE TREATMENT OF

NEUROLOGICAL DISORDERS"

Technical Field

The present application relates to novel inhibitors of β- amyloid cleavage enzyme (BACE, transmembrane aspartyl protease beta-secretase, beta-site APP cleavage enzyme, memapsin2, BACE-1, EC 3.4.23.46), pharmaceutical compositions containing them, and to a new methodology to inhibit BACE1 and its use in the treatment of Alzheimer' s disease (AD) and in other neurological disorders such as Parkinson's disease, Vascular Dementia, Dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, Down's Syndrome, head trauma, and stroke. These pathological conditions have amyloid deposits or act as a risk factor for AD.

Background

Dementia affects a growing number of individuals, mainly aged 60 and over. The number of affected individuals is predicted to be over 100 million in 2050. Alzheimer^'s disease (AD) is the most common dementia worldwide. It is a chronic debilitating neurodegenerative disease of the central nervous system (specifically brain) that impairs the ability to conduct a normal life since it affects cognitive functions like short-term memory, attention, and language, and patients often show as well disorientation and behavioural problems. The neuropathological hallmarks are the amyloid plaques, which are composed of fibrillary beta amyloid peptide (Αβ), and neurofibrillary tangles mainly composed of abnormal tau that are associated with many neurological disorders commonly called tauopathies (Karren et al . , 2011; Selkoe 2011) . Amyloid plaques are unique to AD, however, the presence of Αβ in the brain of patients affected by other neurological conditions namely, Parkinson's disease, Vascular Dementia, Dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, and Down's syndrome has been described.

It is generally accepted that accumulation of Αβ in the brain parenchyma represents an early incident on a cascade of events that ends in neurodegeneration and dementia, and thus, Αβ is considered as the etiologic agent of the disease (Karren et al . , 2011; Selkoe 2011; Yan & Vassar 2014) . The Αβ has been indicated as being responsible for the hyperphosphorylation of tau, which underlies the formation of neurofibrillary tangles. The formation of Αβ requires the initial cleavage of the β-amyloid protein precursor (APP) by the β-secretase (BACE-1) enzyme followed by the activity of the γ-secretase over the ensuing transmembrane fragment. Normally, depending upon the site of C-terminal processing by γ-secretase, Αβ might have between 39-43 amino acids in length (Karren et al . , 2011) . These peptides have a strong propensity to adopt beta sheet structures and to oligomerize and form protein insoluble aggregates. The Αβ40 is the predominant product of the amyloidogenic APP processing, but Αβ42 tends to oligomerize and aggregate faster and is the major form of Αβ linked to AD pathogenesis, leading to synaptic and neuronal loss. The APP is also processed by -secretase, however, the formation of Αβ is abrogated since the cleavage is between its residues 16-17 resulting in non-amyloidogenic peptides.

The BACE1 is the only β-secretase in the brain and its activity is the limiting step on the formation of Αβ (Ohno et al., 2004; Vassar et al . , 2009; Luo & Yan, 2010; De Strooper et al . , 2010; Yan & Vassar, 2014) . From a drug development point of view, BACEl has the advantage of being a single molecular entity while γ-secretase is a multiple subunit aspartyl protease with a high degree of heterogeneity (De Strooper et al . , 2010) . BACE-1 is a type 1 transmembrane aspartic protease that preferentially localizes in acidic intracellular compartments such as the trans-Golgi network and endosomes, where it cleaves APP, a type 1 transmembrane protein as well (Karren et al . , 2011; Selkoe 2011; Yan & Vassar 2014) . A homologous protein, BACE-2, shares 59 % homology with BACE-1 but has different cleavage specificity for APP, cleaving preferentially within the Αβ region and producing non-amyloidogenic peptides. Noteworthy, a 50 % reduction of the BACE-1 gene expression was shown to diminished Αβ deposition in the brains of PDAPP; Bacel+/- mice, as well as to protect against synaptic deficits, without compromising normal brain function (McColongue et al., 2007) . Also, the recently identified Ala673Thr APP variant which is less efficiently cleaved by BACE-1, leading to a decrease in Αβ production by roughly 20 % in individuals that have one copy of the Ala673Thr mutation, confers protection against AD and cognitive decline in elderly individuals (Jonsson et al . , 2012; Yan & Vassar 2014), pointing out that a slight BACEl inhibition might prevent AD. Thus, a careful dosage titration of a potential BACEl inhibitor allows the decrease in the Αβ production while minimizing mechanism- based adverse effects.

Despite the efforts of the scientific community towards the understanding of AD, at present, an effective therapy is an unmet clinical need (Karran et al . , 2011; Selkoe 2011) . Although several medicines are commercially available they only offer symptomatic improvements without preventing the disease progression. Thus, a successful therapy will have an immense impact on the personal, economic and societal burden of this disease.

BACE1 is a key target in AD (Li et al . , 2004; Zetterberg et al., 2008; De Strooper et al . , 2010; Luo and Yan 2010; Karran et al . , 2011; Selkoe 2011; Yan and Vassar, 2014) . In this application we disclose new peptide inhibitors of BACE1 comprising an active peptide and a cell penetrating peptide (CPP) . Cell penetrating peptides are amino acid sequences used as carriers of other molecules or pharmacological active compounds, named "cargoes". The TAT (48-57) sequence (TAT for transactivator of transcription) is a CPP enriched in positively charged residues that corresponds to the domain responsible for the cell penetrating properties of the TAT protein. The TAT positive charge is crucial to promote receptor-independent cellular uptake, mainly by the endocytic pathway (Chauhan et al . , 2007; Jarver et al . , 2010) . The uptake of a drug by endocytosis takes particular relevance within the context of an AD therapy targeting BACE1 since this protease preferentially localizes in acidic compartments such as the endosomes. Moreover, the activation of endocytosis is a specific and early event in sporadic AD, therefore the use of TAT allows an enhancement of the drug cellular uptake and efficacy in the most common form of AD (Cataldo et al . , 2000) . The ability of TAT to translocate the plasma membrane facilitates blood-brain barrier (BBB) permeation and cargo delivery to the cytoplasm of cells (Aarts et al . , 2002; Borsello et al . , 2003; Chauhan et al . , 2007; Taghibiglou et al . , 2009; Ittner et al . , 2010; Jarver et al., 2010; Tu et al . , 2010; Cook et al . , 2012; Bach et al . , 2012; Plattner et al . , 2014) .

The stability of CPP-delivery systems in vivo might be compromised by the action of proteolytic enzymes. To overcome proteolytic degradation the peptide sequences submitted in this application might include D-amino acids in their composition. Often, it is used the non-native D retroinverso (RI) sequence of the L-amino acid (native) peptide (Borsello et al . , 2003; Snyder et al . , 2004; Michod et al . , 2009; Vaslin et al . , 2011) . This double inversion of peptide structure increases the stability and consequently the half-life of biologically active peptides, which allows a decrease of the frequency of drug administration (Michod et al . , 2009) .

Several BACE1 inhibitors have been developed during the last decade (Chang et al . , 2004; Hussain et al . , 2007; Gosh et al., 2008; Fukomoto et al . , 2010; Chang et al., 2011; May et al . , 2015; Thakker et al . , 2015) and US 2011/0275619 Al, US 2006/0063717 Al , W02011 / 119465 , EP 0692490 Bl, EP 2172208 Al, W02009/ 131974 Al ) , mainly small molecules or peptides unrelated to the present application. Moreover, contrary to the molecules of the present application, these BACE1 inhibitors are not coupled to a cell penetrating peptide. In general, they have a poor performance regarding oral bioavailability, potency, selectivity and permeability across the BBB, which frequently made them unsuitable drug candidates. At present, there is no BACE1 inhibitor in clinical use although recently one BACE1 inhibitor reached the phase II/III clinical trials, the small molecule MK- 8931 (Yan and Vassar, 2014) . This small molecule is different from the molecules disclosed in this application, since it is not a peptidic compound neither a molecule based on the use of peptides including cell penetrating peptide carriers.

On the other hand, there are some ongoing clinical trials with promising candidates breaking down or immunoblocking the amyloid plaques formed throughout the disease progression. However, contrary to an efficient BACE1 inhibition, these strategies target preformed amyloid plaques and do not prevent its formation, which is crucial to control AD, and therefore are not the most suitable to prevent the onset neither the progression of the disease.

Currently the symptomatic therapeutics in use for AD include acetylcholinesterase inhibitors, and a NMDA receptor antagonist (memantine) , which allow a better function of the cholinergic and glutamatergic neurotransmission in AD patients. However, these compounds are not disease-modifying drugs and therefore do not prevent or delay disease progression.

Summary

The present application discloses peptide inhibitors of BACE1 comprising an APP amino acid sequence or an APP amino acid derived sequence coupled to a cell penetrating peptide sequence . one embodiment, the APP ammo acid sequence comp ID NO: 1 or SEQ ID NO : 2.

In another embodiment, the peptide inhibitors have a homology equal or higher than 70% with the APP amino acid sequence . In another embodiment, the APP amino acid derived sequence comprises the sequence SEQ ID NO: 3.

In a further embodiment, the cell penetrating peptide sequence comprises the peptide TAT sequence SEQ ID NO: 4 or a TAT variant sequence.

In another embodiment, the amino acids of the APP, APP derived and TAT sequences comprises D-amino acids.

In a further embodiment, the D-amino acids are in retroinverso form.

In another embodiment, the peptide inhibitor of BACE1 comprises one of the following sequences:

SEQ ID NO: 5,

SEQ ID NO: 6,

SEQ ID NO: 7,

SEQ ID NO: 8,

SEQ ID NO: 9; or

SEQ ID NO: 10.

The present application also relates to a method for the inhibition of BACE1 comprising the use of the peptide inhibitors above described.

Further disclosed in the present application is a pharmaceutical composition comprising a peptide inhibitor of BACE1 according to the herein disclosed, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents. The present application further relates to the use of the peptides in a method for the treatment or prevention of a disorder associated with amyloid deposits or with a disorder that constitutes a risk factor for dementia.

Additionally, the present application discloses the use of the peptides in a method for the treatment or prevention of Alzheimer's disease, Parkinson's disease, Vascular Dementia, Dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, Down's Syndrome, head trauma, and stroke.

General description

The present application discloses new drugs comprising peptides designed to inhibit BACE-1. The peptides include an active peptide (cargoe) based on the APP amino acid sequence flanking Asp⁶⁷², as well as on innovative variations of the APP sequence which have never been used, and a cell penetrating peptide, which in a preferred embodiment is the internalization peptide TAT or a TAT variant sequence that promotes the cellular uptake of the peptide. The drugs are composed of L-amino acids or composed, in part or exclusively, of D-amino acids in retroinverso sequence (D-RI) . The peptides are conceived to be used in AD, but can also be used within the context of other neurological disorders characterized by amyloid deposition or that may be a risk factor for AD. The present application provides as well a new methodology to inhibit BACE-1 based on the use of a cell penetrating peptide coupled to a peptide inhibitor of BACE1. This approach to design BACE1 inhibitors has never been addressed yet.

The approach with the peptides of the present application overcomes some of the limitations of the BACE1 inhibitors previously developed, since it presents the following ground-breaking features: i) Facilitated crossing through the BBB and cellular uptake, favouring the bioavailability of the compound in the brain cells where it must act. Indeed, in vivo fluorescence imaging allowed to determine the presence of a peptide herein disclosed (peptide 6) in the mice brain after intraperitoneal administration (i.p.) . ii) Favouring the location of the BACE1 inhibitor in the endosomal compartment, where it co-localizes with BACE1, thus increasing the likelihood to inhibit BACE1. iii) The activation of endocytosis is a specific and early event in sporadic AD, therefore we achieve an enhancement of the drug cellular uptake and efficacy. Possibly this feature also contributes to the selective targeting of the diseased neurons, decreasing potential side-effects. iv) The use of a D-RI-peptide increases the half-life of the inhibitor, allowing for a possible reduction in the frequency of administration. Indeed, in vivo fluorescence imaging allowed to determine the presence of a peptide herein disclosed (peptide 6) in the mice brain 24 h after drug administration (i.p.) . v) A careful dosage titration of these BACE1 inhibitors allows the decrease in the Αβ production while minimizing mechanism-based adverse effects since it prevents a total BACE1 inhibition allowing the enzyme to act upon other endogenous substrates. The above features are particularly relevant when considering a chronic treatment as in the case of AD and other neurodegenerative diseases.

The BACEl inhibitors included in the present application aim to overcome the caveats of the existing drugs in clinical use which do not act as disease modifying therapies and only moderately improve some of the symptoms of AD. Indeed, they will allow for a delay on the onset and progression of AD since the inhibition of BACEl will decrease Αβ production thus abrogating the amyloid pathology, which is due to Αβ accumulation in the brain parenchyma .

New peptide inhibitors of BACEl were developed which overcome some of the limitations of previous BACEl inhibitors that hindered their clinical use. The new peptide inhibitors of BACEl herein disclosed comprise both active peptides (based on the molecular structure of the substrate (APP) , as well as on new artificial variations of the APP sequence that have never been used) and a cell penetrating peptide, which in a preferred embodiment is the TAT(48-57) sequence (SEQ ID NO:4), or a related variant of the TAT peptide, which facilitates cellular membrane permeation and allows the inhibitor to reach effective concentrations in the central nervous system, where it must act. These new peptides constitute an innovative strategy to design an inhibitor of BACEl.

The active peptide includes the human APP sequence (SEQ ID NO:l) flanking the Met-Asp⁶⁷² cleavage site. The application provides as well the human APP sequence (SEQ ID NO: 2) flanking the Leu-Asp⁶⁷² cleavage site, present on the APP- Swedish mutation (APPsw) version, which has an increased affinity for BACE1. Also, it is provided the active peptide of the sequence SEQ ID NO: 3, which is a new artificial variation of the APPswe sequence. The sequences of the active peptides might include a variant sequence with more or less amino acids. The COOH-termini of all peptides are modified by amidation to increase proteolytic resistance.

The number of amino acids in the active peptides was chosen bearing in mind that the BACE1 active site pocket accommodates eight side chains, and considering three or more residues to work as a spacer between the active peptides and cell penetrating peptide sequences. The active peptides should be selectively recognized by BACE1 without interfering with the APP cleavage mediated by a-secretase and BACE2. Indeed, it was selected the APP sequence flanking Asp⁶⁷² instead of the sequence flanking the Tyr- Glu⁶⁸² cleavage site also recognized by BACE1, in order to use an APP sequence distant from the cleavage site recognized by a-secretase and from the in vivo preferential cleavage sites of BACE2. The preferred cell penetrating peptide sequence is a TAT sequence.

To overcome proteolytic degradation the TAT-APP peptides were designed employing protease-resistant D-amino acids and, to best mimic the structure of the natural peptide, it was considered the use of the retroinverso form (RI) of the D-peptides .

The preferred sequences of the peptides of the present application (both native L-form and D-RI-form) are:

• Peptide 1- TAT-APP: SEQ ID NO: 5; • Peptide 2- D-RI-TAT-APP : SEQ ID NO: 6;

• Peptide 3- TAT-APPsw: SEQ ID NO: 7;

• Peptide 4- D-RI-TAT-APPsw : SEQ ID NO: 8;

• Peptide 5- TAT-artificialvariantAPPsw: SEQ ID NO: 9;

• Peptide 6- D-RI-TAT-artificialvariantAPPsw: SEQ ID NO : 10.

These sequences of the peptides might include related variant sequences, with more or less amino acids, so that the peptides have equal or higher than 70 % of homology with the sequences of the peptides included in the present application .

Brief description of drawings

Without intent to limit the disclosure herein, this application presents attached drawings of illustrated embodiments for an easier understanding.

Figure 1 illustrates the BACEl activity in the presence of the new putative BACEl inhibitor peptides: PEP#1 (A), PEP#2

(B) , PEP#3 (C) , PEP#4 (D) , PEP#5 (E) and PEP#6 ( F) . BACEl activity was determined using the BACEl Activity Assay Kit

(Sigma) based on a FRET assay in which the fluorescence signal enhancement is observed after BACEl cleavage of the substrate. Briefly recombinant BACEl was incubated for 2 hours at 37°C with different concentrations of the inhibitors in the presence of the substrate according to the manufacturer protocol (n=3-5 independent experiments) . Fluorescence was recorded in a plate reader fluorometer at 320 nm (excitation) and 405 nm (emission) . Figure 2 illustrates the effect of the new BACE1 inhibitor peptide 5 on Αβ40 and Αβ42 levels in the conditioned medium of Neuroblastoma-2A cells expressing APPswe. (A and B) Effect of PEP#5 on secreted amyloid 40/42 levels. Twenty- four hours after plating, neuroblastoma-2A cells constitutively expressing the APPswe (N2A-APPswe cells) were incubated in FBS free medium with 12.5 to 300 μΜ of the peptide for 24 h, at 37 °C, in a humidified incubator with 5 % CO₂ · At the end of the incubation period, the conditioned medium was collected and stored at -80 °C until analysis of Αβ40 and Αβ42 levels by sandwich ELISA

(Invitrogen kit), according to manufacturer's protocol. Control cells were subjected to the same experimental procedures in the absence of peptide treatment. The results

(pg Αβ per mL) represent the mean ± SEM of n= 2-5 independent experiments performed in duplicate, and are expressed as percentage of control. *p<0.05, ****p<0.0001, significantly different compared to control group, as determined by ANOVA, followed by Dunnet' s post test.

Figure 3 illustrates the effect of the new BACE1 inhibitor peptide 6 on Αβ40 and Αβ42 levels in the conditioned medium of Neuroblastoma-2A cells expressing APPswe. (A and B) Effect of PEP#6 on secreted amyloid 40/42 levels. Twenty- four hours after plating, neuroblastoma-2A cells constitutively expressing the APPswe (N2A-APPswe cells) were incubated in FBS free medium with 12.5 to 300 μΜ of the peptide for 24 h, at 37 °C, in a humidified incubator with 5 % CO₂ · At the end of the incubation period, the conditioned medium was collected and stored at -80 °C until analysis of Αβ40 and Αβ42 levels by sandwich ELISA (Invitrogen kit), according to manufacturer's protocol. Control cells were subjected to the same experimental procedures in the absence of peptide treatment. The results (pg Αβ per mL) represent the mean ± SEM of n= 2-5 independent experiments performed in duplicate, and are expressed as percentage of control. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 significantly different compared to control group, as determined by ANOVA, followed by Dunnet's post test.

Figure 4 illustrates the effect of the new BACE1 inhibitors on Αβ40 and Αβ42 levels in the conditioned medium of Neuroblastoma-2A cells expressing APPswe. (A to D) Effect of PEP#5 and its D-retroinverso form peptide (PEP#6) on secreted amyloid 40/42 levels. Twenty-four hours after plating, neuroblastoma-2A cells constitutively expressing the APPswe (N2A-APPswe cells) were incubated in FBS free medium with 12.5 to 300 μΜ of the peptides for 24 h, at 37 °C, in a humidified incubator with 5 % CO₂. At the end of the incubation period, the conditioned medium was collected and stored at -80 °C until analysis of Αβ40 and Αβ42 levels by sandwich ELISA (Invitrogen kit), according to manufacturer's protocol. Control cells were subjected to the same experimental procedures in the absence of peptide treatment. The results (pg Αβ per mL) represent the mean ± SEM of n= 2-5 independent experiments performed in duplicate, and are expressed as percentage of control.

Figure 5 illustrates that the new BACE1 inhibitor peptides PEP#5 and PEP#6 at the IC50 concentration do not change Neuro2A-APPswe cells viability. The cells were incubated with 50 μΜ (pep#5) or 75 μΜ (pep#6) of the BACE1 inhibitors, in FBS free culture medium for 24 h, at 37 °C, in a humidified incubator with 5 % CO₂. Untreated cells were used as control. Cell viability was assessed by determining LDH (Cytotox 96 Non-Radioactive Cytotoxicity Assay, Promega) and Caspases 3/7 (Caspase 3/7- Glo assay, Promega) activity at the end of the incubation period. The caspase3/7 results represent the mean ± SEM of 2-4 independent experiments (n=2 for 15 min and n=4 for the 30- 180 min time points) and were normalized to control cells caspase activity (A, B) . The LDH results are expressed as percentage of total LDH (C, D) and represent the mean ± SEM of 4-5 independent experiments. The new BACE1 inhibitors at a concentration near the IC50 did not induce N2A-APPswe cells toxicity since no statistical significant differences between control and the experimental treatment conditions were observed, (p>0.05), as determined by ANOVA, followed by Dunnet's post test.

Figure 6 illustrates the new BACE1 inhibitor pep#6 reaches the mice brain and its levels remain high until 24 h after administration in 3xTg-AD mice. Four months old 3xTg-AD mice were treated with a single i.p. injection of 10 mg/kg of PEP#6 labelled with the fluorescent dye Cy5.5. In vivo fluorescence imaging of the brain was performed immediately before treatment and 1-48 hours post-drug administration using the Perkin Elmer IVIS Lumina XR equipment (A) . For that purpose mice were anaesthetized and non-invasive in vivo brain fluorescence imaging performed in animals submitted to depilation in the brain area. Quantification of brain signal was determined by measuring radiant efficiency in a specified region of interest (ROI) . For each mouse, the ROI was delimited in the brain area and the integrated density of the signal [ (p/sec/cm²/sr ) / (μΐΛί/cm²) ] determined by using the Living Image version 4.5 (Perkin Elmer) software. The graph represents the mean ± SEM of n=3 mice per group (B) . Statistical analysis was performed by t test, ****p<0.0001 significantly different compared to time zero .

Figure 7 illustrates that the new BACE1 inhibitors PEP#5 and PEP#6 decrease plasmatic Αβ40/42 levels in 3xTg-AD mice. A single i.p. treatment with the new BACE1 inhibitors PEP#5 and PEP#6 reduced plasma Αβ40 (A) and Αβ42 (B) levels in 4-month old 3xTg-AD mice. Twenty-four hours after administration, mice were sacrificed with anesthesia followed by cervical dislocation and the blood was collected into EDTA-treated tubes. After centrifugation, the plasma was collected and stored at -80 °C until analysis of Αβ40 and Αβ42 levels by sandwich ELISA (Invitrogen kit), according to manufacturer's protocol. Control mice were injected with the vehicle (saline) in the absence of peptides. The results (pg Αβ per mL) represent the mean ± SEM of n= 3 mice per group. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 significantly different compared to control group, as determined by ANOVA, followed by Dunnet's post test.

Figure 8 illustrates that the new BACE1 inhibitors PEP#5 and PEP#6 decrease brain-soluble Αβ40/42 levels in 3xTg-AD mice. A single i.p. treatment with new BACE1 inhibitors PEP#5 and PEP#6 reduced brain-soluble Αβ40 and Αβ42 levels in 4-month old 3xTg-AD mice. Twenty-four hours after administration, mice were sacrificed with anesthesia followed by cervical dislocation and the brain was collected and stored at -80 °C. Brain lysates in RIPA buffer were used to analyse Αβ40 and Αβ42 levels by sandwich ELISA (Invitrogen kit), according to manufacturer's protocol. Control mice were injected with the vehicle (saline) in the absence of peptides. Lysate protein level was assessed by the BCA method. The results (pg Αβ per mg protein) represent the mean ± SEM of n= 3 mice per group. ***p<0.001 significantly different compared to control, as determined by ANOVA, followed by Dunnet's post test.

Figure 9 illustrates the new BACE1 inhibitor pep#5 decreases εΑΡΡβ brain levels whereas sAPP levels remain unchanged in 3xTg-AD mice. A single i.p. treatment with the new BACE1 inhibitors PEP#5 decreased brain εΑΡΡβ without altering sAPPa levels in 4-month old 3xTg-AD mice. Twenty- four hours after administration, mice were sacrificed with anesthesia followed by cervical dislocation and the brain was collected and stored at -80 °C. Brain lysates in RIPA buffer were used to determine εΑΡΡβ (A) , sAPPa (B) , and actin (loading control) levels through Western blotting analysis. Lysate protein was assessed by the BCA method. Control mice were injected with the vehicle (saline) in the absence of peptides. Representative Western blots for each protein are presented above the graph. The results represent the mean ± SEM of, at least, n= 5-9 mice per group, and are expressed as percentage of control. Statistical analysis was performed by t test. *p<0.05 and ***p<0.001 significantly different compared to control.

Figure 10 illustrates the new BACE1 inhibitor pep#6 decreases εΑΡΡβ brain levels whereas sAPPa levels remain unchanged in 3xTg-AD mice. A single i.p. treatment with the new BACE1 inhibitors PEP#6 decreased brain εΑΡΡβ without altering sAPPa levels in 4-month old 3xTg-AD mice. Twenty- four hours after administration, mice were sacrificed with anesthesia followed by cervical dislocation and the brain was collected and stored at -80 °C. Brain lysates in RIPA buffer were used to determine εΑΡΡβ (A) , sAPP (B) , and actin (loading control) levels through Western blotting analysis. Lysate protein was assessed by the BCA method. Control mice were injected with the vehicle (saline) in the absence of peptides. Representative Western blots for each protein are presented above the graph. The results represent the mean ± SEM of, at least, n= 3-5 mice per group, and are expressed as percentage of control. Statistical analysis was performed by t test. *p<0.05 and ***p<0.001 significantly different compared to control.

Figure 11 illustrates the new BACE1 inhibitor pep#5 does not decrease brain APP and BACE1 levels in 3xTg-AD mice. A single i.p. treatment with the new BACE1 inhibitor PEP#5 does not change brain APP and BACE1 levels in 4-month old 3xTg-AD mice. Twenty-four hours after administration, mice were sacrificed with anesthesia followed by cervical dislocation and the brain was collected and stored at -80 °C. Brain lysates in RIPA buffer were used to determine APP

(A), BACE1 (B) and actin (loading control) levels through Western blotting analysis. Lysate protein was assessed by the BCA method. Control mice were injected with the vehicle

(saline) in the absence of peptides. Representative Western blots for each protein are presented above the graph. The results represent the mean ± SEM of, at least, n= 6-10 mice per group, and are expressed as percentage of control. Statistical analysis was performed by t test, no significant differences were found.

Figure 12 illustrates the new BACE1 inhibitor pep#6 does not decrease brain APP and BACE1 levels in 3xTg-AD mice. A single i.p. treatment with the new BACE1 inhibitor PEP#6 does not change brain APP and BACE1 levels in 4-month old 3xTg-AD mice. Twenty-four hours after administration, mice were sacrificed with anesthesia followed by cervical dislocation and the brain was collected and stored at -80 °C. Brain lysates in RIPA buffer were used to determine APP

(A), BACE1 (B) and actin (loading control) levels through Western blotting analysis. Lysate protein was assessed by the BCA method. Control mice were injected with the vehicle

(saline) in the absence of peptides. Representative Western blots for each protein are presented above the graph. The results represent the mean ± SEM of, at least, n= 4 mice per group, and are expressed as percentage of control. Statistical analysis was performed by t test, no significant differences were found.

Detailed description

In the present disclosure are described new peptide inhibitors of BACE1 which overcome some of the limitations of previous BACE1 inhibitors that hindered their clinical use .

The new peptide inhibitors of BACE1 comprise an active peptide based on the molecular structure of the substrate (APP) , as well as on new artificial variations of the APP sequence that have never been used, and a cell penetrating peptide, preferably the TAT₍₄₈-57) sequence (SEQ ID NO:4), or a related variant of the TAT peptide, which facilitates cellular membrane permeation and allows the inhibitor to reach effective concentrations in the central nervous system, where it must act. These new peptides constitute an innovative strategy to design an inhibitor of BACE1 that has never been addressed before. The COOH-termini of all the peptides are modified by amidation to increase proteolytic resistance. The active peptides include the sequence SEQ ID NO: 3, which is a new artificial variation of the APPswe sequence that has never been used before.

The active peptides include the human APP sequence (SEQ ID NO:l) flanking the Met-Asp⁶⁷² cleavage site. The present application provides as well the human APP sequence (SEQ ID NO: 2) flanking the Leu-Asp⁶⁷² cleavage site, present on the APP-Swedish mutation (APPsw) version, which has an increased affinity for BACEl.

To overcome proteolytic degradation we designed TAT-APP peptides employing protease-resistant D-amino acids and, to best mimic the structure of the natural peptide, we considered to use the retroinverso form (RI) of the D- peptides .

The number of amino acids in the active peptide was chosen bearing in mind that the BACEl active site pocket accommodates eight side chains, and considering three or more residues to work as a spacer between the APP and TAT sequences .

The active peptides should be selectively recognized by BACEl without interfering with the APP cleavage mediated by a-secretase and BACE2. It was selected the APP sequence flanking Asp⁶⁷² instead of the sequence flanking the Tyr- Glu⁶⁸² cleavage site also recognized by BACEl, in order to use an APP sequence distant from the cleavage site recognized by a-secretase and from the in vivo preferential cleavage sites of BACE2. Therefore, the disclosed peptides present the following functional innovative features considering previous BACE1 inhibitors :

- Facilitation of the crossing through the BBB and the cellular uptake, favouring the bioavailability of the compound in the brain cells where it must act. Indeed, in vivo fluorescence imaging allowed to determine the presence of a peptide herein disclosed (peptide 6) in the mice brain after intraperitoneal administration (i .p. ) .

- Favouring the location of the BACE1 inhibitor in the endosomal compartment, where it co-localize with BACE1, thus increasing the likelihood to inhibit BACE1.

- The activation of endocytosis is a specific and early event in sporadic AD, allowing for an enhancement of the peptides cellular uptake and efficacy. Possibly this feature also contributes to the selective targeting of the diseased neurons, decreasing possible side-effects .

- The use of a D-RI-peptide increases the half-life of the inhibitor, allowing a reduction in the frequency of administration. Indeed, in vivo fluorescence imaging allowed to determine the presence of a peptide herein disclosed (peptide 6) in the mice brain 24 h after drug administration (i.p.) .An important feature of TAT is that it displays low toxicity. The use of TAT coupled to a BACE1 inhibitor based on the APP amino acid sequence, or in a variation of that sequence, has never been addressed in AD. - On the other hand, a careful dosage titration of a potential BACEl inhibitor allows decreasing Αβ production while minimizing mechanism-based adverse effects, since it will prevent a total BACEl inhibition allowing the enzyme to act upon other cellular substrates.

The above features are particularly relevant when considering a chronic treatment as in the case of AD and other neurodegenerative diseases.

The experimental data that support the disclosed peptides as new BACEl inhibitors is described below.

Firstly, it was determined the efficacy of the peptides to inhibit BACEl activity using a cell free in vitro assay (BACEl Activity Assay Kit, Sigma) following the manufacturer instructions. The in vitro assays allowed to determine the peptides IC50 (Table 1, Figure 1) .

Table 1. Peptides IC50 determined using a cell free assay system. The IC50 refers to the peptide concentration that inhibits BACEl activity by 50%.

Peptide IC50 (M)

Peptide 1 8.316 X 10"'

Peptide 2 7.64 X 10^~6

Peptide 3 1.485 X 10^~6

Peptide 4 3.713 X 10^~6

Peptide 5 9.803 X 10^~6

Peptide 6 6.490 X 10^~7 Thereafter, the peptides 5 and 6 were selected to initiate the studies in a cellular model of AD, the neuroblastoma cell line Neuro-2A overexpressing APPswe (N2A-APPswe) , and the ability of the new BACE1 inhibitors to reduce endogenous Αβ40 and Αβ42 production, as assessed by sandwich ELISA, was determined. It was observed that, after a 24 h incubation period, 100 μΜ peptide 5 (Figure 2) inhibited maximally Αβ40 and Αβ42 formation by 74.0 ± 4.51 % and 84.2 ± 6.6 % while 100 μΜ peptide 6 (Figure 3) inhibited maximally Αβ40 and Αβ42 formation by 73.8 ± 7.7 % and 74.1 ± 5.5 %, respectively. The dose response curves (Figure 4) allowed for the determination of the IC50 for peptide 5 and peptide 6 in N2A-APPswe cells (Table 2) .

Table 2. Peptides IC50 determined in a cellular assay. The IC50 refers to the peptide concentration that inhibits endogenous Αβ40 and Αβ42 production in N2A-APPswe cells by 50%.

Peptide Αβ40 - IC50 (M)

Peptide 5 6. 408 X 10^" -5

Peptide 6 7. 562 X 10^" -5

Peptide Αβ42 - IC50 (M)

Peptide 4. 560 X 10^" -5

Peptide 6 7. 823 X 10^" -5

Importantly, the incubation for 24 h with the new BACE1 inhibitors (peptide 5 and 6) , at concentrations close to the IC50, did not induce caspase-3 and caspase-7 activation (Figure 5 A and B) , neither increased the release of LDH (Figure 5 C and D) . These results indicate that, at the IC50 concentration, peptides 5 and 6 did not cause apoptosis neither necrosis of N2A-APPswe cells. Thus, the new BACE1 inhibitors reduced N2A-APPswe cells endogenous Αβ production in the absence of a cytotoxic effect. In vivo studies were performed using a triple transgenic mouse model of AD (3xTg-AD) harboring PS1M146V, APPswe, and TauP301L transgenes (Oddo et al . , 2003) to evaluate whether the new BACE1 inhibitors, peptide 5 and peptide 6, ameliorate Αβ pathology.

To demonstrate that the peptides herein disclosed have the potential to reach the brain we labelled peptide 6 with the fluorescent dye Cy 5.5 and performed an in vivo fluorescence imaging study in 3xTg-AD mice injected (i.p.) with 10 mg/kg of the compound. As shown in Figure 6, peptide 6 has the ability to cross the blood brain barrier and to penetrate the mouse brain. Its brain levels remained high until 24 h after peptide administration and at 48 h the compound brain levels were identical to basal. Afterwards the brain and plasma levels of Αβ40 and Αβ42 were assessed 24 h after a single administration (i.p. injection) of the compounds in 4 months old 3xTg-AD mice. The results pointed out that both peptides at 1.25 mg/kg reduced plasma Αβ40 by 30% and, at a dose of 5.0 mg/kg, reduced plasma Αβ42 by at least 30 %, as assessed by sandwich ELISA (Figure 7 A, B) . Regarding brain soluble Αβ levels, peptide 5 and 6 at 1,25 mg/kg reduced Αβ40 by 50% and 28 %, respectively, while both peptides reduced Αβ42 by about 28 % (Figure 8 A e B) .

Accordingly, peptide 5 decreased the soluble APP cleavage fragment εΑΡΡβ that ensues from APP cleavage by BACE1 by about 11 % (Figure 9 A), without altering the levels of the sAPP fragment, which arises from APP cleavage by - secretase (Figure 9 B) , as assessed by western blot. Likewise, peptide 6 decreased εΑΡΡβ levels by about 24 % whereas the amount of the sAPP fragment was not significantly changed (Figure 10 A and B) . These results indicate that peptides 5 and 6 selectively inhibit BACE1 activity in 3xTg-AD mice without inhibiting the activity of -secretase .

Moreover, it was observed that the administration of peptides 5 and 6 did not decrease APP (Figure 11 A and Figure 12 A) and BACE1 protein levels (Figure 11 B and Figure 12 B) , which demonstrates that the reduction in Αβ and εΑΡΡβ levels is due to the inhibition of BACE1 activity and not because of a drop on the enzyme or its substrate levels .

RE FERENCE S

- Aarts et al . , 2002, Treatment of Ischemic Brain Damage by Perturbing NMDA Receptor-PSD-95 Protein Interactions, Science 298: 846-850.

- Bach et al . , 2012, A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage. PNAS 109: 3317-3322.

- Borsello et al . , 2003, A peptide inhibitor of c-Jun N- terminal kinase protects against excitotoxicity and cerebral ischemia, Nature Medicine 9: 1180-1186.

- Cataldo et al . , 2000, Endocytic Pathway Abnormalities Precede Amyloid β Deposition in Sporadic Alzheimer's Disease and Down Syndrome Differential Effects of APOE Genotype and Presenilin Mutations. AJP 157: 277-286.

- Chang et al . , 2004, In vivo inhibition of Αβ production by memapsin 2 ( β-secretase ) inhibitors. J Neurochem 89: 1409-1416. - Chang et al . , 2011, Secretase inhibitor GRL-8234 rescues age-related cognitive decline in APP transgenic mice. FASEB J. 25, 775-784.

- Chauhan et al . , 2007, The taming of the cell penetrating domain of the HIV Tat: myths and realities. J Controlled Release 117:148-162.

- Cook et al., 2012, Treatment of stroke with a PSD-95 inhibitor in the gyrencephalic primate brain. Nature 483: 213-217.

- De Strooper et al . , 2010, The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat Rev Neurol. 6: 99-107.

- Fukumoto et al . , 2010, A noncompetitive BACE1 inhibitor TAK-070 ameliorates Αβ pathlogy and behavioural deficits in a mouse model of Alzheimer's disease. J Neurosci 30: 11157- 11166.

Ghosh et al . , 2008, Potent memapsin 2 (b-secretase ) inhibitors: Design, synthesis, protein-ligand X-ray structure, and in vivo evaluation. Bioorganic & Medicinal Chemistry Letters 18 (2008) 1031-1036.

- Hussain et al . , 2007, Oral administration of a potent and selective non-peptidic BACE1 inhibitor decreases β-cleavage of amyloid precursor protein and amyloid-β production in vivo. J Neurochem 100: 802-809.

Ittner et al . , 2010, Dendritic function of Tau mediates amyloid-β toxicity in Alzheimer's disease mouse models. Cell 142: 387-397.

- Jarver et al . , 2010, In vivo biodistribution and efficacy of peptide mediated delivery. TIPS 31: 528-535.

- Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, Hoyte K, Gustafson A, Liu Y, Lu Y, Bhangale T, Graham RR, Huttenlocher J, Bjornsdottir G, Andreassen OA, Jonsson EG, et al . (2012) A mutation in APP protects against Alzheimer's disease and age-related cognitive decline. Nature 488:96 -99.

- Karran et al . , 2011, The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics. Nature Reviews Drug Discovery 10:698-712.

- Li et al . , 2004, Amyloid β peptide load is correlated with increased β-secretase activity in sporadic Alzheimer's disease patients. PNAS 101: 3632-37.

- Luo and Yan, 2010, Inhibition of BACE1 for therapeutic use in Alzheimer's disease. Int J Clin Exp Pathol 3:618- 628;

- May et al . , 2015, The potent BACE1 inhibitor LY2886721 elicits robust central Αβ pharmacodynamic responses in mice, dogs, and humans. J Neuroci 35:1199-1210.

- McColongue et al . , 2007, Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP transgenic mice. J Biol Chem 282: 26326-334.

- Michod et al . , 2009, Effect of RasGAP N2 fragment-derived peptide on tumor growth in mice. J Natl Cancer Inst 101: 828-832.

- Oddo et al . , 2003, Triple-Transgenic Model of Alzheimer's Disease with Plaques and Tangles: Intracellular A_ and Synaptic Dysfunction, Neuron, Vol. 39, 409-421.

Ohno et al., 2004, BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease. Neuron 41: 27-33.

- Plattner et al . , 2014, Memory Enhancement by Targeting Cdk5 Regulation of NR2B. Neuron 81, 1070-1083

Selkoe 2011, Resolving controversies on the path to Alzheimer's therapeutics. Nature Medicine 17:1060-1065; - Snyder et al . , 2004, Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide. PLoS Biology 2: 0186-0193.

Suhorutsenko et al . , 2011, Cell-Penetrating Peptides, PepFects, Show No Evidence of Toxicity and Immunogenicity In vitro and In vivo. Bioconjugate Chem. 22: 2255-2262.

- Taghibiglou et al . , 2009, Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nature Medicine 15: 1399-1406.

- Thakker et al . , 2015, Centrally delivered BACEl inhibitor activates microglia, and reverses amyloid pathology and cognitive deficit in aged Tg2576 mice. J Neurosci 35:6931- 6936.

- Tu et al., 2010, DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke, Cell 140: 222-234.

- Vaslin et al . , 2011, Excitotoxicity-induced endocytosis mediates neuroprotection by TAT-peptide-linked JNK inhibitor. J Neurochem 119: 1243-1252.

- Vassar et al . , 2009, The β-Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential. The Journal of Neuroscience 29:12787-12794;

- Yan and Vassar, Lancet Neurol 2014; 13: 319-29.

Zetterberg et al . , 2008, Elevated cerebrospinal fluid BACEl activity in incipient Alzheimer disease. Arch Neurol 65: 1102-1106.

SEQUENCE LISTING

<110> Universidade de Coimbra

<120> PEPTIDE INHIBITORS OF BACEl FOR THE TREATMENT OF NEUROLOGICAL DISORDERS <130> PPI 51891/15

<160> 10

<170> Patentln version 3.5

<210> 1

<211> 11

<212> PRT

<213> Homo sapiens

<400> 1

Glu He Ser Glu Val Asn Leu Asp Ala Glu 1 5 10

<210> 2

<211> 11

<212> PRT

<213> Homo sapiens

<400> 2

Glu He Ser Glu Val Lys Met Asp Ala Glu 1 5 10

<210> 3

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 3

Leu Glu He Ala Val Ser Asn Glu Phe Glu Asp 1 5 10

<210> 4

<211> 10

<212> PRT <213> Human immunodeficiency virus

<400> 4

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg

1 5 10

<210> 5

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 5

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Glu lie Ser Glu Val Lys

1 5 10 15

Met Asp Ala Glu Phe

20

<210> 6

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 6

Phe Glu Ala Asp Met Lys Val Glu Ser He Glu Arg Arg Arg Gin Arg

1 5 10 15

Arg Lys Lys Arg Gly

20

<210>

<211> <212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 7

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Glu lie Ser Glu Val Asn

1 5 10 15

Leu Asp Ala Glu Phe

20

<210> 8

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 8

Phe Glu Ala Asp Leu Asn Val Glu Ser lie Glu Arg Arg Arg Gin Arg

1 5 10 15

Arg Lys Lys Arg Gly

20

<210> 9

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 9

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Leu Glu lie Ala Val Ser 1 5 10 15

Asn Glu Phe Glu Asp

20

<210> 10

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 10

Asp Glu Phe Glu Asn Ser Val Ala lie Glu Leu Arg Arg Arg Gin Arg

1 5 10 15

Arg Lys Lys Arg Gly

20

Claims

A peptide inhibitor of BACEl comprising an APP amino acid sequence or an APP amino acid derived sequence coupled to a cell penetrating peptide sequence.

The peptide inhibitor of BACEl according to claim 1, wherein the APP amino acid sequence comprises SEQ ID NO: 1 or SEQ ID NO : 2.

The peptide inhibitor of BACEl according to the previous claim, comprising a homology equal or higher than 70% with the APP amino acid sequence.

The peptide inhibitors of BACEl according to the claim 1, wherein the APP amino acid derived sequence comprises the artificial sequence SEQ ID NO: 3.

The peptide inhibitors of BACEl according to claim 1, wherein the cell penetrating peptide sequence comprises the peptide TAT sequence SEQ ID NO: 4 or a TAT variant sequence or other suitable cell penetrating peptide.

The peptide inhibitors of BACEl according to the previous claims, wherein the amino acids of the APP and TAT sequences comprise D-amino acids.

The peptide inhibitor of BACEl according to the previous claim, wherein the D-amino acids are in retroinverso form.

8. The peptide inhibitor of BACE1 according to the previous claims, wherein it comprises one of the following sequences :

SEQ ID NO: 5;

SEQ ID NO: 6;

SEQ ID NO: 7;

SEQ ID NO: 8;

SEQ ID NO: 9; or

SEQ ID NO: 10.

9. A method for the inhibition of BACE1 comprising the use of the peptide inhibitor of claims 1-8.

10. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 8 optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents.

11. A peptide according to any one of claims 1 to 8 for use in a method for the treatment or prevention of a disorder associated with amyloid deposits or with a disorder that constitutes a risk factor for dementia.

12. A peptide according to any one of claims 1 to 8 for use in a method for the treatment or prevention of Alzheimer's disease, Parkinson's disease, Vascular Dementia, Dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, Down's Syndrome, head trauma, and stroke.