Screening β-secretase Inhibitors Through Random Peptide Phage Display Technique

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in the central nervous system. It's characterized by the neuropathologic findings of intracellular neurofibrillary tangles (NTF) and extracelluar amyloid plaques (senile plaque) that accumulate in vulnerable brain regions. AD patients have some common clinical symptoms like progressive memory loss and progressive deterioration of specific cognitive functions. It's reported that there is 510 millions AD patients in USA and this number will increase three times by 2050 year. Up to now, the morbidity rate in China is very close to America. According to the age, AD patients are classified into two parts, age below 65 called'early onset'while up 65 is called'late onset'.Amyloid precursor protein (APP) is the most popular protein which plays an important role in the pathogenic mechanism of AD. APP has three isoforms that is APP770, APP752 and APP695. APP is a typeⅠtansmembrane glycoprotein which was cleaved byβ-andγ-secretases sequentially, then generates three peptide:1) sAPPβ, a soluble peptide released to the extracellular; 2) Aβ, the main toxicity peptide which can accumulate in neurons and deposited as amyloid plaque; 3) AICD (APP intracellular domain). According to the amyloid hypothesis, accumulation of Aβis the primary factor driving AD pathogenesis. Reducing of Aβsecretion can be achieved by decreasingβ-secretase activity and y-secretase activity, these two enzymes have been believed as important drug targets in AD.P-secretase is a typeⅠtransmembrane protein and belongs to pepsin family with its homolog BACE2. BACE1 gene is located on chromosome-11 at 11q23.2-q23.3 and encode a.501 amino acid(aa) protein with an N-terminal signal peptide of 21 amino acids followed by a proprote in domain spanning amino acids 22-45. The lumenal domain of the mature protein extends from residues 46 to 460 and is followed by a transmembrane domain of 17 residues and a short cytosolic tail of 24 amino acids. BACE1 contains two active site motifs at amino acids 93-96 and 289-292 in the lumenal domain, each containing the highly conserved signature sequence of aspartic proteases DT/SGT/S. BACElalso has four putative N-linked glycosylation sites and six lumenal cysteines, which would allow the formation of up to three intramolecular disulfide bonds. Evidence showed no obvious deficits in basal neurological and physiological functions in BACE1 gene knockout mice.γ-secretase is a protease complex consisting of four transmembrane proteins, presenilin, nicastrin, APH1 and PEN2, that together catalyze the intramembrane cleavage of APP at the y-secretase sites. In addition to generating Aβduring the pathogenesis of Alzheimer's disease, presenilin-dependent cleavage is also intimately involved in several physiological signal-transduction processes, such as Notch signals. So inhibition ofγ-secretase would produce some unwanted side effects. In contrast,β-secretase inhibitor seems safer thanγ-secretase inhibitor. So far, there has been noβ-secretase inhibitor used to AD treatment in clinical.Among various pathways to screen the enzyme inhibitors, the phage display technique has its own advantage. Phage display describes a selection technique in which a peptide or protein is expressed in fusion with a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the virion and its encoding DNA inside the virion. In its simplest form, panning is carried out by the following steps:1) Constructing the original phage library or amplification of an existing library; 2) Incubating a library of phage-displayed peptides with a plate coated with the target, washing away the unbound phage, and eluting the specifically-bound phage; 3) amplified the eluted phage and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences. After 3-4 rounds, individual clones are characterized by DNA sequencing; 4) Synthesized and analyzed the affinity or specificity of these proteins or peptides.It's reported that scientists have successfully selected peptide inhibitors of MurC, MurD and MurF enzymes through phage display technique, which are essential enzymes of the antibiotic resistant pathogen Pseudomonas aeruginosa. This technique also was used to pan the inhibitors ofβ-lactamase.Based on the data above, we decided to use phage display technique to pan theβ-secretase inhibitor which could provide a new therapeutic agent to AD treatment.Before the panning procedure, we need to set up a cell model to test the activity ofβ-secretase inhibitor. Firstly, cDNA-coding sequences (BACE1) was modified by the addition of a HindⅢsite and a enterokinase cleavage site at the 5'end, and a 6xHis Tag together with a BamHI site at 3'end by PCR, then the amplified fragment was ligated into the pEGFP-c3 expression vector and transformed into the E.coli DH5a competent cell. After the single clones were identified by PCR and DNA sequencing, the recombinant plasmid was transfect into the HEK293 cell. After 24 hours, the cell was harvested and sonicated, the cell lysis was centrifuged and the supernatant was passed through the TALON Mental Affinity Resins column to purify the EGFP-BACE1 fusion protein. Then the fusion protein was cleaved by tenterokinase at room temperature. The BACE1 was obtained by transferring the digestion to the resins again to remove EGF protein. The activity of purified BACE1 was tested with BACE1 Activity Assay Kit. The assay is based on a convenient method of fluorescence resonance energy transfer (FRET) in which the fluorescence signal is released by BACE1 cleavage. The procedures was run by adding BACE1 to the 96 well plate contain the fluorescence substrate, read the fluorescence value immediately and then incubate the plate at 37℃for two hours. Read the fluorescence value again. Statistics results showed that the fluorescence value of purified BACE1 was 1587.115±138.4868 (n=3),P=0.247, versus group of BACE1 standard. That verified our purified BACE1 has enzyme activity in vitro.Then we co-tansfected the recombinant BACE1/pEGFP-c3 plasimid and APP/pDsRed-Monomer-N1 plasmid to the HEK293 cell,24 hours later, harvest the cell and observe the cleavage activity of BACE1 in cell by western blot. We found that the CTFAPPβwith 12KD band appear. This result suggests that BACE1 cleaved APP in the HEK293 cell. All this evidences show that we successfully set up a cell model, which can be used to test the activity ofβ-secretase inhibitor.The small peptide has several advantages because of its small molecular, such as they can permeable the cell wall easily and possess small immunogenicity when use on the animal models. So we selected the Ph.D.-C7CTM random peptide library to panning BACE1 inhibitors. After three rounds panning, we sequenced the DNA of the selected 11 clones and found five of them have the same sequence. We synthesized the peptide-1(CPLHARLLC) and observed its inhibition activity at five different concentration that is 100nM,1000nM,10,000nM,100,000nM和1,000,000nM. However, there is no obvious effect on the enzyme activity detected in the test. Then we had two more screening and found the preponderance clone sequence that is Peptide-2 (CGYPSLHAC). We synthesized the Peptide-2 in three different structures and test their activity. None of them showed inhibition activity. Now another screening has been done under improved steps referred to the previous experiments. The rest bound clones from the previous screening were analyzed and the following step is still going on.