Novel non-peptide b-secretase inhibitors

This missive describes an efficient attack by incorporating practical showing with bio-assay engineering for happening little organic inhibitors aiming & A ; beta ; -secretase ( BACE-1 ) . 15 hits with repressive authorities runing from 2.8 to 118 & A ; mu ; M ( IC50 ) against & A ; beta ; -secretase were successfully identified. Compound 12 with IC50 of 2.8 & A ; mu ; M is the most powerful hit against BACE-1. Docking simulation from GOLD 3.0 suggests putative adhering manner of 12 in BACE-1 and possible key pharmacophore groups for farther designing of non-peptide compounds as more powerful inhibitors against BACE-1.

Keywords: b-secretase ; Virtual showing ; Bioassay ; FRET

Alzheimer ‘s disease ( AD ) , a neurodegenerative upset, is the most common signifier of dementedness and histories for two tierces of all instances. The disease is acquiring worse and worse as the population of aging people is acquiring higher worldwide. Today it is the sixth-leading cause of decease in the United States and has become a major societal and economic load for both the society and family1. Remedy for this disease is presently unavailable although extended research has been focused on the development of curative attacks. Therefore, probes on new drug find and development for AD are of pressing necessity. AD is pathologically characterized by the presence of intracellular neurofibrillary tangles and extracellular senile plaques in the brain2-4. The major constituents of the plaques remained unknown until a little peptide termed beta amyloid ( A & A ; beta ; ) was purified from neuritic plaques5. This peptide consists of 39-43 residues that are endo-proteolytically derived from a transmembrane amyloid precursor glycoprotein ( APP ) 6. Following the find and study of A & A ; beta ; , a dominant trademark of pathogenesis known as amyloid cascade hypothesis was developed to suggest that the overrun and collection of a 42-amino acerb signifier of A & A ; beta ; is followed by its deposition in the plaques in the brain7-9. The endoproteolytic cleavage of the APP to acquire A & A ; beta ; involves the consecutive actions of two peptidases, the & A ; beta ; -secretase ( BACE-1, hereinafter ) and & A ; beta ; -secretase10.Therefore, BACE-1 has become an attractive curative mark and its inhibitors are possible drug campaigners for the intervention of AD. In the past decennary, the major attempt in planing BACE-1 inhibitors was the production of passage province isosteres such as hydroxyethylamines, reduced amides, statine-based peptidometic inhibitors countering the catalytic aspartyl groups. However, no non-peptidic inhibitors were reported and none of the already reported BACE-1 inhibitors has been marketed as efficient drug so far due to the complication by the demand for cardinal nervous system penetration11, 12. Hence, designation of fresh little non-peptide inhibitors is necessary to do the pharmacokinetic belongingss of chemicals more favourable for farther development and enlarge the infinite of drug lead find every bit good as to convey the leads into pre-clinical and clinical tests.

While peptidomimetic passage province isostere based inhibitors, such as statine, homostatine, norstatine, and hydroxyethylamine, have dominated the major attempt in the design of powerful inhibitors of human BACE-1. Li ‘s group employed a combinative chemical science attack to develop homostatine based inhibitor which had an IC50 value of 143 nanometer in an enzymatic assay13 and Shering-Plough Corp presented a hydroxyethylamine based inhibitor with an IC50 of 4 nM14. Merely till late, some non-peptide compounds were identified as inhibitors of BACE-1. Astex research workers highlighted their work in detecting aminopyridine and cyclic amidine categories as BACE-1 inhibitors15. Barrow et al reported the designation of spiropiperidine inhibitor templet for BACE-116. Although the inhibitors from others have been demonstrated potent in enzymatic checks, this has non discouraged us from researching new BACE-1 inhibitors with alternate structural scaffolds. These inhibitors rarely enter the encephalon due to their unfavourable physicochemical belongingss, such as their high polar surface countries and high figure of H-bond givers and acceptors as they are peptides in nature. Therefore, placing selective nonpeptidic BACE-1 inhibitors with ideal hydrophobicity for CNS incursion and good pharmacokinetic belongingss would be demanding.

To detect novel little molecule inhibitors with new chemical skeleton as possible drug leads, we applied a receptor-based practical showing attack to seek the compound database Specs ( ) incorporating ~280,000 chemicals and identified 42 hit compounds. All computations were performed on IBM bunch equipped with 64 processors. Crystal construction of BACE-1 complexed with an inhibitor OM00-3 ( PDB entry: 1M4H ) resolved at 2.1 & A ; Aring ; 17 was extracted from Brookhaven Protein Data Bank ( PDB ) ( ) . Hydrogen atoms were added and H2O molecules co-crystallized with the protein were removed from the original construction utilizing Sybyl 8.0 ( Tripos associate inc. , St. Louis, MO, USA ) . The modified crystal construction of BACE-1 was used as the mark for practical showing on commercial chemical databases Specs by utilizing GOLD 3.0 package ( CCDC, Cambridge, U.K. ) . Chemical database Specs was edited from its original sdf file format to mol2 format. The default parametric quantities in GOLD 3.0 were used. The active site radius is 15A* ‘ from atom 1846 OD2 of Asp228, which is one of the cardinal amino acid residues in the aspartyl peptidase. The GoldScore fittingness map was applied and top 3000 molecules with the highest GOLDscores from initial practical showing were so re-submitted for multiple moorage of 10 conformations for each ligand. Finally, the top 1000 hits were selected for farther ocular review of their binding conformation and geometrical matching quality with the active sites of BACE-1. Based on the predicted putative H-bonds formed by the hits and active site residues of BACE-1, the possible hydrophobic and aromatic-aromatic interactions, every bit good as predicted clogP values of 4~6 for blood encephalon barrier, 42 compounds among the top 1000 hits were selected for biological checks. Among them, 15 new possible BACE-1 inhibitors ( Shown in Table 1 ) were discovered to be active through bio-assay with FRET technology18, showing that the applied attack is a extremely efficient manner to detect active compounds with new scaffold different from current peptidic BACE-1 inhibitors. In this survey, about tierce of the compounds ( 12/42 ) demonstrated their repressive authorities of greater than 50 % of BACE-1 at 100 & A ; mu ; M and the most active compound 12 identified from this work has an IC50 value of 2.8 & A ; mu ; M. Although it is weaker than the positive control, a statine-based peptide with an IC50 value of 120 nanometer from our trial ( mention IC50 value is 30 nanometer ) 19, it is fresh in footings of its organic construction with smaller molecular weight that makes it possible to perforate the encephalon barrier. The coevals of several different structural scaffolds as fresh pharmacophores of BACE-1 inhibitors implies the possibility and importance of the fast, economic computer-assisted attack in modern drug find and design.

From our docking survey, all 15 inhibitors were proposed to adhere with BACE-1 within the enzyme active pocket. Due to the fact that BACE-1 consists of more sub-pockets ( S4′-S4 ) than other aspartyl peptidases in its active site, it is expected that inhibitors capable of interacting with more sub-sites could take to stronger repressive consequence. Such outlook is consistent with our bio-assay consequences, as exemplified by repressive differences among all inhibitors. Compounds 12 and 13 with higher activity about occupy the whole active pocket of BACE-1 while all the remainder bind with BACE-1 chiefly via S3-S3 ‘ interaction. Notably, 12 exhibited low micromolar authority against BACE-1 in the FRET check. As shown in Figure 1, molecular docking derived from GOLD suggested a sensible binding manner of compound 12 in BACE-1. Bing the cardinal “ span ” of 12, the benzothiazole pealing occupies S1 sub-pocket, doing aromatic-aromatic interaction with Tyr71. The linker S group fits the little, shallow S3 and S4 pocket is occupied by di-methoxy phenyl group, lending to hydrophobic and Van der Waals force within the site. The right ( premier ) side of the active site is chiefly occupied by thiazine together with two piperidine groups on it. Several H bond interactions were observed from the docking simulation ( Figure 2 ) . Among them the most of import interaction is the H-bond formed between the linker NH with O in Asp228, thereby miming the isostere payloads in antecedently reported synthetically optimized inhibitors. Besides, Gly34 and Tyr198 besides form two H bonds with the triazine and piperidine in 12 severally. Interestingly, a similar molecule 13 with IC50 of 10.2 & A ; mu ; M was besides identified from the practical showing. However, the alteration from dimethoxyphenyl group to fluorobenzne in 13 rendered the activity lose by 5 creases. Hence, the Van der Waals interactions between the Ala231 to the methoxy group on the P4 phenyl ring in the inhibitors might be indispensable for strong suppression of the enzymatic activity. As small is known about the usage of compound 12 elsewhere earlier, the chemical scaffold in 12 might stand for a new category for farther drug lead optimisation aiming BACE-1.

Albeit compound 12 is a fresh moderate inhibitor of BACE-1, farther hunt of compounds bearing of import pharmacophores in 12 will be continued. Furthermore, execution of multiple hiting maps in preliminary computational anticipations of possible hits would lend to better enrichment rates during practical showing and molecular moorage procedure. Recently, Vijayan et al reported a intercrossed structure-based practical showing for designation of several prospective BACE-1 inhibitors and the survey ensured the high quality of the modified methodological analysis over conventional docking methods in giving higher enrichment rates20.

In drumhead, structure-based practical showing in combination with bio-assay resulted in designation of multiple fresh non-peptide inhibitors of human BACE-1. This method provided an efficient and high hit-rate attack for inhibitor find against BACE-1. The inhibitors reported herein are largely hydrophobic in nature with moderate molecular sizes ( ~500-600 Da ) . Therefore, they are perchance developed to be penetrants of blood encephalon barrier and able to accomplish the terminal consequence of turn toing the implicit in neuropathology. The most powerful molecule, compound 12 has a benzothiazole ring which docks into the S1 pocket of the enzyme and spans the interaction through about all the sub-sites of BACE-1. The docking airs of compound 12 in the active site of BACE-1 is utile in steering lead optimisation and structure-activity relationships survey in future. Encouraged by current cognition, our attempt in optimising present sub-micromolar hits into more powerful nanomolar leads will be continued based on the molecular hints from this research.


We appreciatively acknowledge fiscal support from pupil Toteboard undertaking funding 11-27801-45-2345, Singapore Polytechnic, 863 plan of China ( 2006AA02Z336 ) and international coaction plan of China ( 20061334 ) .

Mentions and notes

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