Introduction BACE2 Virtual Screening

Conclusions

Structure of BACE1 vs BACE2

On the Pursuit of New Beta-Secretase 2 Inhibitors Using Structure-

Based De-Novo Design MethodsBoobalan Pachaiyappan, Hongbin Yuan, Pavel A. Petukhov*

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago IL 60612

University of Illinois at Chicago College of Pharmacy UIC* pap4@uic.edu

-Secretase 2 (BACE2), a homolog of -Secretase 1 (BACE1), belongs to the family of aspartic

protease and predominantly is expressed in peripheral tissues. There exists a broad consensus in the

Alzheimers disease (AD) research community that the key to successful treatment of AD lies in the

specific inhibition of BACE1. BACE1 is crucial for release of the amyloidogenic fragments that later form

the extracellular neuritic amyloid plaques one of the factors in the pathogenesis of AD. Most of the

known BACE1 inhibitors are relatively non-selective and also target other aspartic proteases including

the highly homologous BACE2. It has been shown that BACE2 may serve as an alternative -secretase,

another crucial enzyme that plays an important role in degradation of the amyloid precursor protein

(APP), inhibition of which is highly undesirable. To better understand the role of BACE2 in human

physiology and APP processing, we believe that selective inhibitors of this enzyme are needed as

research tools. We hypothesize that it should be possible to design and synthesize inhibitors for BACE2

using an integrated multidisciplinary approach based on the combination of computer-aided drug design,

medicinal chemistry, and biology. Herein we present the results of validation of docking and scoring

procedures and highlight the structural differences of the binding sites of BACE1 and BACE2 useful for

further design of active and selective BACE2 inhibitors.

The homologues share 51% overall primary sequence identity and fold in a nearly similar manner. The

side chains of amino acids forming the binding pockets of BACE1 and BACE2 are not oriented exactly

towards the ligand binding region thereby making the selective inhibition a real challenge.

Figure 1: (Left) Overlay of BACE1 and BACE2 protein structures complexed with ligands. (Right) The superimposed binding

regions of BACE1 and BACE2 complexed with ligands. Only heavy atoms are shown here. Green carbon, red = oxygen,

blue nitrogen, yellow sulfur.

Until now, there are no specific inhibitors for BACE2. The ones that are reported are actually BACE1

inhibitors that display BACE2 activity as well, or bind to other members of aspartic protease family.

Experimental Methods

Protein Structures

BACE1 (2b8l.pdb,1 2irz.pdb,2 2is0.pdb,3 1w51.pdb4 and 2g94.pdb5)

BACE2 (2ewy.pdb6 and homology model developed in our lab)

Simple Docking:

FlexX,7 FRED8 and GOLD9

Scoring: FlexX score, CScore, GOLD score, Chemgauss3

and Chemscore

Virtual screening (VS) experiments using FRED

Conformer generation: Omega2

Scoring functions: PLP, Chemgauss3, Screenscore, Shapegauss, CGO, CGT, OEChemscore and

consensus.

Unless indicated, tripos software (Sybyl7.3)11 was used for modeling and visualization purposes

All root mean square deviation (r.m.s.d) calculations were done using the tool match in Sybyl7.3

Docking Results

Inhibitors

VolSurf Results

BACE-1 inhibitors must pass BBB. Our modeling results suggest none of the inhibitors reported cross

BBB

BACE-2 inhibitors must be cell permeable. Our modeling results suggest that none of the inhibitors

reported permeate CACO2 cell model.

Figure 2: (Top left) BACE1 inhibitors projected on BBB model. Red points refer to high ability to cross BBB, whereas the blue points

have low BBB penetrating ability. Black points are BACE1 inhibitors published in literature. (Top right) BACE2 inhibitors projected on

CACO2 model. Red and blue points refer to high and low permeability. Black points are BACE1 inhibitors published in literature.

We evaluated the docking accuracy of BACE1 (5 crystal structures) and BACE2 (2 structures) using

FlexX, FRED and GOLD. The results are summarized in Table 2.

Figure 3: Comparison of poses from crystal structure

(cyan) and GOLD results for a BACE1 inhibitor (2b8l_lig).

Lowest r.m.s.d was less than 1.5).

Figure 4: Comparison of poses from crystal structure (cyan)

and GOLD results for a BACE2 inhibitor (2ewy_lig). Lowest

r.m.s.d was less than 1.5).

GOLD FlexX FRED

2b8l.pdb 0.34 1.80

0.96*

-

1w51.pdb 1.23 1.82 -

2g94 2.76

0.78*

- 3.68

2irz.pdb 0.60 0.90 2.36

2is0.pdb 0.68 0.82 -

2ewy.pdb 0.41

0.39*

0.46 1.19

Bace2_homology NA NA NA

In order to validate the scoring accuracy, virtual screening was performed on two BACE2 structures

(2EWY.pdb and homology model developed in our lab). A library containing 10,009 non-binders

(obtained from NIH database) and 10 BACE2 binders (see figure) was inputted in omega2 to create

~500 conformers for all ligands. These conformers were rigidly docked to both the BACE2 models

using FRED and analyzed using six scoring functions.

Simple docking

Except in one case, original binding modes of ligands are reproduced best using GOLD. The

decreasing order of efficiency of docking programs was found to be: GOLD > FlexX > FRED.

Inclusion of constraints in GOLD or FlexX resulted in better binding modes

Docking using FRED is dependent upon number of conformers given as input. Because the

number of conformers increases exponentially with the linear increase of number of rotatable

bonds, conformational sampling is difficult and hence FRED gives inconsistent results.

Virtual Screening

BACE2 homology model developed in our lab produced better results than the X-ray crystal

structure itself. This model will be used in future in silico experiments

Scoring accuracy of Shapegauss was found to be better than any other FRED scoring

functions for both BACE2 models

New scaffolds appeared in top 5% during BACE2 VS will be re-docked (using GOLD and FlexX )

and scored to further explore its utility

VS of Chembridge library of ~200,000 drug-like compounds will be performed to further diversify

our pursuit.

VS of Virtual focused combinatorial library will be carried out using the same protocol (alternate

CADD strategy)

Future Directions

Acknowledgments

This research is supported by the National Institute of Health and the National Institute of Aging

grant R21 AG027454 and Hans Vahlteich endowment program grant of College of Pharmacy at

University of Illinois at Chicago.

BACE 2 (2ewy.pdb)

0

200

400

600

800

1000

Ran

k

2ewy_lig 2b8l_lig M-22 M-23 M-24

M-25 M-26 M-27 M2 M3

CGO PLPScreen

score

OEChem

score

Shape

gauss

Chem

gauss3

ConsensusCGO

BACE2_Homology_Model

0

100

200

300

400

500

600

700

800

900

1000

Ran

k

2ew y_lig

2b8l_lig

M-22

M-23

M-24

M-25

M-26

M-27

Merck2

Merck3CGO PLP Screenscore

OEChem

score

Shape

gauss

Chem

gauss3ConsensusCGO

Figure 5: (top) VS results using 2ewy.pdb.

All the ten binders are ranked within top

171 out of 10,019 compounds using

when score using shapegauss. (Bottom)

VS results using BACE2 homology

model developed in our lab. The

weighted r.m.s.d between the

backbones of X-ray structure and our

homology model is 1.8. Major

conformational difference exists only at

the loop regions. Shapegauss performed

best as all the ten binders are ranked

within top 35 out of 10,019 compounds.

The pose of BACE2 inhibitor (2ewy_lig)

was inspected and found to be similar to

the X-ray structure of the ligand.

Table 2: Comparison of r.m.s.d (in ) between X-crystal poses to

the ones generated by docking programs. * represents docking

under constraints.

Shape H-bonds Metal Aromatic Desolvation

Shapegauss Yes No No No No

Chemgauss3 Yes Yes Yes No Yes

OEchemscore Yes Yes Yes No No

PLP Yes Yes Yes No No

Screenscore Yes Yes Yes No No

M-26

Table 1: Energy terms used in structure-based FRED scoring

functions.

Figure 4. Chemical Structures of Inhibitors1,6,12-13

References:

1. Bioorg.Med.Chem.Lett. v16 pp.641, 2006

2. J Med.Chem. v49 pp.7270, 2006

3. J.Med.Chem. v49 pp.7270, 2006

4. J.Mol.Biol. v343 pp.407, 2004

5. J.Am.Chem.Soc. v128 pp.5310, 2006

6. J.Mol.Biol. v355 pp.249, 2006

7. www.biosolveit.de

8. www.eyesopen.com

9. www.ccdc.cam.ac.uk

10. NIH molecular libraries small molecule repository

(http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=375)

11. www.tripos.com

12. J Med Chem. V47 pp6447, 2004

13. J Med Chem. V47, pp6117, 2004

NS

O O

O

NH

HN

OPh

OHHN O

HN

O Ph

HO HN

2b8l_lig 2ewy_lig

NRS

O O

O

NH

HN

OPh

OHHNF

M-22 (R=H),

M-23 (R=CH3)

M-25

M-2

M-24

M-27

NS

O O

O

NH

HN

OPh

OH

NH2F

NS

O O

O

NH

HN

OPh

OH

NHEtF

NS

O O

O

NH

HN

OPh

OHHN

NS

N

O O

O

NH

HN

OPh

OHHN

OS

PhH2C

O O

O

NH

OFHN

O

NH2

5

M-3

NS

O O

O

NH

F

ON

N

NH2Ph