Supplementary Information A steered molecular dynamics ...3 quality of the HDAC1 model. Further, a Verify-3D score of 99.18% and an ERRAT score of 84.68% validate the accuracies of

1

Supplementary Information

A steered molecular dynamics mediated hit discovery for histone deacetylases

Subha Kalyaanamoorthy, Yi-Ping Phoebe Chen

Email: [email protected]

Electronic Supplementary Material (ESI) for Physical Chemistry Chemical PhysicsThis journal is © The Owner Societies 2013

mailto:[email protected]

2

Text S1

The recently solved crystal structure of the human HDAC2 protein (PDB ID:

3MAX)(6) is retrieved from the PDB and used in this work. On the other hand, the

experimental structure of HDAC1 is not available and thus the homology based molecular

modeling technique is used for constructing its three-dimensional (3D) structure. Initially, the

protein sequence of the human HDAC1 with 482 amino acid residues (Swissprot id: Q13547)

is downloaded from the SWISS-PROT protein sequence database [www.expasy.ch/sprot/]

and a BLAST search is carried out to identify a potential homolog for building its 3D

structure. The BLAST search performed using BLOSUM62 similarity matrix and an

expectation value of 10 finds that HDAC1 sequence to show 93% of identity against HDAC2

enzyme. Until recently, the HDLP protein and HDAC8 protein structures were used to model

HDAC1 enzyme with their sequence identities of 35.2% with HDLP and 43% with

HDAC8.(17) However, HDAC2, whose experimental structure is currently available, has

93% of identity against HDAC1. Hence, HDAC2 is used as a template to model HDAC1

structure. While modeling HDAC1, the penta-coordinated zinc (Zn2+

) metal ion and the LLX

ligand are extracted from the HDAC2 structure in order to retain those features in the

modeled HDAC1 structure. Further, the modeling is carried out with a non-bonded zinc

method that includes van der Waals and electrostatic terms. This non-bonded method is

identified to be appropriate for the divalent zinc ion (Zn2+

) and also a few other divalent

metal ions such as Ca2+

, Mg2+

and Cd2+

with closed shells.(18) R. H. Stote and M. Karplus

have shown that this model can be used for the simulations involving zinc binding sites in

proteins and solution.(18) A recent molecular dynamics study of HDAC enzymes also

employs the non-bonded zinc method.(2) The superimposed structures of HDAC1 (modeled)

and HDAC2 (experimental template) enzymes displayed an RMSD value < 1 Å.

The quality of the modeled HDAC1 structure is verified using the SAVES structure

analysis program [http://nihserver.mbi.ucla.edu/SAVES/] which integrates five different tools

such as Procheck,(19) What Check,(20) Errat,(21) Verify 3D,(22) Prove(23) to evaluate the

protein structure. The quality of the modeled HDAC1 structure is evaluated using the SAVES

structure verification program. The Ramachandran plot given in fig.4.3 identifies 89.4% of

the amino acid residues in the core region, 10% of the residues in the allowed region and

0.6% within the generously allowed regions. However, none of the residues exist in the

disallowed region of the Ramachandran plot, therefore, confirming the stereo-chemical


http://www.expasy.ch/sprot/

http://nihserver.mbi.ucla.edu/SAVES/

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quality of the HDAC1 model. Further, a Verify-3D score of 99.18% and an ERRAT score of

84.68% validate the accuracies of the structure-sequence compatibility and the non-bonded

interactions respectively in the modeled structure. Similarly, the atomic volumes of the

modeled HDAC1 residues are evaluated for their consistencies with the equivalent residues in

the PDB database for which, a satisfactory Z-score mean value of 0.44 is obtained for the

HDAC1 model.

The 3D structures of the targets HDAC1 (model), HDAC2 (PDB: 3MAX), HDAC3

(PDB: 4A69) and HDAC8 (PDB: 1T67) are prepared by correcting the missing side chain

atoms, if any, assigning the bond orders and adding the polar hydrogen atoms to the

receptors. The prepared structures are energy minimized using OPLS2001 force field(24) to

obtain the stable structures for subsequent molecular dynamics studies. Prime module of the

‘state-of-the-art’ Schrodinger drug discovery package (25, 26) is used in all the modeling and

structure refinement procedures described above.

Missing atoms in the residues of 1T67 structure that were reconstructed using the Prime

module are listed below,

ASP 92, GLU 95, LYS 249, LYS 221, THR 105, ALA 104, GLU 330, LYS 374, THR 69,

GLU 358, LYS 60,LYS 370, LYS 168, GLN 77, ALA 32, GLN 253, HIS 360, LYS 81, LEU

14, GLU 106, GLN 84, VAL 377

Equilibration Procedure

The selected hit-target complexes from vHTS are solvated in a TIP3P water box and electro-

neutralized by adding 0.15 mol L-1

of sodium chloride ions. The solvated systems are energy

minimized and subsequently equilibrated for 1 ns at 300 K temperature and 1 atm pressure,

those are maintained using the Langevin Piston method. The electrostatic interactions in the

complexes are treated using the fast particle mesh Ewald summation method. A cut-off of 12

Å is applied for the van der Waals and short-range electrostatic interactions. The

CHARMM22 force field parameters are used for the enzymes, while the hits are

parameterized using the Swissparm server. Under these conditions, the MD simulations are

carried out with a time-step of 2fs using the NAMD 2.73b package.


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Fitness Score- Pharmacophore based vHTS

A fitness score represents a numerical value to measure the level of alignments between the

pharmacophores of the query (i.e. datasets) and templates, which can be written as,

( ) ( ( )

( )) ( ) ( )

( ) ( ) ( ) ( )

where, ‘site’ represents the pharmacophore position in the dataset and/or template, ‘vec’

denotes the vector features (donors and aromatic rings, for instance), ‘vol’ denotes the

volume features that are computed using van der Waals radius of all non-hydrogen atoms and

‘ivol’ is the overlapping volume regions between the template and the dataset. A fitness score

is useful to match all the pharmacophore features in the templates to screen the datasets.

However, we used a flexible matching criterion to match at least n-2 of the total (n)

pharmacophore features included in the templates. This flexible matching approach is

generally facilitated by implying a penalty to the calculated fitness score, which will give a

new score for matching as,

( ) √ ( ) ( ) ( ) ( )

Here, Weight(old) and Weight(new) are the weightage scores calculated before and after the

matching procedures. Penalty(align) is the penalty score to account the unmatched alignment

of the pharmacophore features.

Table S1. Number of filtered compounds in the docking-based vHTS protocol.

Protocol HDAC1 HDAC2 HDAC3 HDAC8

Glide HTVS 4702 7988 13685 11566

Glide SP 109 261 186 323

Glide XP 79 170 133 173


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Table S2. List of known HDAC actives with their biological activity as reported in the

literature. IC50 values were obtained from www.bindingdb.com


http://www.bindingdb.com/

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Table S3: Actives and Decoys used for SMD force profiling.

Target Active 2D structure

HDAC1

HDAC2

HDAC3

HDAC8

Decoy 2D structure

HDAC1

HDAC2

HDAC3

HDAC8

Decoy 1 Decoy 2 Decoy 3`

Decoy 4 Decoy 5 Decoy 6`

Decoy 12 Decoy 11 Decoy 10

Decoy 9 Decoy 8 Decoy 7


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Table S4: Hits identified from the vHTS for the HDAC1* enzyme.

S.No. Structure Name Docking

score Compound ID

vdW Coul

H1a

N-benzyl-2-(2-fluorophenyl)-5-(hydroxymethyl)triazole-4-carboxamide

-13.608 ZINC21149066 -40.42 -29.11

H1b

5-phenyl-2-C-[4-(pyrrolidine-1-sulfonyl)benzene]thiophene-2,3-diamido

-13.343 LC02206 -52.14 -26.94

H1c

N'-[2-(4-chlorophenoxy)acetyl]quinoline-2-carbohydrazide

-12.230 ZINC15891190 -45.82 -25.32

H1d

2-fluoro-N-(2-hydroxy-5-nitro-phenyl)benzamide

-13.594 ZINC35974033 -41.47 -23.24

H1e

N1-(2-hydroxy-4-methylphenyl)-4-[(2-pyridylthio)methyl]benzamide

-13.416 ZINC01037880 -40.94 -28.70

H1f

N'-[2-(4-fluorophenoxy)acetyl]quinoline-2-carbohydrazide

-12.451 ZINC06208429 -50.16 -23.50

H1g

2-[(1,3-benzodioxol-5-ylmethyl)carbamoyl]cyclohexanecarboxylic acid

-11.732 ZINC00276485 -19.86 -26.34

H1h

5-amino-1-(2-chlorobenzyl)-N-(3-fluoro-4-methylphenyl)-1H-1,2,3-triazole-4-carboxamide

-12.080 ZINC04852462 -44.92 -13.28

H1i

N-(2-thienylsulfonyl)tryptophan

-11.302 ZINC28167340 -32.19

-24.77

H1j

(2S)-3-(4-hydroxyphenyl)-2-[[(2S)-2-hydroxy-3-phenyl-propanoyl]amino]propionate

-11.742 ZINC02561150 -39.23 -21.98

H1k

N'-[(1E)-(2-hydroxyphenyl)methylidene]-5-(naphthalen-1-yl)-1H-pyrazole-3-carbohydrazide

-11.543 LCT02557 -45.30 -22.08

H1l

3-hydroxy-N-{4-[(6-methoxypyridazin-3-yl)sulfamoyl]phenyl}naphthalene-2-carboxamide

-11.861 LCT01039 -45.45 -30.32

* Note that the hits presented in the table are not ordered based on their scores. The vHTS resulted in almost

similar hits for HDAC1 and HDAC2, due to their high level of homology (93%). Therefore, structurally different hits were manually chosen for HDAC1 and HDAC2 and are presented in the table.


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Table S5: Hits identified from the vHTS for the HDAC2† enzyme.


score Compound ID

vdW Coul

H2a

(S)-3-(1H-indol-3-yl)-2-(3-(m-tolyl)ureido) propanoic acid

-15.749 ZINC04029782

-32.94 -28.89

H2b

2-fluoro-N-(2-hydroxy-5-nitro-phenyl)benzamide

-15.084 ZINC35974033

-40.81 -22.17

H2c

2-(2-fluorophenyl)-5-(hydroxymethyl)-N-[(3-methoxyphenyl)methyl]triazole-4-carboxamide

-14.066 ZINC21149087 -42.35 -25.07

H2d

N-(2-hydroxy-5-nitrophenyl)-3-phenylpropanamide

-14.709 ZINC05773304

-42.71 -22.22

H2e

(2R)-4-(4-chloroanilino)-4-oxo-2-(p-tolylmethyl)butanoic

-14.707 ZINC35141851

-36.73 -26.83

H2f

N-(2-hydroxy-5-phenylphenyl)-3-phenylpropanamide

-14.581 ZINC05773306

-49.12 -28.91

H2g

N-(3'-chloro-3-biphenylyl)-1-(3-pyridinylmethyl)-4-piperidinecarboxamide

-13.720 ZINC12203871 -38.63 -19.25

H2h

N1-(2-hydroxy-4-methylphenyl)-4-[(2-pyridylthio)methyl]benzamide

-13.622 ZINC01037880 -40.23 -25.28

H2i

N-(2-fluoro-5-methyl-phenyl)-3-(3-imidazol-1-ylpropanoylamino)benzamide

-13.148 ZINC58226601 -40.28 -26.57

H2j

3-hydroxy-N-{4-[(6-methoxypyridazin-3-yl)sulfamoyl]phenyl}naphthalene-2-carboxamide

-13.125 LCT01039 -44.55 -29.84

H2k

3-amino-N'-(3-methoxy-4-methyl-benzoyl)-5-phenyl-thiophene-2-carbohydrazide

-12.869 ZINC14229725 -48.33 -21.79

H2l

N-(4-methoxyphenyl)-4-oxo-1-(m-tolyl)-1,4-dihydropyridazine-3-carboxamide

-12.414 ZINC05483118 -49.52 -22.09

† Note that the hits presented in the table are not ordered based on their scores. The vHTS resulted in almost

similar hits for HDAC1 and HDAC2, due to their high level of homology (93%). Therefore, structurally different hits were manually chosen for HDAC1 and HDAC2 and are presented in the table.


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Table S6: Hits identified from the vHTS for the HDAC3 enzyme.


score Compound ID

vdW Coul

H3a

2-hydroxy-N'-[(1E)-(2-hydroxy-5-methoxyphenyl)methylidene]benzohydrazide

-10.948 F1355-0048 -13.67 -23.72

H3b

N-(cyclopropylcarbamoyl)-2-[(4,8-dimethyl-2-quinolyl)sulfanyl]acetamide

-9.327 ZINC05581530 -36.84 -19.96

H3c

3-(3-tert-butyl-1,2,4-oxadiazol-5-yl)-N-(4-hydroxy-3-pyridyl)propanamide

-9.518 ZINC40555059 -21.08 -22.19

H3d

(2R)-N-[4-[1-[[(2S)-tetrahydrofuran-2-yl]methylcarbamoyl]cyclohexyl]phenyl]tetrahydrofuran-2-carboxa

-12.119 ZINC40454878 -26.94 -20.31

H3e

N-(cyclopropylcarbamoyl)-2-[6-(3,5-dimethylpyrazol-1-yl)pyridazin-3-yl]sulfanyl-propanamide

-11.495 ZINC08748503 -37.33 -19.96

H3f

N-isopropyl-2-(4-{[4-methyl-5-(piperidin-4-ylmethyl)-4H-1,2,4-triazol-3-yl]methyl}piperazin-1-yl)acetamide

-11.259 ZINC55036339 -25.06 -22.74

H3g

N-(2-hydroxyphenyl)-1-[3-(2-oxo-3H-benzimidazol-1-yl)propyl]piperidine-4-carboxamide

-11.007 ZINC15043891 -18.60 -29.45

H3h

3-[(2S)-5-oxospiro[3,4-dihydro-1,4-benzoxazepine-2,3'-piperidine]-1'-yl]propanamide

-9.279 ZINC19374799 -25.25 -17.25

H3i

2-(2,4-dimethyl-6-oxo-1H-pyrimidin-5-yl)-N-[(6-pyrazol-1-yl-3-pyridyl)methyl]acetamide

-12.115 ZINC42361338 -27.26 -18.35

H3j

4-benzyl-1-[(4-oxo-5-{[(propan-2-yl)carbamoyl]methoxy}-4H-pyran-2-yl)methyl]piperazin-1-ium

-11.440 LCT00721 -26.44 -24.09

H3k

2-[2-(2-benzamido-1,3-thiazol-4-yl)acetamido]benzamide

-11.352 LCT01327 -32.41 -17.73

H3l

ethyl 3-{3-[(3,5-dichloro-2-hydroxybenzylidene)amino]-4-hydroxyphenyl}propanoate

-11.158 ZINC02160662 -40.12 -10.71

H3m

5-(1-{[(3-chlorophenyl)carbamoyl]methyl}-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-3-yl)-N-(propan-2-yl)pentanamide

-11.049 LCT07224 -50.01 -16.54

H3n

2-{3-hydroxy-4-[4-(2-methoxyphenyl)-1H-pyrazol-5-yl]phenoxy}acetohydrazide

-10.962 F3385-1581

-32.52 -17.53


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Table S7: Hits identified from the vHTS for the HDAC8 enzyme.


score Compound ID

vdW Coul

H8a

2-hydroxy-N-[(1-{[5-(1H-pyrazol-3-yl)-2-thienyl]methyl}pyrrolidin-3-yl)methyl]acetamide

-11.599 ZINC67909338 -27.66 -23.85

H8b

(1R,2R,3S)-1-[5-(p-tolylamino)-1,3,4-thiadiazol-2-yl]butane-1,2,3,4-tetrol

-11.836 ZINC15775177 -13.70 -30.19

H8c

2-oxo-3-(3-phenylpropyl)-1H-quinoxaline-6-carbohydroxamic

-10.899 ZINC45245682 -29.64 -11.87

H8d

N-(2,3-dihydroxypropyl)-2-[(quinolin-8-yloxy)methyl]-1,3-oxazole-4-carboxamide

-12.428 ZINC67674713 -32.02 -25.53

H8e

N-(2-oxo-1,3-dihydrobenzoimidazol-5-yl)-2-(1-piperidyl)acetamide

-12.786 ZINC07785247 -18.02 -15.41

H8f

N-(2-hydroxyethyl)-4-{[(4-hydroxyquinazolin-2-yl)sulfanyl]methyl}benzamide

-12.281 ZINC09045156 -36.30 -22.09

H8g

N-cyclopropyl-6-[(3S)-3-[(2-hydroxy-5-methyl-phenyl)methylamino]pyrrolidin-1-yl]pyridine-3-carboxami

-12.126 ZINC49421010 -35.65 -17.69

H8h

N-(3-hydroxypropyl)-1-[1-(1H-indol-2-ylmethyl)-3-piperidinyl]-1H-1,2,3-triazole-4-carboxamide

-11.698 ZINC14983085 -25.96 -27.44

H8i

4-({3-[4-(ethoxycarbonyl)phenoxy]-7-hydroxy-2-methyl-4-oxo-4H-chromen-8-yl}methyl)-1-methylpiperazine-1,4-diium

-11.276 LCT02657 -39.95 -15.58

H8j

ethyl 2-(2-{[5-(1H-indol-3-yl)-4-(3-methoxyphenyl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetamido)acetate

-10.584 LCT00137 -43.87 -16.54

H8k

N-(cyclopropylcarbamoyl)-2-[[5-(1H-indol-3-yl)-4-[[(2S)-tetrahydrofuran-2-yl]methyl]-1,2,4-triazol-3

-10.542 ZINC21462567 -46.12 -17.21

H8l

6-{3-[1-(2,5-difluorophenyl)-1,3,4,9-tetrahydro-2H-beta-carbolin-2-yl]-3-oxopropyl}-4,5-dihydro-3(2H)-pyridazinone

-10.053 ZINC12433123 -33.09 -9.58

H8m

4-acetamido-N-[2-[(3,4-dimethylphenyl)sulfonylamino]ethyl]benzamide

-12.523 ZINC21682204 -37.06 -17.06


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Figure S1: Hits-HDAC1 interactions


H1a- HDAC1 H1b- HDAC1 H1c- HDAC1



12






13

Figure S5: Relationship between IC50 values, unbinding force and time in pico seconds is

represented as a 3D scatter plot. Unbinding forces of the selected actives against HDAC1,2,3

and 8 and inactive MS-275 against HDAC8 are obtained by sampling different

combinations of spring constant (k) and velocity (vel).


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Figure S6: Unbinding force profiles for the active-HDAC complexes and decoy-HDAC8

complex obtained for different spring constants and velocity values.

MS275-HDAC1 LBH- HDAC2

MS275-HDAC3 B3N-HDAC8

MS275-HDAC8


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Figure S7: Relationship between the number of hydrogen bonds (H-bonds) and the

unbinding force profiles of the three selected hits for HDAC2 enzymes, such as H2a (a) ,

H2b (b) and H2c (c), respectively, during the 100 ps of SMD simulation.

(a)

(b)

(c)


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Figure S8: Evolution of the number of H-bonds between the selected hits from vHTS

and the respective HDAC enzymes, such as H1a/H1b/H1c with HDAC1 enzyme (a),

H3a/H3b/H3c with HDAC3 enzymes (b) and H8a/H8b/H8c with HDAC8 enzyme (c).

(a)

(b)

(c)


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Figure S9. Interaction energies for the selected hits against four active site residues

(TYR, PHE and two HIS) of the class I HDACs during the unbinding simulation.


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Documents

Supplementary Information A steered molecular dynamics ...3 quality of the HDAC1 model. Further, a Verify-3D score of 99.18% and an ERRAT score of 84.68% validate the accuracies of