Supplementary Information

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Supplementary Information

Effect of N-1 arylation of monastrol on kinesin Eg5 inhibition in Glioma cell lines.

Itamar Luís Gonçalves1¥_{, Liliana Rockenbach}1¥_{, Gustavo Machado das Neves}1_{, Gabriela Göethel}2_, Fabiana Nascimento1_{, Luciano Porto Kagami}1_{, Fabrício Figueiró}3_{, Gabriel Oliveira de Azambuja}1_, Amanda de Fraga Dias3_{, Andressa Amaro}3_{, Lauro Mera de Souza}4_{, Ivan da Rocha Pitta}5_{, Daiana Silva} Avila6_{, Daniel Fábio Kawano}7_{, Solange Cristina Garcia}2_{, Ana Maria Oliveira Battastini}3_{, Vera Lucia} Eifler-Lima*1_.

1

_{Laboratório de Síntese Orgânica Medicinal/LaSOM, Faculdade de Farmácia, Universidade}

Federal do Rio Grande do Sul. Avenida Ipiranga, 2752, Porto Alegre / RS, Brazil.

2

_{Laboratório de Toxicologia –LATOX, Faculdade de Farmácia, Universidade Federal do Rio}

Grande do Sul, Porto Alegre/RS, Brazil.

3

_{Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Porto}

Alegre/RS, Brazil

4

_{Instituto de Pesquisa Pelé Pequeno Príncipe, Faculdades Pequeno Príncipe, Curitiba-PR, Brazil}

5

_UFPE

6

_{Grupo de Pesquisa em Bioquímica e Toxicologia em Caenorhabditis elegans (GBToxCE),}

Universidade Federal do Pampa-UNIPAMPA, BR 592, Km 472, CEP 97500-970 Uruguaiana,

RS, Brazil

7

_{Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas, Campinas-SP,}

Brazil

¥ Author Contributions: These authors contributed equally to this work.

* Corresponding authors:

[email protected]

Electronic Supplementary Material (ESI) for MedChemComm.

This journal is © The Royal Society of Chemistry 2018

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Table S1. Redocking and cross-docking validation procedures. The ligands are represented according

their own PDB ligand code:.. ...S3

Table S2 - Atomic interaction contact list between monastrol and the allosteric binding site of kinesin

eg5...S4

Table S3 - Atomic interaction contact list between compound 19e and the allosteric binding site of

kinesin eg5...S5

Table S4 - Atomic interaction contact list between compound 20e and the allosteric binding site of

kinesin eg5...S6

Table S5 - Atomic interaction contact list between Fluorastrol and the allosteric binding site of

kinesin eg5...S7

Table S6 - Atomic interaction contact list between compound 19h and the allosteric binding site of

kinesin eg5...S8

Table S7 - Atomic interaction contact list between compound 20h and the allosteric binding site of

kinesin eg5...S9

Table S8 - Ligands employed in the cross-docking method with their respective crystallographic

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Table S1. Redocking and cross-docking validation procedures. The ligands are represented according their own PDB ligand code: monastrol (NAT); enastron (KZ9); dimethylenastron (EGB); fluorastrol (X7E); S-Trityl-L-Cysteine analogue (V02); Triphenylbutanamine analogue (DQ6); Sb743921 (6LX); ispinesib (G7X).

Redocking procedure Ligand Goldscore RMSD NAT 57.5355 1.2281 NAT 58.5463 0.363294 NAT 59.9759 0.383424 NAT 60.8637 0.445574 NAT 59.0829 0.542652 NAT 59.6923 0.496567 NAT 52.7531 4.5459 NAT 58.1197 0.590268 NAT 60.0128 0.375302 NAT 59.5296 0.364134 Cross-docking procedure

Ligand Goldscore RMSD Ligand Goldscore RMSD Ligand Goldscore RMSD KZ9 60.5191 0.533382 EGB 60.6326 0.325158 X7E 65.0821 0.952493 KZ9 60.3019 0.465757 EGB 59.9178 0.494828 X7E 62.962 0.983904 KZ9 60.0803 0.476939 EGB 60.0052 0.396757 X7E 58.1434 0.876413 KZ9 60.1796 0.558188 EGB 38.1831 5.66487 X7E 55.2356 0.945662 KZ9 60.5311 0.421172 EGB 49.4489 4.82599 X7E 62.8119 0.952558 KZ9 59.449 0.552352 EGB 55.6293 1.51596 X7E 62.136 1.0316 KZ9 60.4149 0.481185 EGB 58.3139 0.414888 X7E 62.9822 0.922604 KZ9 60.2135 0.454473 EGB 60.4513 0.343691 X7E 62.8465 0.976689 KZ9 59.1877 0.35397 EGB 57.9536 0.619277 X7E 60.2577 0.869159 KZ9 60.2812 0.485319 EGB 55.8278 0.383717 X7E 63.1844 0.912852 V02 73.3863 1.55954 DQ6 62.8583 5.64975 6LX 80.8343 4.24354 V02 72.1936 4.14156 DQ6 69.1778 1.00392 6LX 77.0745 4.68741 V02 66.799 4.10456 DQ6 65.231 0.82211 6LX 77.7239 4.70337 V02 64.7888 1.58429 DQ6 69.8302 0.916334 6LX 57.9574 7.18968 V02 57.7391 5.00004 DQ6 69.8569 0.913953 6LX 80.8536 4.30281 V02 67.2968 4.22044 DQ6 64.5179 6.06321 6LX 81.0215 4.29079 V02 63.7971 1.5211 DQ6 59.1428 5.69687 6LX 81.8666 3.80857 V02 67.3363 4.22237 DQ6 68.3803 1.04275 6LX 80.8115 4.21378 V02 70.8312 4.13633 DQ6 64.844 6.0185 6LX 80.9271 4.02956 V02 70.4996 4.25362 DQ6 67.0231 0.89036 6LX 80.7774 4.23032 G7X 59.2981 6.69215 G7X 63.4052 4.33776 G7X 60.6351 4.22554 G7X 66.0923 4.15514 G7X 61.2297 4.00019 G7X 66.697 4.35491 G7X 60.1856 7.68457 G7X 67.2378 4.3279 G7X 66.9509 4.22816 G7X 64.0892 4.23902

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4 3 2 1 6 5 7 12 11

_N

H

109

NH

8

S

20 18 14

O

19

O

15 16 17

HO

13 Table S2 - Atomic interaction contact list between monastrol and the

allosteric binding site of kinesin eg5.

Atomic Interactions Monastrol (1)

Atom 1 Atom 2 Distance (Å) Type

A:801:NAT:N8 A:116:GLU:O 2.8 Hydrogen Bond A:801:NAT:O13 A:118:GLU:O 2.7 Hydrogen Bond A:801:NAT:C1 A:137:PRO:CB 3.7 Hydrophobic A:801:NAT:C1 A:137:PRO:CG 3.7 Hydrophobic A:801:NAT:C1 A:211:TYR:CE2 3.8 Hydrophobic A:801:NAT:C3 A:118:GLU:C 3.7 Hydrophobic A:801:NAT:C3 A:119:ARG:CD 3.5 Hydrophobic A:801:NAT:C4 A:119:ARG:CD 3.3 Hydrophobic A:801:NAT:C4 A:133:ALA:CA 3.6 Hydrophobic A:801:NAT:C4 A:133:ALA:CB 3.6 Hydrophobic A:801:NAT:C5 A:127:TRP:CB 3.8 Hydrophobic A:801:NAT:C5 A:133:ALA:CB 3.7 Hydrophobic A:801:NAT:C6 A:137:PRO:CB 3.5 Hydrophobic A:801:NAT:C6 A:137:PRO:CG 3.8 Hydrophobic A:801:NAT:C6 A:211:TYR:CD2 3.8 Hydrophobic A:801:NAT:C6 A:211:TYR:CE2 3.7 Hydrophobic A:801:NAT:C9 A:116:GLU:CB 3.9 Hydrophobic A:801:NAT:C16 A:119:ARG:CG 3.7 Hydrophobic A:801:NAT:C18 A:214:LEU:C 3.8 Hydrophobic A:801:NAT:C18 A:218:ALA:CB 3.8 Hydrophobic

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22 21 20 25 24 23 10 11 12

_N

7 8

NH

9

S

13 14 15

O

17

O

16 18 19 6 5 4 3 2 1

N

O

26 27 28

Table S3 - Atomic interaction contact list between compound 19e and the allosteric binding site of kinesin eg5.

Atomic Interactions compound 19e

A:19e:N9 A:GLU116:O 2.0 Hydrogen Bond

A:19e A:GLU116:OE2 4.7 π-anion

A:19e:C1 A:214:LEU:CA 3.5 Hydrophobic A:19e:C1 A:214:LEU:C 3.8 Hydrophobic A:19e:C1 A:214:LEU:CB 3.1 Hydrophobic A:19e:C1 A:214:LEU:CG 3.5 Hydrophobic A:19e:C1 A:214:LEU:CD2 3.1 Hydrophobic A:19e:C2 A:214:LEU:CA 3.4 Hydrophobic A:19e:C2 A:214:LEU:C 3.7 Hydrophobic A:19e:C2 A:214:LEU:CB 3.7 Hydrophobic A:19e:C2 A:214:LEU:CD2 3.4 Hydrophobic A:19e:C3 A:160:LEU:CD2 3.7 Hydrophobic A:19e:C3 A:217:GLY:CA 3.7 Hydrophobic A:19e:C3 A:217:GLY:C 3.6 Hydrophobic A:19e:C4 A:217:GLY:C 3.8 Hydrophobic A:19e:C4 A:218:ALA:CA 3.6 Hydrophobic A:19e:C4 A:218:ALA:CB 3.6 Hydrophobic A:19e:C4 A:221:ARG:CZ 3.5 Hydrophobic A:19e:C5 A:218:ALA:CB 3.7 Hydrophobic A:19e:C8 A:116:GLU:CB 3.6 Hydrophobic A:19e:C10 A:116:GLU:C 3.8 Hydrophobic A:19e:C14 A:218:ALA:CB 3.5 Hydrophobic A:19e:C18 A:118:GLU:CA 3.8 Hydrophobic A:19e:C18 A:119:ARG:CG 3.5 Hydrophobic A:19e:C19 A:118:GLU:CA 3.8 Hydrophobic A:19e:C20 A:137:PRO:CG 3.8 Hydrophobic A:19e:C21 A:117:GLY:C 3.8 Hydrophobic A:19e:C21 A:118:GLU:C 3.6 Hydrophobic A:19e:C21 A:133:ALA:CA 3.8 Hydrophobic A:19e:C22 A:118:GLU:C 3.8 Hydrophobic A:19e:C22 A:119:ARG:CD 3.7 Hydrophobic A:19e:C22 A:133:ALA:CA 3.3 Hydrophobic A:19e:C22 A:133:ALA:CB 3.4 Hydrophobic A:19e:C23 A:133:ALA:CA 3.8 Hydrophobic A:19e:C23 A:133:ALA:CB 3.4 Hydrophobic A:19e:C24 A:137:PRO:CB 3.5 Hydrophobic A:19e:C24 A:137:PRO:CG 3.6 Hydrophobic A:19e:C25 A:137:PRO:CB 3.5 Hydrophobic A:19e:C25 A:137:PRO:CG 3.4 Hydrophobic

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22 21 20 25 24 23 10 11 12

_N

7 8

NH

9

S

13 14 15

O

17

O

16 18 19 6 5 4 3 2 1

N

O

27 28 29

HO

26 Table S4 - Atomic interaction contact list between compound 20e and

the allosteric binding site of kinesin eg5.

Atomic Interactions compound 20e

A:20e:N9 A:116:GLU:O 2.1 Hydrogen bond A:20e:O26 A:118:GLU:O 1.5 Hydrogen bond

A:20e A:116:GLU:OE2 4.7 π-anion

A:20e:C1 A:214:LEU:CA 3.5 Hydrophobic A:20e:C1 A:214:LEU:C 3.9 Hydrophobic A:20e:C1 A:214:LEU:CB 3.2 Hydrophobic A:20e:C1 A:214:LEU:CG 3.6 Hydrophobic A:20e:C1 A:214:LEU:CD2 3.1 Hydrophobic A:20e:C2 A:214:LEU:CA 3.5 Hydrophobic A:20e:C2 A:214:LEU:C 3.8 Hydrophobic A:20e:C2 A:214:LEU:CB 3.7 Hydrophobic A:20e:C2 A:214:LEU:CD2 3.4 Hydrophobic A:20e:C3 A:160:LEU:CD2 3.7 Hydrophobic A:20e:C3 A:217:GLY:CA 3.8 Hydrophobic A:20e:C3 A:217:GLY:C 3.7 Hydrophobic A:20e:C4 A:217:GLY:C 3.8 Hydrophobic A:20e:C4 A:218:ALA:CA 3.5 Hydrophobic A:20e:C4 A:218:ALA:CB 3.6 Hydrophobic A:20e:C4 A:221:ARG:CZ 3.5 Hydrophobic A:20e:C5 A:218:ALA:CB 3.7 Hydrophobic A:20e:C6 A:214:LEU:CB 3.9 Hydrophobic A:20e:C8 A:116:GLU:CB 3.6 Hydrophobic A:20e:C14 A:218:ALA:CB 3.5 Hydrophobic A:20e:C18 A:119:ARG:CG 3.7 Hydrophobic A:20e:C19 A:118:GLU:CA 3.6 Hydrophobic A:20e:C19 A:118:GLU:C 3.8 Hydrophobic A:20e:C19 A:119:ARG:CG 3.5 Hydrophobic A:20e:C20 A:137:PRO:CG 3.9 Hydrophobic A:20e:C21 A:117:GLY:C 3.8 Hydrophobic A:20e:C21 A:118:GLU:C 3.6 Hydrophobic A:20e:C21 A:133:ALA:CA 3.7 Hydrophobic A:20e:C22 A:119:ARG:CD 3.8 Hydrophobic A:20e:C22 A:133:ALA:CA 3.1 Hydrophobic A:20e:C22 A:133:ALA:CB 3.3 Hydrophobic A:20e:C23 A:133:ALA:CA 3.7 Hydrophobic A:20e:C23 A:133:ALA:CB 3.3 Hydrophobic A:20e:C24 A:137:PRO:CB 3.6 Hydrophobic A:20e:C24 A:137:PRO:CG 3.7 Hydrophobic A:20e:C25 A:137:PRO:CB 3.6 Hydrophobic

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CAT CAM CAX CAK CAH CAI CAY CAS CAP NAZ

_N

CAR

NH

NAO

S

SAE CAA

OH

OAC

HO

CAB OAD CAU CAJ CAL CAV CAW

F

CAQ CAN FAF FAG

Table S5 - Atomic interaction contact list between Fluorastrol and the allosteric binding site of kinesin eg5.

Atomic Interactions Fluorastrol

A:1365:X7E:NAO A:117:GLY:O 2.5 Hydrogen Bond A:1365:X7E:OAD A:118:GLU:O 2.6 Hydrogen Bond

A:1365:X7E A:GLU116:OE1 3.7 π-anion A:1365:X7E:CAN A:214:LEU:CB 3.7 Hydrophobic A:1365:X7E:CAV A:221:ARG:CZ 3.8 Hydrophobic A:1365:X7E:CAJ A:116:GLU:CG 3.8 Hydrophobic A:1365:X7E:CAJ A:116:GLU:CD 3.8 Hydrophobic A:1365:X7E:CAU A:116:GLU:CG 3.6 Hydrophobic A:1365:X7E:CAQ A:116:GLU:CG 3.8 Hydrophobic A:1365:X7E:CAA A:214:LEU:C 3.8 Hydrophobic A:1365:X7E:CAA A:214:LEU:CB 3.6 Hydrophobic A:1365:X7E:CAY A:116:GLU:C 3.8 Hydrophobic A:1365:X7E:CAM A:118:GLU:C 3.7 Hydrophobic A:1365:X7E:CAM A:119:ARG:CD 3.7 Hydrophobic A:1365:X7E:CAK A:137:PRO:CB 3.8 Hydrophobic A:1365:X7E:CAK A:137:PRO:CG 3.6 Hydrophobic A:1365:X7E:CAH A:137:PRO:CB 3.4 Hydrophobic A:1365:X7E:CAH A:137:PRO:CG 3.6 Hydrophobic A:1365:X7E:CAH A:211:TYR:CE2 3.7 Hydrophobic A:1365:X7E:CAT A:119:ARG:CD 3.4 Hydrophobic A:1365:X7E:CAT A:133:ALA:CA 3.8 Hydrophobic A:1365:X7E:CAT A:133:ALA:CB 3.8 Hydrophobic A:1365:X7E:FAF A:221:ARG:CZ 2.9 Halogen A:1365:X7E:FAF A:117:GLY:C 3.5 Halogen

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19 20 8 16 17 18 6 1 2

_N

3 4

NH

5

S

7 14 9

O

11

O

10 12 13 15 21 22 23 24 25

O

26 27

Table S6 - Atomic interaction contact list between compound 19h and the allosteric binding site of kinesin eg5.

Atomic Interactions compound 19h

A:19h::N5 A:117:GLY:O 2.6 Hydrogen Bond A:19h::S7 A:119:ARG:N 3.1 Hydrogen Bond A:19h::C6 A:117:GLY:C 3.9 Hydrophobic A:19h::C12 A:116:GLU:CB 3.9 Hydrophobic A:19h::C12 A:116:GLU:CG 3.9 Hydrophobic A:19h::C12 A:136:ILE:CG2 3.8 Hydrophobic A:19h::C12 A:214:LEU:CD2 3.8 Hydrophobic A:19h::C13 A:116:GLU:CG 3.8 Hydrophobic A:19h::C13 A:116:GLU:CD 3.6 Hydrophobic A:19h::C14 A:214:LEU:C 3.7 Hydrophobic A:19h::C14 A:214:LEU:CB 3.5 Hydrophobic A:19h::C16 A:118:GLU:C 3.4 Hydrophobic A:19h::C16 A:119:ARG:CD 3.7 Hydrophobic A:19h::C16 A:133:ALA:CA 3.7 Hydrophobic A:19h::C17 A:119:ARG:CD 3.7 Hydrophobic A:19h::C17 A:133:ALA:CA 3.2 Hydrophobic A:19h::C17 A:133:ALA:CB 3.3 Hydrophobic A:19h::C18 A:119:ARG:CD 3.9 Hydrophobic A:19h::C18 A:133:ALA:CB 3.5 Hydrophobic A:19h::C19 A:127:TRP:CB 3.7 Hydrophobic A:19h::C19 A:211:TYR:CD2 3.9 Hydrophobic A:19h::C19 A:211:TYR:CE2 3.7 Hydrophobic A:19h::C20 A:211:TYR:CE2 3.7 Hydrophobic A:19h::C21 A:218:ALA:CB 3.3 Hydrophobic A:19h::C22 A:218:ALA:CB 3.2 Hydrophobic A:19h::C24 A:215:GLU:CG 3.6 Hydrophobic A:19h::C25 A:211:TYR:CZ 3.8 Hydrophobic

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19 20 8 16 17 18 6 1 2

_N

3 4

NH

5

S

7 14 9

O

11

O

10 12 13 15 21 22 23 24 25

O

26 27

HO

28 Table S7 - Atomic interaction contact list between compound 20h and

the allosteric binding site of kinesin eg5.

Atomic Interactions compound 20h

A:20h::C4 A:119:ARG:CG 3.9 Hydrophobic A:20h::C8 A:119:ARG:CD 3.4 Hydrophobic A:20h::C12 A:116:GLU:CA 3.6 Hydrophobic A:20h::C12 A:116:GLU:C 3.7 Hydrophobic A:20h::C12 A:116:GLU:CB 3.7 Hydrophobic A:20h::C12 A:137:PRO:CG 3.6 Hydrophobic A:20h::C12 A:137:PRO:CD 3.3 Hydrophobic A:20h::C13 A:116:GLU:CB 3.8 Hydrophobic A:20h::C13 A:116:GLU:CG 3.9 Hydrophobic A:20h::C13 A:136:ILE:CG2 3.8 Hydrophobic A:20h::C14 A:214:LEU:C 3.5 Hydrophobic A:20h::C14 A:214:LEU:CB 3.8 Hydrophobic A:20h::C14 A:218:ALA:CB 3.7 Hydrophobic A:20h::C15 A:218:ALA:CB 3.6 Hydrophobic A:20h::C16 A:118:GLU:CA 3.7 Hydrophobic A:20h::C16 A:118:GLU:C 3.2 Hydrophobic A:20h::C16 A:119:ARG:CD 3.4 Hydrophobic A:20h::C17 A:118:GLU:C 3.7 Hydrophobic A:20h::C17 A:119:ARG:CD 3.7 Hydrophobic A:20h::C17 A:133:ALA:CA 3.2 Hydrophobic A:20h::C17 A:133:ALA:CB 3.4 Hydrophobic A:20h::C18 A:133:ALA:CA 3.7 Hydrophobic A:20h::C18 A:133:ALA:CB 3.3 Hydrophobic A:20h::C19 A:127:TRP:CB 3.9 Hydrophobic A:20h::C19 A:211:TYR:CE2 3.8 Hydrophobic A:20h::C20 A:119:ARG:CD 3.8 Hydrophobic A:20h::C20 A:211:TYR:CD2 3.8 Hydrophobic A:20h::C20 A:211:TYR:CE2 3.3 Hydrophobic A:20h::C21 A:218:ALA:CB 3.4 Hydrophobic A:20h::C22 A:218:ALA:CB 3.2 Hydrophobic A:20h::C23 A:218:ALA:CB 3.2 Hydrophobic A:20h::C24 A:215:GLU:CA 3.7 Hydrophobic A:20h::C24 A:215:GLU:CG 3.8 Hydrophobic A:20h::C24 A:218:ALA:CB 3.5 Hydrophobic A:20h::C25 A:215:GLU:CA 3.8 Hydrophobic A:20h::C25 A:215:GLU:CG 3.6 Hydrophobic A:20h::C25 A:218:ALA:CB 3.6 Hydrophobic A:20h::C27 A:218:ALA:C 3.8 Hydrophobic A:20h::C27 A:219:ALA:CA 3.7 Hydrophobic

A:218:ALA:C,O

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Table S8 - Ligands employed in the cross-docking method with their respective crystallographic structure identification code(PDBID)

Structure ID Name PDBID Chemical structure

NAT Monastrol 1X88 N H NH S O O OH KZ9 Enastron 2X7C N H NH S O OH EGB Dimethylenastron 2X7D N H NH S O OH

X7E Fluorastrol 2X7E

N H N S CH3 CH3 HO HO F F V02 S-Trityl-L-Cysteine _analogue 4BBG S OH NH2

DQ6 Triphenylbutanamine _analogue 4A50

CH3

H₂N

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6LX Sb743921 4BXN O O Cl N O NH₂ G7X Ispinesib 4AP0 N N O Cl N O NH₂