Supporting Information

Identification of Novel Interleukin-2 Inhibitors through Computational Approaches

Sobia Ahsan Halim,a,b Omer Mohamed Abdalla,a,eM. Ahmed Mesaik,aAbdul Wadood,a. cZaheer

Ul-Haq,*a,d and Maria Kontoyiannib

a. Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan.

b. Department of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University Edwardsville, Edwardsville, Illinois 62026-2000.

c. Department of Chemistry, University College of Science, Shankar Campus, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.

d. Heidelberg Institute for Theoretical Studies (HITS) gGmbH, Schloss-Wolfsbrunnenweg 35, 69118 Heidelberg, Germany.

e. Institute of Radiobiology, Ministry of Science and Technology, Khartoum-11111, Sudan, P.O.Box 3001.

*Corresponding Authors:

Email: zaheer.qasmi@iccs.edu; (Z. Ul-Haq); mkontoy@siue.edu; (M. Kontoyianni).

Supporting Information Content

Figure S1. Structures of Known Inhibitors 1M48_L (1), 1PW6_L (2) and 1PY2_L (3) used in this study for the Assessment of Docking Programs.Figure S2. Superimposed view of four IL-2 X-ray structures: 1M48 (cyan), 1M49 (green), 1PW6 (yellow), and 1PY2 (pink). The inhibitors possess similar binding patterns. Figure S3. Docking results for (a) 1M48,(b) 1PW6, and (c) 1PY2 generated by four different docking/scoring schemes. The experimental (yellow) and docked (cyan) conformationsare presented as stick models.Figure S4.Cross Docking results for (a) 1M48,(b) 1PW6, and (c) 1PY2 generated by GOLD. It can be seen that all three ligands are accommodated in the ligand binding pocketof 1M48.Table S1: Root mean square deviations of 1M49, 1PW6, and 1PY2 with respect to 1M48. Table S2. Protein-ligand complexes used in this study for assessment of docking programsTable S3: Cross docking results of 1M48, 1PW6, and 1PY2 predicted by GOLD.Table S4: Inhibitory activities of selected VS hits (1-23) Table S5: Docking scores of Compounds 1-23, obtained by GOLD, FlexX, LigandFit and Glide. GOLD was used with Gold Score, ChemScore, and ASP. FlexX and Glide were used with FlexX and Glide SP scores, respectively.LigandFit was combinedwith LigScore 1, LigScore 2, PLP1, PLP2, Jain, and PMF.

HNH3CO

O

ON

HN NH2

NHN

N NHO

ClCl

O HN NH2

NH

N N

ClCl

O N NH

O

O HN NH2

NHOHO

O

1

2

3

Figure S1. Structures of known inhibitors 1M48_L (1), 1PW6_L (2) and 1PY2_L (3) used in this study for the

assessment of docking programs

Figure S2. Superimposed view of four IL-2 X-ray structures: 1M48 (cyan), 1M49 (green), 1PW6 (yellow), and

1PY2 (pink). The inhibitors possess similar binding patterns.

Table S1: Root mean square deviations of 1M49, 1PW6, and 1PY2 with respect to 1M48.

RMSD (Å)Reference Protein: 1M48

1M49 0.5521PW6 0.7641PY2 1.040

Figure S3. Docking results for (a) 1M48,(b) 1PW6, and (c) 1PY2 generated by four different docking/scoring

schemes. The experimental (yellow) and docked (cyan) conformationsare presented as stick models.

Table S2. Protein-ligand complexes used in this study for assessment of docking programs

S#

Protein

Ligand Ligand name IC50 (µM) Resolution

(Å)References

RMSD (Å)GOLD

FlexX

Glide LigandFit

1 1M48 FRG

2-[3-methyl-4-(N-methyl-guanidino)-butyrylamino]-3-(4-phenylethynyl-phenyl)-propionic acid methyl ester

3 1.95 [1] 0.88 0.79 0.63 0.82

2 1PW6 FRB

2-cyclohexyl-N-(2-{4-[5-(2,3-dichloro-phenyl)-2H-pyrazol-3-yl]-piperidin-1-yl}-2-oxo-ethyl)-2-guanidino-acetamide

6 2.60 [2] 0.69 0.82 0.51 0.53

3 1PY2 FRH

5-[2,3-dichloro-4-(5-{1-[2-(2-guanidino-4-methylpentanoylamino)-acetyl]-piperidin-4-yl}-1-methyl-1H-pyrazol-3-yl)-phenoxy methyl]-furan-2-carboxylic acid

0.06 2.80 [2] 0.60 0.72 0.61 0.66

Table S3: Cross docking results of 1M48, 1PW6, and 1PY2 predicted by GOLD.

RANK

1M48 1PW6 1PY2

1M48 1 3 3

1PW6 3 1 2

1PY2 2 2 1

(a)

(b)

(c)

Figure S4. Cross Docking results for (a) 1M48,(b) 1PW6, and (c) 1PY2 generated by GOLD. It can be seen

that all three ligands are accommodated in the ligand binding pocketof 1M48.

Table S4: Inhibitory activities of selected VS hits (1-23)

Compounds # Structures

T-Cell proliferation IC50

(µM)

IL-2 IC50

(µM)IL-2Rα IC50

(µM) References

1

N

HN NH

CH3O

35.8 ± 3.2 > 72 NT [3]

2 O

HO

OH

O

36 ± 0.3 > 42.5 NT [4]

3N

O

N

O

N S NO

O

8.6 ± 4.4 > 46 NT [5]

4N

O

O

N NO 26.4 ± 10 > 66.8 NT [5]

5N

O

O

N NO N

O

O

2.1 ± 0.1 11.3 ± 0.4 58.2 ± 2.5 [5]

6

NH

O

N NHNH

SN

O

O

< 1.5 140 ± 3.8 140 ± 3.8 [6]

7

S

NNN

HN

O

N

O O

S

Cl

3.2 ± 0.18 > 36 NT [7]

8S N

NNHN

ON

OO

NO

O 10.7 ± 1.3 > 43.8 NT [7]

9S N

NN

HN

O

N

OO

NN 4.2 ± 0.06 > 42 NT [7]

10S N

NN

HN

O

N

OO

N

32.7 ± 4.4 > 48.5 NT [7]

11N

O

N

< 1.8 6.56 ± 0.1 5.8 ± 0.18 [8]

12N Cl

OS

Cl

< 1.4 > 57.4 NT [9]

13 O

O

HO O

O

O

O

O

OH > 76.6 NT NT [10]

14 NHNNH

O

OCH3

H3CO

H3CO

> 116 NT NT [3]

15 O

O

HOO

> 107 NT NT [4]

16 N

N

OO

NO

> 178 NT NT [5]

17

NH

HN

NNN

NH2

HS

S OO

> 121 NT NT [6]

18S N

NN

HN

O

N

OO

Cl > 112 NT NT [7]

19S N

NN

HN

O

N

OO

N > 121 NT NT [7]

20S N

NN

HN

O

N

OO

OCH3

OCH3

> 106 NT NT [7]

21S N

NN

HN

O

N

OO

NH

O > 107 NT NT [7]

22

N N

S NO

HN

OO

N

NCl > 98 NT NT [7]

23 NO

O

H3CO

> 201 NT NT [11]

NT= not tested. The isolation and the synthesis of all the compounds has been characterized earlier [3-11].

Table S5: Docking scores of Compounds 1-23, obtained by GOLD, FlexX, LigandFit, and Glide. GOLD was used with Gold Score, ChemScore and ASP. FlexX and Glide were used with FlexX and Glide SP scores, respectively.LigandFit was combined with LigScores 1 and2, PLP1, PLP2, Jain, and PMF.

S#GOLD

FlexXLigandFit

GlideGoldScore ChemScore ASP LigScore1 LigScore2 PLP1 PLP2 Jain PMF

1 57.91 61.17 72.38 -21.50 6.55 4.06 56.45 54.66 6.63 52.87 -10.132 58.35 59.06 68.56 -22.70 7.46 5.65 53.87 45.51 5.23 59.89 -12.763 55.29 68.97 66.75 -21.50 7.64 5.86 69.77 67.08 5.02 50.92 -12.364 52.04 66.76 66.39 -28.00 7.38 5.16 63.74 57.02 5.56 59.97 -13.455 73.24 80.29 88.56 -22.70 8.56 8.65 64.38 59.76 8.24 73.52 -13.616 71.38 77.86 68.98 -29.50 9.29 8.49 79.07 64.23 8.16 64.58 -12.427 62.59 60.86 72.45 -20.90 7.64 5.74 69.47 78.53 5.21 61.31 -12.58 54.67 61.56 58.88 -26.70 8.24 7.35 51.42 46.97 6.17 74.08 -12.379 65.49 63.45 57.60 -23.30 7.83 5.37 50.48 49.78 5.71 64.47 -12.6810 56.35 60.89 57.89 -20.10 6.39 6.69 77.69 67.83 6.94 68.77 -14.0911 76.82 86.73 84.32 -29.50 9.81 9.48 71.21 71.48 9.87 84.43 -14.8912 66.45 56.76 65.45 -25.44 6.52 6.71 67.48 65.4 5.71 54.21 -11.8413 64.23 54.41 60.76 21.26 6.94 6.41 55.23 36.01 6.71 56.29 -10.0514 61.76 55.54 60.46 -23.50 7.64 7.06 57.66 48.86 6.35 56.44 -11.6615 60.36 56.68 58.93 -21.20 7.81 6.29 55.34 34.99 5.35 65.11 -12.2916 59.28 67.71 70.01 -23.90 6.64 6.23 55.68 55.78 4.84 64.22 -10.1417 60.56 60.98 66.38 -23.10 7.39 6.07 53.05 44.31 4.37 65.98 -10.3118 67.38 63.65 62.98 -21.60 7.66 3.35 50.37 57.16 4.81 56.69 -10.3719 65.65 63.45 60.04 -20.00 6.46 6.63 53.53 43.7 4.35 60.57 -11.1320 68.76 63.77 72.11 -22.60 6.59 6.34 54.97 52.31 4.65 57.03 -12.8721 61.10 61.89 65.65 -20.70 6.59 5.65 52.75 55.16 4.48 50.53 -112622 65.09 60.97 68.89 -23.10 7.16 5.39 48.5 50.98 4.83 54.65 -11.3423 62.13 60.58 60.95 -20.22 5.04 5.4 42.57 52.25 4.38 51.06 -11.94

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