ROLE OF COMPUTER AIDED DRUG DESIGNING IN THE IDENTIFICATION OF CYTOKINES INHIBITORS 1 Halim,

1 Zaheer-ul-Haq, Abdul

Sobia Ahsan 1 2 Mesaik, and Maria Kontoyianni

1 Wadood, Omer

Mohamed

1 Abdalla, Mohammed

Ahmed

1Dr. Panjwani

Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan 2Department of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University Edwardsville, Edwardsville, Illinois 62026-2000, USA [email protected], [email protected]

Abstract Interleukin-2 (IL-2) is now emerging as a target in drug discovery for novel therapeutic approaches in several autoimmune disorders. In an attempt to identify new ligands for IL-2, virtual screening (VS) was performed. Four different docking/scoring programs (GOLD, FlexX, Glide and LigandFit) were used to identify novel IL-2 inhibitors. VS of a database of 6, 000 compounds resulted in the identification of three novel and potent active hits. The newly found inhibitors possess moderate potency with IC50 values ranging from 1.8 to 15 µM. These results demonstrate our VS protocol can identify novel chemical scaffolds for IL-2. Optimization of these molecules could lead to better anti-inflammatory therapy.

Introduction

O H3CO

H N O

Interleukin-2 (IL-2) is a 15.5 kDa cytokine that has a predominant role in the growth of activated T-helper 1(TH1) cells. Binding of IL-2 to the trimeric IL-2 receptor (IL-2R) consisting of α (IL-2Rα), β (IL-2Rβ), and γ (IL-2Rγ) causes proliferation and clonal expansion of activated T-cells. Abnormal Th1 immune responses have been implicated in graft rejection, and certain neoplastic diseases. Hence, IL-2 holds considerable promise as a therapeutic target for the treatment of autoimmune disorders. For the development of new immunomodulators, current therapies target IL-2 production or the IL-2 signaling pathway. A small molecule inhibitor of the IL-2/IL-2Rα interaction could offer a significant improvement in immunosuppressive therapy. Computational drug design approaches can be effectively carried out resulting in lower cost and less screening time at onset. Stemming from our interest and efforts in the development of novel and potent immunomodulators, we effectively used VS protocol in order to identify IL-2 inhibitors. Molecular docking was performed using four different docking/scoring approaches: GOLD, FlexX, Glide and LigandFit, along with eleven scoring functions. GOLD was used with GOLD score, ASP and ChemScore, FlexX and Glide were used with their default scoring functions, and LigandFit with six different scoring functions: LigScore1, LigScore2, PLP1, PLP2, Jain and PMF. A set of 6, 000 compounds from our in-house database of drug-like molecules served as the screening library. We feel this is even more exciting given that no published data exist to-date pertaining to the use of computer-guided VS for the identification of IL-2 inhibitors.

N HN

1M48_L

NH2

N NH O N

Cl

Cl

H N

N H

O

NH2 NH

1PW6_L N N O

HO

Assessment of Docking Programs

N Cl

O

Results

O

O

Cl

O

H N

N H

NH2 NH

1PY2_L

Figure 1. Chemical Structures of Reference Compounds

Prior to docking of our in-house compound library, an initial validation of the docking protocol was performed by re-docking of the reference ligands (1M48_L, 1PW6_L and 1PY2_L) into the binding site of their cognate receptors 1M48, 1PW6 and 1PY2 by GOLD, FlexX, Glide and LigandFit. RMSD (Table 1) was calculated between experimental and docked conformations. Poses of all three ligands predicted by each program are in good agreement with those found in the experimentally determined crystal structures. Consequently, all docking algorithms were well-suited for the experiments conducted here. The best docking poses generated by each program compared with the X-ray structures are shown in Figure 2.

High-Throughput Docking The database of 6, 000 compounds was docked in the crystal structure of IL-2 (1M48) by GOLD, FlexX, Glide and LigandFit and a total of eleven scoring functions (GOLD score, ASP, and ChemScore implemented in GOLD, the default FlexX and Glide scoring function, LigScore1, LigScore2, PLP1, PLP2, Jain and PMF implemented in LigandFit) were employed to rank those compounds. A consensus strategy was adopted to improve the screening accuracy. Three co-crystallized reference ligands were included as positive control. Examination of ranks for these three positive controls showed that reference ligands were among the top 5% predicted by all scoring functions. The top 5% of the database, ranked by eleven different scoring functions were selected for further examination. On the basis of the scoring functions of each program and visual inspection of docked poses of top 5% compounds led us to choose 23 compounds for biological testing.

T-Cell Proliferation and Regulation of IL-2 Production from PBMNCs The selected VS hits were tested for their ability to inhibit T-cell proliferation. Figure 3 shows the effect of varying concentrations of the 23 compounds on the mean proliferative response. Among the experimentally tested compounds, at least twelve were thought to be promising in inhibiting human T-cell proliferation, three of which showed inhibitory potency at <0.5 µg/ml; six molecules inhibited proliferation in a dose- dependent manner with IC50 ranging from 0.7± 0.0 to 5.00± 0.0 µg/ml; finally, three compounds exhibited IC50’s ranging from 10.2 ± 0.9 to 15.8 ± 0.0 µg/ml. The results are summarized in Table 2. These twelve active compounds were subsequently assessed for their ability to inhibit IL-2 production from activated PBMNs. For this purpose, human PBMNCs were incubated with varying concentrations (1.0 – 20 µg/mL) of the compounds, 2.5 µg/mL PHA and 20 ng/mL PMA for 18 hours and the supernatants were analyzed by ELISA for IL-2 production from activated PBMNs. The 18 hour time point was chosen based on preliminary results that showed IL-2 up-regulation by both, PHA and PMA, at that time (data not shown). Control cultures were incubated in the absence of PHA and PMA. Treatment of cells with the twelve compounds resulted in a significant and dose-dependent inhibition of IL-2 production by three compounds, specifically; compound 11 was inhibitory at 1.8 ± 0.033 µg/mL, compound 5 at 3.7±0.14 µg/mL and compound 6 at 15.01 ± 0.3 µg/mL ).

Figure 2. Docking results for (a) 1M48 (b) 1PW6 and (c) 1PY2 generated by four different docking/scoring approaches. The experimental conformation (yellow) and docked conformation (cyan) are presented in stick model Table 1. Protein-ligand Complexes used in this study for Assessment of Docking Programs RMSD (Å) S# Protein Ligand IC50 (µM) Resolution (Å)

GOLD FlexX Glide

LigandFit

1

1M48

FRG

3

1.95

0.88

0.79

0.63

0.82

2

1PW6

FRB

6

2.60

0.69

0.82

0.51

0.53

3

1PY2

FRH

0.06

2.80

0.60

0.72

0.61

0.66

Conclusion Figure 3. The Structures of Compounds 1-12

Figure 4. (a). Influence of compounds 1-23 on PHA-induced PBMC Proliferation (b) Effects of compounds 1-12 on IL-2 production.

Table 2:The Inhibitory Activities of Compounds 1-12 T-Cell proliferation

IL-2

IC50 (µg/ml)

IC50 (µg/ml)

1

10.2±0.9

>20

2

15.8±0.0

>20

3

3.66±1.89

>20

4

5.00±0.0

>20

5

0.7±0.0

3.7±0.14

6

< 0.5

15.1 ± 0.3

7

1.8±0.1

>20

8

4.9±0.6

>20

9

2.0±0.0

>20

10

13.5±1.8

>20

11

< 0.5

1.8 ± 0.033

12

< 0.5

>20

Compounds

Using structure-based VS with multiple docking/scoring schemes, novel scaffolds for IL-2 were identified from our in-house compound collection. The binding mode analysis of these hits reveals that the compounds are able to block ligand binding site of IL2. We believe these compounds can function as a starting point for the discovery of promising new immunomodulatory drug candidates.

Figure 4. The binding modes of compounds (a) 5 (b) 6 and (c) 11. The compounds are shown in Yellow, while the interacting residues are represented as orange sticks. The hydrogen bonds are represented as black dotted lines.

References 1. 2. 3.

M. Ahmed Mesaik, Almas Jabeen, Sobia A. Halim, Afshan Begum, A. Shukralla Khalid, Muhammad Asif, Beenish Fatima, Zaheer Ul-Haq and M. Iqbal Choudhary, In Silico and In Vitro Immunomodulatory Studies on Compounds ndelofia stylosa Chemical Biology and Drug Design, 79, 2012, 290–299. M. Ahmed Mesaik, Sobia A. Halim, Zaheer Ul-Haq, M. Iqbal Choudhary, Salma Shahnaz, S. A. M Ayatollahi, Shahnaz Murad and Aqeel Ahmad Immunosuppressive Activity of Buxidin and E-Buxenone from Buxus hyrcana, Chemical Biology and Drug Design, 75, 2010, 310–317. M. Ahmed Mesaik, Zaheer-ul-Haq, Shahnaz Murad, Zakiah Ismail, Noor Rain Abdullah, H. Kauar Gill, Atta-ur-Rahman, Muhammad Yousaf, R. Ali Siddiqui, Aqeel Ahmad, M. Iqbal Choudhary, Biological and molecular docking studies on coagulin-H: Human IL-2 novel natural inhibitor, Molecular Immunology, 43, 2006, 1855-1863.

Acknowledgement Financial sponsor from São Paulo School of Advanced Sciences (ESPCA) to attend the course “Advanced topics in computational Biology Agrochemical and Drug Design” is highly appreciated. We are also grateful to Higher Education Commission (H.E.C) Pakistan for the financial support to conduct this research.