Simultaneous targeting of MyD88 and Nur77 as an effective approach for the treatment of inflammatory diseases

Figures & data

Figure 1 Strategy of dual-target inhibitor discovery against MyD88 and Nur77.

Notes: The MyD88 homodimer and Nur77 structure were subjected to virtual screening against the TCM database using the iScreen server. The resulting hits were shortlisted to CP1 and CP2, after an in-depth analysis based on common binding pattern to both the targets. Further, interaction mapping and pharmacophore model development has been done after probing critical receptor–ligand interactions in both the complexes.
Abbreviations: TCM, traditional Chinese medicine; CP1, Compound 1; CP2, Compound 2.

Figure 2 Superimposed structures of the docked MyD88 dimers showing complete overlap of the docked complexes.

Notes: (A) The ClusPro docked complex is depicted in pink ribbons, while the GRAMM-X docked complex is shown in green ribbons. (B) Zoomed interfaces of the complexes showing the position of BB-loop residues in ball and stick.
Abbreviation: GRAMM-X, Global Range Molecular Matching.

Table 1 The top 25 virtual screening hits and their scores for MyD88 from TCM database integrated iScreen program

Rank	Ligand name	Score
1	Rosmarinic_acid_ethyl_ester	−82.1139
2	Cuneataside_C	−80.9749
3	Indole_glycoside	−80.486
4	(–)-Epigallacatechin_3-O-gallate	−80.2361
5	(3S)-3-Acetoxy-1-(3_4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-(6E)-6-heptene	−79.9856
6	Phenethyl_alcohol_xylopyranosyl(1–6)glucopyranoside	−79.7394
7	Kuwanon_E	−79.3575
8	(3S)-1-(3_4-Dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol	−79.1834
9	Ethyl_rosmarinate	−78.8894
10	(3S)-3-Acetoxy-1-(3_4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptanes	−78.7793
11	(4-Hydroxyphenyl)-ethyl-6-O-(E)-caffeoyl-β-d-glucopyranoside	−78.5771
12	Prim-O-β-d-glucosylcimifugin	−78.4834
13	β-Methoxylferruginoside_B	−78.3034
14	10-O-benzoylscandoside_methyl_ester	−77.9669
15	Icariside_D1	−77.9542
16	(3R)-3-Acetoxy-1-(3_4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptanes	−77.7939
17	Kushenol_T	−77.7503
18	(–)-Epicatechin-3-O-gallate	−77.3899
19	Emodin-8-β-d-glucoside	−77.1649
20	(5R)-5-Hydroxy-1-(3_4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-3-heptanone	−77.0339
21	(–)-Epicatechin__gallate	−76.5479
22	(3R)-1-(3_4-Dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol	−76.4823
23	Moralbanone	−75.8924
24	Junipediol_A_2-O-beta_-d-glucopyranoside	−75.83
25	6-O-β-glucosylaucubin	−75.6496

Abbreviation: TCM, traditional Chinese medicine.

Table 2 The top 25 virtual screening hits and their scores for Nur77 from TCM Database integrated iScreen program

Rank	Ligand name	Score
1	Cornuside	−103.704
2	Kukoamine_A	−103.481
3	3_4-Dihydroxyphenethyl_alcohol-6-O-caffeoyl-β-d-glucoside	−101.89
4	Lobetyolin	−101.599
5	(2S)-3-O-(9Z_12Z-octadecadienoyl)glyceryl-β-d-galactopyranoside	−99.9174
6	Emodin-8-β-d-glucoside	−97.7977
7	Vicenin-2	−95.8652
8	4_O-methyl_myricetin_3-O-(6-O-α-l-rhamnopyranosyl)-β-d-glucopyranoside	−95.7478
9	Sanggenol_A	−95.5011
10	11-Hydroxy-sec-O-β-d-glucosylhamaudol	−95.3443
11	3_4_5-Trihydroxyl_3-methoxyl_4-caffeoyl_lignan	−95.2761
12	β-Methoxylferruginoside_B	−95.2541
13	Manninotriose	−95.1534
14	Melittoside	−94.6846
15	trans-Caffeic_acid_stearyl_ester	−94.6059
16	Moralbanone	−94.3901
17	Cordyceamides_B	−93.6693
18	Desrhamnosyl_isoacteoside	−93.6493
19	Clinopodic_acid_E	−93.4
20	Kaempferol-3-O-galactoside	−93.1492
21	Regaloside_C	−93.1124
22	Apigenin_6_8-C-di-β-glucopyranoside	−92.9904
23	Litherospermic_acid_monomethyl_ester	−92.6758
24	Prim-O-β-d-glucosylcimifugin	−92.5737
25	Trifolirhizin	−92.4467

Abbreviation: TCM, traditional Chinese medicine.

Table 3 Four compounds selected on the basis of common binding to both MyD88 and Nur77 targets

Compound	Name	Structure
CP1	β-Methoxylferruginoside_B
CP2	Emodin-8-β-d-glucoside
CP3	Moralbanone
CP4	Prim-O-β-d-glucosylcimifugin

Figure 3 Mode of inhibitor docking in the MyD88 binding site.

Notes: (A) CP1, (B) CP2, (C) CP3, and (D) CP4 are shown in magenta-colored sticks, while the binding site residues are depicted as grey (carbon), blue (nitrogen) and red (oxygen) sticks. Van der Waals interactions are shown as light green dotted lines at some places. For better picture clarity, the hydrogen atoms of both the ligand and the receptor have been omitted. The conventional H-bonds are depicted as green dotted lines, while alkyl and π–alkyl bonds are shown as pink and magenta dotted lines, respectively.
Abbreviations: MyD88, myeloid differentiation primary response protein 88; CP1, Compound 1; CP2, Compound 2; CP3, Compound 3; CP4, Compound 4.

Table 4 Corresponding sequence numbers of residues in Uniprot database as well as in the docked poses for Myd88 and Nur77

S no	Residue name	Residue number
		(Myd88 model)	(Uniprot: Q99836)
1	RDVLPG	49–54	196–201
2	Q	29	176
3	P	14	158
4	Q	26	173
5	H	12	156
6	R	16	160
7	P	22	169
8	E	30	177
9	I	25	172
		(4RZG: PDB)	(Uniprot: P22736)
1	L	106	437
2	S	110	441
3	D	263	594
4	T	264	595
5	D	105	436
6	D	103	434
7	L	187	518
8	G	186	517
9	P	266	597
10	M	262	593
11	H	185	516
12	L	265	596
13	R	184	515
14	A	111	442
15	I	260	591
16	A	102	433
17	K	259	590
18	E	109	440
19	Q	188	519
20	L	265	596

Abbreviation: PDB, Protein Data Bank.

Figure 4 Mode of inhibitor docking in the Nur77 binding site.

Notes: (A) CP1, (B) CP2, (C) CP3, and (D) CP4 are shown in magenta-colored sticks while the binding site residues are depicted as grey (carbon), blue (nitrogen) and red (oxygen) sticks. Van der Waals interactions are shown as light green dotted lines at some places. For better picture clarity, the hydrogen atoms of both the ligand and the receptor have been omitted. The conventional H-bonds are depicted as green dotted lines, while alkyl and π–alkyl bonds are shown as pink and magenta dotted lines respectively.
Abbreviations: CP1, Compound 1; CP2, Compound 2; CP3, Compound 3; CP4, Compound 4.

Figure 5 Two-dimensional representation of inhibitor binding in the MyD88 binding site.

Notes: (A) CP1, (B) CP2, (C) CP3, and (D) CP4 are shown as magenta atom color lines and the binding site residues are depicted as green balls for H-bond interactions and pink for alkyl and π–alkyl interactions and light green balls for C–H/Van der Waals (VdW) interactions. Similarly, the dotted lines are colored correspondingly. It may be noted that many of the VdW interactions have not been shown here for clarity. Also, the program does not automatically depict all possible VdW interactions.
Abbreviations: CP1, Compound 1; CP2, Compound 2; CP3, Compound 3; CP4, Compound 4.

Figure 6 Two-dimensional representation of inhibitor binding in the Nur77 binding site.

Notes: (A) CP1, (B) CP2, (C) CP3, and (D) CP4 are shown as magenta atom color lines and the binding site residues are depicted as green balls for H-bond interactions and pink for alkyl and π–alkyl interactions and light green balls for C–H/Van der Waals (VdW) interactions. Similarly, the dotted lines are colored correspondingly. It may be noted that many of the VdW interactions have not been shown here for clarity. Also, the program does not automatically depict all possible VdW interactions.
Abbreviations: CP1, Compound 1; CP2, Compound 2; CP3, Compound 3; CP4, Compound 4.

Figure 7 Pharmacophore mapping of the docked complexes.

Notes: Overall representation of (A) CP1 and (B) CP4 in the MyD88 binding site and (C) CP1 and (D) CP4 in the Nur77 binding site is shown. The compounds are shown as magenta atom-colored sticks and the binding site residues of MyD88 and Nur77 are shown as green atom-colored sticks. Both CP1 and CP4 have been atom-numbered in white labels for easy reference. Residue numbers are shown as green labels, while the pharmacophore features are labeled with yellow color. Pharmacophore features have been attributed to various substituents. The dark blue arrowed ball and blue meshed spheres refer to H-bond donor groups in the compound, while the red arrowed balls and red meshed spheres refer to H-acceptor features. Similarly, the aromatic ring for π–alkyl interactions is shown as orange arrowed ball and orange meshed spheres are shown with the ring plane depicted in green rectangle. The alkyl hub for C–H/Van der Waals interactions is shown in green ball and cyan meshed sphere.
Abbreviations: CP1, Compound 1; CP4, Compound 4.

Table 5 The similarity between CP1 and CP4 for binding same residues in MyD88 and Nur77 targets, respectively, via a similar set of atoms

S no	MyD88	CP1 (atom number)	CP4 (atom number)	Type of interaction
1	Pro53	13–18	1–6	π–alkyl ring
2	Gln29	52	45	H-donor
3	Gln29	19	15	H-acceptor
4	Asp50	59	54	H-donor
5	Asp50	60	58	H-donor
6	Gln26, Leu52, etc	62, 63, 64	36, 37, 38	Van der Waals/C–H interaction
	Nur77
1	Ser110	13–18	1–6	π–alkyl ring
2	Ser110, Thr264	52	45	H-donor
3	Ser110, Thr264	19	15	H-acceptor
4	Asp263	59	54	H-donor
5	Asp263	60	58	H-donor
6	Ser110, Leu106, etc	62, 63, 64	36, 37, 38	Van der Waals/C–H interaction

Abbreviations: CP1, Compound 1; CP4, Compound 4.

Figure 8 The final pharmacophore model based on MyD88 and Nur77.

Notes: Pharmacophore model (A) with CP1 (magenta) and CP4 (orange) and (B) without CP1 and CP4 in the background. The six major pharmacophore points have been numbered in black labels. The radii of the spatial constraints have been assigned after comparing the spatial features of CP1 and CP4.
Abbreviations: CP1, Compound 1; CP4, Compound 4.

Table 6 Distances between various pharmacophore points in the final pharmacophore model

Pharmacophore points	MyD88		Nur77
	CP1	CP2	CP1	CP2
1 and 3	2.7	2.7	2.7	2.7
1 and 4	10.4	8.3	9.5	8.9
4 and 5	2.9	2.5	2.9	2.5
1 and 6	3.8	5.10	4	5.2
2 and 3	2.7	2.9	2.7	2.7
2 and 4	11.9	11.8	10.6	11.1
4 and 6	8.5	7	8.3	7

Abbreviations: CP1, Compound 1; CP4, Compound 4.