Selective Mechanism of Inhibitors to Two Bromodomains of BRD4 Revealed by Multiple Replica Molecular Dynamics Simulations and Free Energy Analyses

1.
School of Physics and Electronics, Shandong Normal University, Jinan 250358, China
2.
School of Science, Shandong Jiaotong University, Jinan 250357, China

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Corresponding author: E-mail: liuxinguo@sdnu.edu.cn; jzchen@sdjtu.edu.cn

摘要

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Abstract: Bromodomain-containing protein 4 (BRD4) is critical in cell cycle regulation and has emerged as a potential target for treatment of various cancers. BRD4 contains two bromodomains, namely BD1 and BD2. Research suggests that selectively inhibiting BD1 or BD2 may provide more effective treatment options. Therefore, understanding the selective mechanism of inhibitor binding to BD1 and BD2 is essential for development of high selective inhibitors to BD1 and BD2. Multiple replica molecular dynamics (MRMD) simulations are utilized to investigate the binding selectivity of inhibitors SG3-179, GSK778, and GSK620 for BD1 and BD2. The results show that BD1 has stronger structural flexibility than BD2, moreover BD1 and BD2 exhibit different internal dynamics. The analyses of free energy landscapes reveal significant differences in the conformational distribution of BD1 and BD2. Binding free energy predictions suggest that entropy changes, electrostatic interactions, and van der Waals interactions are key factors in the selective binding of BD1 and BD2 by SG3-179, GSK778, and GSK620. The calculations of the energy contributions of individual residues demonstrate that residues (W81, W374), (P82, P375), (Q85, K378), (V87, V380), (L92, L385), (N93, G386), (L94, L387), (C136, C429), (N140, N433), (K141, P434), (D144, H437) and (I146, V439) corresponding to (BD1, BD2) generate significant energy difference in binding of SG3-179, GSK778, and GSK620 to BD1 and BD2, and they can serve as effective targets for development of high selective inhibitors against BD1 or BD2. The related information may provide significant theoretical guidance for improving the selectivity of inhibitors for BD1 and BD2.
- Bromodomain /
- Binding selectivity /
- Multiple replica molecular dynamics simulation /
- Free energy prediction

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Figure 1. Molecular structures of inhibitors and proteins: (A) the superimposed structures of BD1 (cyan) with BD2 (orange); (B) the superimposed structures of inhibitor in the binding pocket; (C), (D), and (E), the molecular structures of the inhibitors SG3-179, GSK778, and GSK620 with their potencies (IC₅₀) to BD1 and BD2 of BRD4, respectively.

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Figure 2. Root-mean-square fluctuations (RMSFs) of the Cα atoms in BD1 and BD2. (A) SG3-179, GSK778, and GSK620 binding to BD1, (B) SG3-179, GSK778, and GSK620 binding to BD2, (C) and (D) are the ZA and BC loops in BD1 and BD2, respectively.

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Figure 3. Frequency distribution of molecular surface areas of BD1 and BD2. (A) BD1 and (B) BD2.

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Figure 4. Dynamic cross-correlation matrix reflects the relative motion between residues in BD1 and BD2. (A), (C), and (E) SG3-179, GSK778, and GSK620 bound to BD1, respectively; (B), (D), and (F) SG3-179, GSK778, and GSK620 bound to BD2, respectively.

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Figure 5. Collective motions of BD1 and BD2 correspond to the first eigenvector PC1. (A), (C), and (E) respectively correspond to SG3-179, GSK778, and GSK620 complexed with BD1; (B), (D), and (F) respectively correspond to SG3-179, GSK778, and GSK620 complexed with BD2.

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Figure 6. Free energy landscapes of BD1 and BD2 constructed by projecting the MRMD trajectories onto PC1 and PC2: (A) SG3-179 bound to BD1, (B) SG3-179 bound to BD2, (C) GSK778 bound to BD1, (D) GSK778 bound to BD2, (E) GSK620 bound to BD1, and (F) GSK620 bound to BD2.

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Figure 7. Energy contribution of key residues in BD1 and BD2. (A) SG3-179, (B) GSK778, and (C) GSK620.

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Figure 8. Energy decomposition of hot spot residues and interactions network in the SG3-179 bound to BD1/BD2. (A, B) SG3-179 bound to BD1, and (C, D) SG3-179 bound to BD2. Hydrogen bonds (green), CH−π (pink) and π−π (hot pink).

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Figure 10. Energy decomposition of hot spot residues and interactions network in the GSK620 bound to BD1 or BD2. (A, B) GSK620 bound to BD1, and (C, D) GSK620 bound to BD2. Hydrogen bonds (green), CH−π (pink) and π−π (hot pink).

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Figure 9. Energy decomposition of hot spot residues and interactions network in the GSK778 bound to BD1 or BD2. (A, B) GSK778 bound to BD1, and (C, D) GSK778 bound to BD2. Hydrogen bonds (green), CH−π (pink) and π−π (hot pink).

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Table I. Binding free energies of inhibitors to BD1 and BD2 calculated by MM-PBSA method. All values are in kcal/mol.

Components	${\Delta E}_{\rm{ele}}$	${\Delta E}_{\rm{vdW}}$	${\Delta G}_{\rm{pol}}$	${\Delta G}_{\rm{nonpol}}$	${\Delta G}_{\rm{ele+pol}}$^a	${\Delta G}_{\rm{vdW+nonpol}}$^b
SG3-179/BD1	−25.19 ± 0.35	−41.63 ± 0.17	37.90 ± 0.32	−4.21 ± 0.01	12.71 ± 0.15	−45.84 ± 0.12
SG3-179/BD2	−20.16 ± 0.23	−43.50 ± 0.15	33.42 ± 0.22	−4.60 ± 0.01	13.26 ± 0.14	−48.10 ± 0.11
GSK778/BD1	−9.22 ± 0.26	−45.53 ± 0.14	26.77 ± 0.25	−4.48 ± 0.01	17.55 ± 0.14	−50.01 ± 0.10
GSK778/BD2	−8.82 ± 0.19	−44.04 ± 0.15	26.96 ± 0.20	−4.53 ± 0.01	18.14 ± 0.12	−48.57 ± 0.11
GSK620/BD1	−18.53 ± 0.18	−31.35 ± 0.12	30.44 ± 0.14	−3.66 ± 0.01	11.91 ± 0.15	−35.01 ± 0.09
GSK620/BD2	−23.84 ± 0.19	−36.07 ± 0.12	34.10 ± 0.14	−3.95 ± 0.01	10.26 ± 0.15	−40.02 ± 0.09
Components	$ \Delta H $	$ -T\Delta S $	${\Delta G}_{\rm{bind}}$	IC₅₀/(nmol/L)	${\Delta G}_{\rm{exp}}$^c
SG3-179/BD1	−33.13 ± 0.19	21.75 ± 0.64	−11.38	21	−10.57
SG3-179/BD2	−34.84 ± 0.15	24.14 ± 0.73	−10.70	−^d
GSK778/BD1	−32.46 ± 0.17	21.85 ± 0.85	−10.61	40	−10.19
GSK778/BD2	−30.43 ± 0.14	22.73 ± 0.67	−7.70	6300	−7.16
GSK620/BD1	−23.10 ± 0.13	16.75 ± 0.67	−6.35	15800	−6.61
GSK620/BD2	−29.76 ± 0.15	19.85 ± 0.66	−9.91	79	−9.78
^a${\Delta G}_{\rm{ele+pol}}={\Delta E}_{\rm{ele}}+{\Delta G}_{\rm{pol}}$. ^b${\Delta G}_{ {\rm{vdW} }+{\rm{nonpol} } }={\Delta E}_{\rm{vdW} }+{\Delta G}_{\rm{nonpol} }$. ^cThe experimental values are calculated from the equation: ${\Delta G}_{\rm{exp} }=-RT\ln({\rm{IC} }_{50}$). ^dThe IC50 value is not available. available.

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Table II. Hydrogen bonds between inhibitors with BD1 and BD2.

Complexes	Hydrogen bonds^a	Distance/Å	Angle/(^ο)	Occupancy/%
SG3-179/BD1	N140-ND2-HD21···SG3-179-N21	2.96	162.9	99.49
	SG3-179-N23-H19···N140-OD1	3.09	160.8	88.96
	SG3-179-N16-H15···P82-O	3.13	145.3	65.25
SG3-179/BD2	N433- ND2-HD21···SG3-179-N21	2.98	162.1	99.87
	SG3-179-N23-H19···N433-OD1	2.98	162.2	99.13
	SG3-179-N16-H15···P375-O	3.26	147.0	30.34
GSK778/BD1	N140-ND2-HD21···GSK778-O26	3.17	154.2	81.38
	N140-ND2-HD22···GSK778-N25	3.18	142.14	74.68
	W81-NE1-HE1···GSK778-O05	2.99	148.7	36.96
GSK778/BD2	N433- ND2-HD21···GSK778-O26	3.18	149.9	73.64
GSK778/BD2	N433- ND2-HD21···GSK778-N25	3.23	140.6	57.46
GSK620/BD1	N140-ND2-HD21···GSK620-O08	2.93	163.3	99.23
GSK620/BD1	GSK620-N34-H14···N140-OD1	3.01	146.8	91.48
GSK620/BD2	N433- ND2-HD21···GSK620-O08	2.96	164.3	99.30
GSK620/BD2	GSK620-N34-H14···N433-OD1	2.93	153.4	99.25
^a Hydrogen bonding is defined as an acceptor···H-donor angle of > 120° and an acceptor···donor distance < 3.5 Å.

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