A Novel Phenanthridionone Based Scaffold As a Potential Inhibitor of the BRD2 Bromodomain: Crystal Structure of the Complex

<div><p>Bromodomain containing proteins recognize the level of histone acetylation and regulate epigenetically controlled processes like gene transcription and chromatin modification. The BET (<u>b</u>romodomain and <u>e</u>xtra-<u>t</u>erminal) family proteins, which are transcriptional co-regulators, have been implicated in the pathogenesis of cancer, neurodegenerative disorders, and defects in embryonic stem cell differentiation. Inhibitors selectively targeting the BET bromodomains can pave the path for new drug discovery against several forms of major diseases. By a rational structure-based approach, we have identified a new inhibitor (NSC127133) of the second bromodomain (BD2) of the BET family protein BRD2 using the NCI Diversity Set III library. A high-resolution crystal structure of the BRD2-BD2 in complex with this compound and in <i>apo</i>- form is refined to 0.91 and 0.94 Å, respectively. The compound, which is a phenanthridinone derivative, binds well to the acetyl-lysine binding pocket of BD2 and displays significant hydrophobic and hydrophilic interactions. Moreover, the atomic resolution data obtained in this study allowed us to visualize certain structural features of BD2 which remained unobserved so far. We propose that the discovered compound may be a potential molecule to develop a new library for inhibiting the BRD2-BD2 function.</p></div

Insights into the crystal structure of BRD2-BD2 – phenanthridinone complex and theoretical studies on phenanthridinone analogs

<p>Bromodomain and extra-terminal family proteins recognize the acetylated histone code on chromatin and participate in downstream processes like DNA replication, modification, and repair. As part of epigenetic approaches, BRD2 and BRD4 were identified as putative targets, for the management of chronic diseases. We have recently reported the discovery of a new scaffold of the phenanthridinone-based inhibitor (L10) of the second bromodomain of BRD2 (BRD2-BD2). Here, we present the crystal structure of the BRD2-BD2, refined to 1.4 Å resolution, in complex with β-mercaptoethanol (a component of the protein buffer). The β-mercaptoethanol covalently links to C425 of BD2 in the acetyl-lysine binding pocket, to form a modified cysteine mercaptoethanol (CME). The CME modification significantly hinders the entry of ligands into the BD2 binding pocket, suggesting that β-mercaptoethanol should be removed during protein production process. Next, to confirm whether phenanthridionone scaffold is a new inhibitor family of BRD2-BD2, we have determined the crystal structure of BD2 in complex with 6(5H)-Phenanthridinone (a core moiety of L10), refined to 1.28 Å resolution. It confirmed that the phenanthridinone molecule, unambiguously, binds to BD2. Moreover, we performed molecular docking and molecular dynamic studies on selected phenanthridinone analogs. The predicted L10 analogs are stable with essential hydrophobic and hydrophilic interactions with BD2 during molecular dynamic simulations. We propose that the predicted phenanthridinone analogs may be potential molecules for inhibiting the BD2 function of acetylated histone recognition.</p

Binding analysis of L10 towards BRD2-BD2.

<p>SPR sensorgrams obtained by running different concentrations of L10 over immobilized BRD2-BD2. The figure in the inset shows fitting of data points at equilibrium (Req) for the interaction of different concentrations of L10 to immobilized BD2.</p

Data reduction and refinement statistics of the <i>apo-</i>form of BRD2-BD2 and <i>BD2-L10</i> structures.

<p>Data reduction and refinement statistics of the <i>apo-</i>form of BRD2-BD2 and <i>BD2-L10</i> structures.</p

Alternate positions of conserved water molecules.

<p><b>(A)</b> Among five conserved water molecules in the <i>apo-</i>form binding pocket, three are observed to have alternate positions. (<b>B)</b> Three out of four conserved water molecules demonstrate alternate positions in the <i>BD2-L10</i> complex. The ligand and selected residues are shown as sticks. Water molecules are shown as spheres. <b>|</b>2Fo|-|Fc| map of water molecules contoured at 1.0σ. The hydrogen bonds are shown as broken lines.</p

Superposition of <i>BD2-H4K5ac</i> (PDB Id: 2E3K) and <i>BD2-L10</i> complex.

<p>The H4K5ac and L10 are represented in grey and yellow, respectively. The binding site residues corresponding to <i>BD2-L10</i> (purple), the ligand L10 (yellow), H433 of <i>BD2-H4K5ac</i> (grey) and the <i>H4K5ac</i> side-chain (grey) are shown as sticks. H433 is swung in towards the binding pocket in the <i>BD2-L10</i> complex.</p

Comparison of water bridged interactions formed in the JQ1 and L10 complexes at the edge of the binding pocket.

<p>Two water molecules (W1 and W2) are demonstrating water bridged hydrogen bonds (red) in the <i>BD2-L10</i> complex. A single water mediated hydrogen bond is observed in the case of JQ1 (grey). Compound L10 is shown in yellow. The residues corresponding to <i>BD2-L10</i> and <i>BD2-JQ1</i> are shown in purple and grey, respectively. The ligands and the interacting residues are shown as sticks. Water molecules are shown as spheres. The hydrogen bonds are shown as broken lines.</p

Superposition of BC and ZA loop regions of <i>BD2-H4K5ac</i> complex (PDB Id: 2E3K) and <i>apo-</i>form.

<p>Binding site residues (purple) in the <i>apo-</i>form demonstrating conformations different to that observed in the <i>BD2-H4K5ac</i> complex structure (grey). The side chain of C425 and M438 are flipped in the <i>apo-</i>form compared to the <i>H4K5ac</i> structure. The residues near the binding site region, which possess alternate conformations, are shown as sticks. |2Fo|-|Fc| map of the residues contoured at 1.0σ. The hydrogen bonds are shown as broken lines.</p

Detailed analysis of the four binding sites of PB on SOD1.

The polder density map and the intermolecular interactions of PB1 (shown as orange sticks) on the dimer interface of chains C (shown as a pink cartoon) and D (shown as a blue cartoon) are shown in (a); right and left panels, respectively. The polder map for the other poses on the lateral region of chains B (shown as a golden yellow cartoon), C, and I (shown as a wheat brown cartoon) are shown in (b) and (c) (left panels). The intermolecular interactions of PB2 (purple sticks), PB3 (pink sticks), and PB4 (green sticks) are shown in (b) and (c) (right panels). The Polder maps are contoured at the 3σ level for PB1 and PB2, 3.3σ level for PB3, and 3.5σ level for PB4. The maps are shown as green mesh. The binding site residues are shown as yellow sticks.</p

MST interaction analysis of fluorescently labeled SOD1 mutants against PB.

The binding of PB to fluorescently Cys-labeled SOD1- variants is quantified in PBS buffer. The concentration of PB varied from 30.5 nM to 250 μM, while the respective mutants were kept constant at 25 nM. MST experiments were conducted at 20% LED excitation and medium MST power at 25 °C. The resulting dose-response curves were fitted and yielded the binding affinity of (a) 15.95 ± 2.65 μM for SOD1G10P, (b) 225.44 ± 4.29 μM for SOD1L42R, (c) weak-binding for SOD1A123F, and (d) no binding for SOD1K9F. Error bars indicate the standard error of the mean, SEM (n = 3).</p