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

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

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

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

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