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

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 <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

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

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

Interplay between Liquid Crystalline Order and Microphase Segregation on the Self-Assembly of Side-Chain Liquid Crystalline Brush Block Copolymers

Herein we investigate the influence of competing self-organizing phenomena on the hierarchical self-assembly of liquid crystalline brush block copolymers (LCBBCs). A library of LCBBCs are synthesized using ring-opening metathesis polymerization (ROMP) of norbornene side-chain functionalized monomers comprising (1) cholesteryl mesogen with nine methylene spacer and (2) semicrystalline poly­(ethylene glycol) (PEG). The self-assembly of LCBBCs with variations in LC block content (7–80 wt %) are investigated in their melt state. All LCBBCs show two distinct thermal transitions corresponding to PEG semicrystalline phase and LC mesophases. Interestingly, the LCBBCs display a multilevel hierarchical structure evidenced by the results from X-ray scattering and transmission electron microscopy (TEM): (1) smectic A (SmA) mesophases (<i>d</i> = 3–7 nm) by the assembly of cholesteryl side chains and (2) microphase segregation into lamellar or cylinder (<i>d</i> = 40–75 nm) resulting from the incompatibility between LC moieties and PEG side chain. Surprisingly, the presence of microphase-segregated domains in LCBBCs prevents the formation of cholesteric mesophase in sharp contrast to side-chain liquid crystalline homopolymer (SCLCP) bearing the same mesogen and the flexible spacer. This could be attributed to very high surface to volume ratio at intermaterial dividing surface (IMDS) in LCBBCs, by which only LC layers (i.e., SmA mesophase) are favored to form at the IMDS. On the fundamental side, these LCBBCs are an interesting scaffold to explore the impact of interactions between LC order and microphase segregation of side-chain polymeric brushes on the self-assembly of LCBBCs. Moreover, these new LCBBC scaffolds will serve as a tool box for rational design of hierarchically organized functional materials for stimuli responsive applications

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

Hierarchically Self-Assembled Photonic Materials from Liquid Crystalline Random Brush Copolymers

Here we report a general methodology to attain novel hierarchical nanostructures using new polymer scaffolds that self-assemble to form cholesteric 1D photonic mesophases existing in conjunction with microphase segregated domains. To achieve this, a series of liquid-crystalline random brush copolymers (LCRBC) consisting of cholesteryl liquid crystalline (LC) mesogen and brushlike PEG as side chain functionality are synthesized. At room temperature, all LCRBCs exhibits microphase segregation of PEG side chains on length scale of 10–15 nm, whereas LC domain forms smectic mesophase (3–7 nm LC layers). Interestingly, upon heating a cholesteric mesophase is exclusively observed for copolymer containing 78 and 85 wt % of LC content (LCRBC78 and LCRBC85, respectively) existing along with microphase segregated PEG domains. Moreover, the phase behavior of these copolymers studied by temperature-controlled small-angle X-ray scattering (SAXS) suggests the order–disorder transition for the microphase segregated structure coincides with the cholesteric–isotropic transition. Remarkably, LCRBC78 and LCRBC85 quenched from cholesteric mesophase exhibits nanoscale hierarchical order consisting of (1) smectic LC ordering with 3–7 nm periodicity, (2) microphase segregation of PEG side chain on 10–12 nm length scale, and (3) periodicities from helical mesophase (cholesteric phase) on optical length scales of 150–200 nm. Thus, by exploiting LCRBC molecular architecture and composition, hierarchical nanostructure can be obtained and preserved which allows for the creation of unique 1D-photonic materials