Epigenetic regulation is known to play a key role in progression of anti-cancer therapeutics. Lysine acetylation is an important mechanism in controlling gene expression. There has been increasing interest in bromodomain owing to its ability to modulate transcription of various genes as an epigenetic ‘reader.’ Herein, we report the design, synthesis, and X-ray studies of novel aristoyagonine (benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one) derivatives and investigate their inhibitory effect against Brd4 bromodomain. Five compounds 8ab, 8bc, 8bd, 8be, and 8bf have been discovered with high binding affinity over the Brd4 protein. Co-crystal structures of these five inhibitors with human Brd4 bromodomain demonstrated that it has a key binding mode occupying the hydrophobic pocket, which is known to be the acetylated lysine binding site. These novel Brd4 bromodomain inhibitors demonstrated impressive inhibitory activity and mode of action for the treatment of cancer diseases.

Aberrant tyrosine-protein kinase Mer (MerTK) expression triggers prosurvival signaling and contributes to cell survival, invasive motility, and chemoresistance in many kinds of cancers. In addition, recent reports suggested that MerTK could be a primary target for abnormal platelet aggregation. Consequently, MerTK inhibitors may promote cancer cell death, sensitize cells to chemotherapy, and act as new antiplatelet agents. We screened an inhouse chemical library to discover novel small-molecule MerTK inhibitors, and identified AZD7762, which is known as a checkpoint-kinase (Chk) inhibitor. The inhibition of MerTK by AZD7762 was validated using an in vitro homogeneous time-resolved fluorescence (HTRF) assay and through monitoring the decrease in phosphorylated MerTK in two lung cancer cell lines. We also determined the crystal structure of the MerTK:AZD7762 complex and revealed the binding mode of AZD7762 to MerTK. Structural information from the MerTK:AZD7762 complex and its comparison with other MerTK:inhibitor structures gave us new insights for optimizing the development of inhibitors targeting MerTK.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a ubiquitous signaling lipid that regulates multiple ion channels. In human cyclic nucleotide-gated (CNG) channels, including the rod photoreceptor channel, PIP2 has been reported to exert inhibitory effects, but the underlying mechanism has remained unclear. Because this inhibition lowers the apparent cGMP sensitivity of rod CNG channels, it can play a key role in controlling the light sensitivity and dynamic range of rod photoreceptors. Here we report how PIP2 modulates the function of human CNGA1 channels, the major subunit of human rod photoreceptor CNG channels. Ensemble ion flux assays with liposome-reconstituted purified CNGA1 channels demonstrated robust inhibition by PIP2 via a reduction in apparent cGMP sensitivity, and single-channel recordings revealed PIP2 reduces the channel open probability without altering unitary conductance. To uncover the structural basis, we determined cryo-EM structures of CNGA1 in lipid nanodiscs under multiple ligand conditions. In PIP2-free conditions, closed, intermediate, and open conformations were observed, whereas in the presence of PIP2, the open state was absent. Density consistent with bound PIP2 was detected at inter-protomer grooves between the voltage-sensing and pore domains indicating that PIP2 binding stabilizes non-conductive conformations by sterically preventing C-linker elevation and outward movement of helix S6, conformational changes needed for pore dilation. Collectively, our results establish a structural mechanism for PIP2-mediated inhibition of rod CNG channels, define a mechanistic framework for phosphoinositide control of ligand-gated channels across the CNG superfamily, and provide an inhibitory allosteric binding site for future drug targeting in this channel family.

Human immunodeficiency virus-1 (HIV-1) exploits the viral gp120 protein and host CD4/CCR5 receptors for the pandemic infection to humans. The host co-receptors of not only humans but also several primates and HIV-model mice can interact with the HIV receptor. However, the molecular mechanisms of these interactions remain unclear. Using Shaik et al. (2019)'s gp120/CD4/CCR5 structure of HIV-1B and human, here, we investigate the molecular dynamics between HIV sub-lineages (B, C, N, and O) and potential hosts in Euarchontoglires (primates and rodents). Although both host genes show similar protein structures conserved in all animals, CD4 gene demonstrates significantly stronger binding affinities in Catarrhini (apes and Old-World monkeys). Its known candidate residues interacted with gp120 fail to explain these affinity variations. Therefore, we identified novel candidate sites under positive selection on the Catarrhini lineage. Among four positively selected sites, residue R58 in humans is located within an antigen-antibody binding domain, exhibiting apomorphic amino acid substitutions as Arginine (R) in Catarrhini, which are mutually exclusive to the other animals where Lysine (K) is prevalent. Applying for artificial mutation test, we validated that K to R substitutions can lead stronger binding affinities of Catarrhini. Ecologically, these dynamics may relate to shared equatorial habitats in Africa and Asia. Our findings suggest a new candidate site R58 driven by the lineage-specific evolution as a molecular foundation on HIV infection.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid that regulates multiple ion channels. In rod cyclic nucleotide-gated (CNG) channels, PIP2 was reported to inhibit activity, controlling light sensitivity and dynamic range. Reports of low PIP2 levels in rod outer segment (ROS) membranes questioned the physiological relevance of such inhibition, and the underlying mechanism remained unclear. Here we define the mechanism of PIP2 inhibition of human CNGA1, the principal rod CNG channel subunit. Flux assays and single-channel electrophysiology of purified, liposome-reconstituted CNGA1 channels demonstrate inhibition at PIP2 concentrations estimated for ROS membranes. Cryo-EM structures of PIP2-free channels in lipid nanodiscs capture closed, intermediate, and open conformations, whereas the open state is absent with PIP2. PIP2 binds at the interface between voltage-sensing, pore, and C-linker domains, stabilizing closed states and sterically preventing channel opening. Our findings establish the physiological relevance and structural mechanism of PIP2 inhibition, and provide an inhibitory allosteric site for therapeutic targeting.