14-3-3 interaction with phosphodiesterase 8A sustains PKA signaling and downregulates the MAPK pathway.

The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.

Structural insight to human Retinoid X receptor alpha-Thyroid hormone receptor beta heterodimer by molecular modelling and MD-simulation studies: role of conserved water molecules.

The Retinoid X receptor alpha-Thyroid hormone receptor beta (RXRα-THRß) heterodimer plays an important role in physiological function of humans specially in the growth and development. Extensive MD-simulation studies on the aquated complexes of modelled RXRα-THRß heterodimer with DNA-duplex have indicated the role of some conserved/semiconserved water molecules in the complexation process in presence or absence of Triiodothyronine (T3) and 9-cis retinoic acid (9CR) in the respective Ligand Binding Domain (LBD) domain. Among the seventeen conserved/semi-conserved water molecules, the W1-W4 water centers have been observed to mediate the interaction between the residues of A-chain (DBD of RXR) to consensus sequence (C-chain) of DNA. The W5-W8 water centers involve in recognition of the residues of B-chain (DBD of THR) to C-chain of DNA. The W9-W13 centers have connected the different residues of B-chain (THR) to D-chain of DNA through H-bonds, whereas W14-W17 water molecules were involved in the interaction of A-chain's (RXR) residues to D-chain of DNA. In our previous study with homodimeric THRß from Rattus norvegicus we have identified fifteen conserved water molecules at the DNA-DBD interface. Moreover, the conformational flexibility of Met313 (in the LBD of THR) from open to close form in presence or absence of T3 molecule in the holo and Apo-protein may provide a plausible rational on the possible role of that residue to acts as gate which could restrict the solvent molecules to enter into the hydrophobic T3-binding pocket of LBD during the absence of ligand molecule and thus could help the stabilization of that domain in THRß structure.Communicated by Ramaswamy H. Sarma.

Molecular modeling and molecular dynamics simulation studies on thyroid hormone receptor from Rattus norvegicus: role of conserved water molecules.

Thyroid hormone receptor (THR) belongs to the nuclear receptor (NR) superfamily that is activated by binding of appropriate ligand molecules (thyroid hormones). These receptors directly bind to specific DNA sequences for gene expression, which is essential for metabolism, homeostasis, and the development of organisms, making it an important drug target. Extensive MD-simulation studies of triiodothyronine (T3) docked modeled rnTHRß1 structures have indicated the presence of twelve conserved water molecules at the DNA-DBD (DNA binding domain) interface. The W1-W5 water centers have been involved in the recognition between the A-chain of DBD to C-chain of DNA, W6 and W7 mediated the interaction between A-chain of DBD and D-chain of DNA, W8 and W9 recognized the B-chain of DBD and C-chain of DNA, and W9-W12 centers conjugated the residues of B-chain of DBD to D-chain of DNA through hydrogen bonds. The conformation flexibility of Phe272 and Met313 residues in the absence of T3 at the LBD (ligand-binding domain) region have been observed and reported.

Structural insight to hydroxychloroquine-3C-like proteinase complexation from SARS-CoV-2: inhibitor modelling study through molecular docking and MD-simulation study.

The spread of novel coronavirus strain, Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) causes Coronavirus disease (COVID-19) has now spread worldwide and effecting the entire human race. The viral genetic material is transcripted and replicated by 3 C-like protease, as a result, it is an important drug target for COVID-19. Hydroxychloroquine (HCQ) report promising results against this drug target so, we perform molecular docking followed by MD-simulation studies of HCQ and modelled some ligand (Mod-I and Mod-II) molecules with SARS-CoV-2-main protease which reveals the structural organization of the active site residues and presence of a conserve water-mediated catalytic triad that helps in the recognition of Mod-I/II ligand molecules. The study may be helpful to gain a detailed structural insight on the presence of water-mediated catalytic triad which could be useful for inhibitor modelling. Communicated by Ramaswamy H. Sarma.

Recognition dynamics of dopamine to human Monoamine oxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity.

The human Monoamine oxidase (hMAO) metabolizes several biogenic amine neurotransmitters and is involved in different neurological disorders. Extensive MD simulation studies of dopamine-docked hMAO B structures have revealed the stabilization of amino-terminal of the substrate by a direct and water-mediated interaction of catalytic tyrosines, Gln206, and Leu171 residues. The catechol ring of the substrate is stabilized by Leu171(C-H)⋯π(Dop)⋯(H-C) Ile199 interaction. Several conserved water molecules are observed to play a role in the recognition of substrate to the enzyme, where W1 and W2 associate in dopamine- FAD interaction, reversible dynamics of W3 and W4 influenced the coupling of Tyr435 to Trp432 and FAD, and W5 and W8 stabilized the catalytic Tyr188/398 residues. The W6, W7, and W8 water centers are involved in the recognition of catalytic residues and FAD with the N+- site of dopamine through hydrogen bonding interaction. The recognition of substrate to gating residues is made through W9, W10, and W11 water centers. Beside the interplay of water molecules, the catalytic aromatic cage has also been stabilized by π⋯water, π⋯C-H, and π⋯π interactions. The topology of conserved water molecular sites along with the hydration dynamics of catalytic residues, FAD, and dopamine has added a new feature on the substrate binding chemistry in hMAO B which may be useful for substrate analog inhibitor design.

Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer.

The Ku protein, a heterodimer of Ku70 and Ku80, binds to chromosomal replication origins maximally at G1-phase and plays an essential role in assembly of origin recognition complex. However, the mechanism regulating such a critical periodic activity of Ku remained unknown. Here, we establish human Ku70 as a novel target of cyclin B1-Cdk1, which phosphorylates it in a Cy-motif dependent manner. Interestingly, cyclin E1- and A2-Cdk2 also phosphorylate Ku70, and as a result, the protein remains in a phosphorylated state during S-M phases of cell cycle. Intriguingly, the phosphorylation of Ku70 by cyclin-Cdks abolishes the interaction of Ku protein with replication origin due to disruption of the dimer. Furthermore, Ku70 is dephosphorylated in G1-phase, when Ku interacts with replication origin maximally. Strikingly, the over-expression of Ku70 with non-phosphorylable Cdk targets enhances the episomal replication of Ors8 origin and induces rereplication in HeLa cells, substantiating a preventive role of Ku phosphorylation in premature and untimely licensing of replication origin. Therefore, periodic phosphorylation of Ku70 by cyclin-Cdks prevents the interaction of Ku with replication origin after initiation events in S-phase and the dephosphorylation at the end of mitosis facilitates its participation in pre-replication complex formation.

Ligand/PTC-free intramolecular Heck reaction: synthesis of pyrroloquinoxalines and their evaluation against PDE4/luciferase/oral cancer cell growth in vitro and zebrafish in vivo.

A series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines has been designed for the potential inhibition of PDE4 without inhibiting luciferase. A ligand/PTC (phase transfer catalyst) free intramolecular Heck cyclization strategy was used to prepare these compounds, some of which showed significant inhibition of PDE4B (IC50≈ 5-14 µM) and growth inhibition of oral cancer cells (CAL 27) but not inhibition of luciferase in vitro. They also showed acceptable safety profiles but no apoptosis in zebrafish embryos.

Novel N-indolylmethyl substituted olanzapine derivatives: their design, synthesis and evaluation as PDE4B inhibitors.

A new strategy for converting antipsychotic drug olanzapine into PDE4 inhibitors is described via the design and Pd/C mediated synthesis of novel N-indolylmethyl olanzapine derivatives. One compound showed good inhibition (IC50 1.1 µM) and >10 fold selectivity towards PDE4B over D that was supported by docking studies. This compound also showed significant inhibition of TNF-α and no major toxicities in cell lines and a zebrafish embryo model except the teratogenic effects to be re-assessed in rodents.

Quinolines: a new hope against inflammation.

Although a number of anti-inflammatory drugs have been discovered and developed to treat diseases associated with acute and chronic inflammation, many anti-inflammatories cause adverse side effects. The quinoline framework has emerged as a new template for the design and identification of novel anti-inflammatory agents. These agents are classified based on the number of substituents present on the quinoline ring or compounds containing a quinoline ring fused to other heterocycles. This review focuses on the discovery of various quinoline derivatives as inhibitors of cyclooxygenase (COX), phosphodiesterase 4 (PDE4) and tumour necrosis factor-α converting enzyme (TACE), along with transient receptor potential vanilloid 1 (TRPV1) antagonists.