Myricetin allosterically inhibits Dengue NS2B-NS3 protease as studied by NMR and MD simulations

Dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here Myricetin, a plant flavonoid, was characterized to non-competitively inhibit Dengue protease. Further NMR study identified the protease residues perturbed by binding to Myricetin, which were utilized to construct the Myricetin-protease complexes. Strikingly, in the active form Myricetin binds a new allosteric site (AS2) far away from the active site pocket and allosteric site (AS1) for binding Curcumin, while in the inactive form it binds both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by Myricetin, we conducted molecular dynamics (MD) simulations on different forms of Dengue NS2B-NS3 protease. Unexpectedly, the binding of Myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal b- hairpin from the active site pocket. By contrast, the binding of Myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore Myricetin represents the first small molecule which allosterically inhibits Dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in Dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as Myricetin and Curcumin. As Myricetin has been extensively used as a food additive, it might be directly utilized to fight the Dengue infections and as a promising starting for further design of potent allosteric inhibitors.

In Silico Based Approach to Investigate Plant Lignans as inhibitor Candidates for Estrogen Receptor in Breast Cancer

Background: In the last decades, growing evidence demonstrates interest in phytoestrogen intake to modulate targets in different types of cancer. Plant lignans have proven efficacious in blocking estrogen receptors of breast cancer cells. Among them, four phytoestrogen lignans: pinoresinol, matairesinol, lariciresinol, and secoisolariciresinol have been most studied. However, available studies have mostly dealt with anti-cancer effects of groups of lignans in certain foods or plants and the effects of specific lignans, especially from a molecular interaction viewpoint, have been rarely addressed in the literature. Objective: We aimed to in silico predict pharmacological properties, binding ability and binding strength of pinoresinol, matairesinol, lariciresinol and secoisolariciresinol as possible inhibitors of estrogen receptor alpha which is the most important biomarker in breast cancer. Methods: Firstly, we evaluated the pharmacological properties of four lignans using SwissADME. Then we investigated the ligand-receptor interactions of these molecules as positively appraised ligands for ER-positive breast cancer targeted therapy using docking method. We finally compared the inhibitory effect possibility of the lignans against endoxifen which is the active metabolite of tamoxifen. Results: The best binding affinity of endoxifen, matairesinol, pinoresinol, lariciresinol and secoisolariciresinol were respectively -9.2, -7.5, -6.7, -6.7, -5.8 kcal/mol. In the meantime, matairesinol showed the minimum binding energy than other studied lignans in addition to the most similar interactions to endoxifen with conserved domain residues of the active site pocket in Leu:391, Ala:350, Met:421, and Phe:404. Conclusion: Among the studied lignans, matairesinol showed the favorable pharmacokinetics and drug-likeliness properties, the least binding energy as well as the most common interactions in conserved residues of the active site pocket with estrogens. This makes it a molecule with low number of nonspecific interactions, better target selectivity, and hence fewer side effects. Thus, our results introduce matairesinol as a possibly effective anti-estrogen receptor inhibitor candidate.

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose–methanol–choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His–Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da.

Structure-based redesign of the bacterial prolidase active-site pocket for efficient enhancement of methyl-parathion hydrolysis

The recruitment and rapid evolution of promiscuous catalysis provide insights into the precise mechanisms underlying enzyme repurposing and help understand molecular determinants of the remarkable adaptability of proteins. Starting from...

Symmetry-related residues as promising hotspots for the evolution of de novo oligomeric enzymes

Symmetry-related residues located at the rotational axes can be promising hotspots for the evolution of de novo oligomeric enzymes even though they are distantly located from the active site pocket.

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

Abstractβ-glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

Structural Basis for the Selective Inhibition of Cdc2-Like Kinases by CX-4945

Cdc2-like kinases (CLKs) play a crucial role in the alternative splicing of eukaryotic pre-mRNAs through the phosphorylation of serine/arginine-rich proteins (SR proteins). Dysregulation of this processes is linked with various diseases including cancers, neurodegenerative diseases, and many genetic diseases. Thus, CLKs have been regarded to have a potential as a therapeutic target and significant efforts have been exerted to discover an effective inhibitor. In particular, the small molecule CX-4945, originally identified as an inhibitor of casein kinase 2 (CK2), was further revealed to have a strong CLK-inhibitory activity. Four isoforms of CLKs (CLK1, CLK2, CLK3, and CLK4) can be inhibited by CX-4945, with the highest inhibitory effect on CLK2. This study aimed to elucidate the structural basis of the selective inhibitory effect of CX-4945 on different isoforms of CLKs. We determined the crystal structures of CLK1, CLK2, and CLK3 in complex with CX-4945 at resolutions of 2.4 Å, 2.8 Å, and 2.6 Å, respectively. Comparative analysis revealed that CX-4945 was bound in the same active site pocket of the CLKs with similar interacting networks. Intriguingly, the active sites of CLK/CX-4945 complex structures had different sizes and electrostatic surface charge distributions. The active site of CLK1 was somewhat narrow and contained a negatively charged patch. CLK3 had a protruded Lys248 residue in the entrance of the active site pocket. In addition, Ala319, equivalent to Val324 (CLK1) and Val326 (CLK2), contributed to the weak hydrophobic interactions with the benzonaphthyridine ring of CX-4945. In contrast, the charge distribution pattern of CLK2 was the weakest, favoring its interactions with benzonaphthyridine ring. Thus, the relatively strong binding affinities of CX-4945 with CLK2 are consistent with its strong inhibitory effect defined in the previous study. These results may provide insights into structure-based drug discovery processes.