Identification of ortho-hydroxy anilide as a novel scaffold for lysine demethylase 5 inhibitors

Fe(II)/α-ketoglutarate-dependent lysine demethylases (KDMs) are attractive drug targets for several diseases including cancer. In this study, we designed and screened ortho-substituted anilides that are expected to function as Fe(II) chelators, and identified ortho-hydroxy anilide as a novel scaffold for KDM5A inhibitors. Treatment of human lung cancer A549 cells with a prodrug form of 4-carboxy-2-hydroxy-formanilide (9c) increased trimethylated lysine 4 on histone H3 level, suggesting KDM5 inhibition in the cells.     

Downregulation of histone deacetylase 1 by microRNA-520h contributes to the chemotherapeutic effect of doxorubicin

Doxorubicin induces DNA damage to exert its anti-cancer function. Histone deacetylase 1 (HDAC1) can protect the genome from DNA damage. We found that doxorubicin specifically downregulates HDAC1 protein expression and identified HDAC1 as a target of miR-520h, which was upregulated by doxorubicin. Doxorubicin-induced cell death was impaired by exogenous HDAC1 or by miR-520h inhibitor. Moreover, HDAC1 reduced the level of γH2AX by preventing the interaction of doxorubicin with DNA. In summary, doxorubicin downregulates HDAC1 protein expression, by inducing the expression of HDAC1-targeting miR-520h, to exacerbate DNA–doxorubicin interaction. The upregulation of HDAC1 protein may contribute to drug resistance of human cancer cells and targeting HDAC1 is a promising strategy to increase the clinical efficacy of DNA damage-inducing chemotherapeutic drugs.     

Design,synthesis and anticancer evaluation of acridine hydroxamic acid derivatives as dual Topo and HDAC inhibitors

Multitarget inhibitors design has generated great interest in cancer treatment. Based on the synergistic effects of topoisomerase and histone deacetylase inhibitors, we designed and synthesized a new series of acridine hydroxamic acid derivatives as potential novel dual Topo and HDAC inhibitors. MTT assays indicated that all the hybrid compounds displayed good antiproliferative activities with IC₅₀ values in low micromolar range, among which compound 8c displayed potent activity against U937 (IC₅₀?=?0.90?μM). In addition, compound 8c also displayed the best HDAC inhibitory activity, which was several times more potent than HDAC inhibitor SAHA. Subsequent studies indicated that all the compounds displayed Topo II inhibition activity at 50?μM. Moreover, compound 8c could interact with DNA and induce U937 apoptosis. This study provides a suite of compounds for further exploration of dual Topo and HDAC inhibitors, and compound 8c can be a new dual Topo and HDAC inhibitory anticancer agent.     

Structural insights into the Plasmodium falciparum histone deacetylase 1 (PfHDAC-1): A novel target for the development of antimalarial therapy

The histone deacetylase (HDAC) enzyme from Plasmodium falciparum has been identified as a novel target for the development of antimalarial therapy. A ligand-refined homology model of PfHDAC-1 was generated from the crystal structures of human HDAC8 and HDLP using a restraint guided optimization procedure involving the OPLS/GBSA potential setup. The model was extensively validated using protein structure checking tools. A predictive docking study was carried out using a set of known human HDAC inhibitors, which were shown to have in vitro antimalarial activity against the chloroquine sensitive D6 and resistant W2 strains of P. falciparum. Pose validation and score-based active/inactive separation studies provided independent validation of the geometric accuracy and the predictive ability of the generated model. Comparative analysis was carried out with the human HDACs to identify differences in the binding site topology and interacting residues, which might be utilized to develop selective PfHDAC-1 inhibitors.     

Molecular simulation studies on the binding activity and selectivity of 3-amino-phenyl-5-chloro-pyrimidine-2, 4-diamine derivatives in complexes with kinases c-Met and ALK

Receptor tyrosine kinases c-Met and ALK have been demonstrated to be important therapeutic targets for cancer therapy. However, selectivity and drug resistance could hinder the development of their corresponding inhibitors. In this study, three compounds with similar scaffold were examined to study activity and selectivity mechanism towards c-Met or ALK by utilising a combined approach of computational techniques, including flexible dock, molecular electrostatic potential (MESP) calculations, molecular dynamic (MD) simulation, and binding free-energy calculation. Molecular simulation provides us new chemical insights into steric and electronic complementarities of these inhibitors to target binding sites. The computed binding free energies were consistent with the changing trend of experimental affinities on c-Met and ALK. H-bond with Asp169 and hydrophobic interaction with Phe36 of c-Met, respectively, could be crucial for the binding affinity of an inhibitor binding to c-Met. Meanwhile, for inhibitor–ALK complex, both H-bond interactions with Arg28 and Met101 and hydrophobic interactions with Leu30, Val38, and Leu158 could enhance the bioactivity and selectivity. The present work may provide a structural understanding of molecular mechanism expected to be valuable for the guidelines of the development of new potent c-Met or ALK selective inhibitors.     

Control of ColE1 plasmid replication. Intermediates in the binding of RNA I and RNA II.

Replication of plasmid ColE1 is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to the precursor (RNA II) of the primer for DNA synthesis and inhibits primer formation. The process of binding of RNA I to RNA II that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability. Formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second RNA I species was firmly established by more extensive studies. This complex is converted to a more stable yet reversible complex that was identified by its RNase sensitivity, which was altered from that of the earlier complex or from that of free RNA I or RNA II. In these complexes, most loops of RNA II interact with their complementary loops of RNA I. The kinetic and structural analyses of the binding process predict formation of a complex interacting at a single pair of complementary loops that precedes formation of these complexes. Thus the process of binding of RNA I to RNA II is seen to consist of a sequence of reactions producing a series of progressively more stable intermediates leading to the final product.     

Structure and activity of membrane receptors: Modeling and computational simulation of ligand recognition in a three-dimensional model of the 5-hydroxytryptamine1A receptor

A three-dimensional molecular model of the transmembrane domain of the 5-HT_1A receptor (5-HT_1AR) is presented in the context of a general strategy for modeling the macromolecular structure of a guanine nucleotide binding, regulatory protein coupled receptor (GPCR). The model of the 5-HT_1AR rests on the definition of the putative residues of the ligand-binding site guided by criteria based on specific models proposed from structure-activity studies and on published results of modifications of GPCRs using methods of molecular biology. The resulting requirements for matching recognition sites in the agonist-binding pocket define the molecular details of the interaction between the agonist 5-HT and the human 5-HT_1AR that includes: (1) the interaction between the protonated amine moiety and the conserved negative Asp-116, located in TMH 3; (2) the hydrogen bond between the hydroxyl group and Thr-199, located in TMH 5; and (3) the interaction complex between the aromatic ring portion of the ligand and the neutral form of His-192, located in TMH 5. Results from quantum mechanical calculations of the interaction between an agonist and the proposed recognition pocket of the 5-HT_1AR model suggest a trigger of the receptor activation mechanism resulting from ligand binding. The antagonist-binding pocket of the human 5-HT_1AR is inferred from the interaction sites of pindolol with the receptor model: (1) the ionic interaction between the protonated amine of the ligand and the side chain of the conserved Asp-116, located in TMH 3; and (2) the hydrogen bonds between the ether oxygen and the hydroxyl group of the ligand and Asn-385, located in TMH 7. Use of the model is proposed to facilitate the identification of the structural elements of agonists and antagonists that are key for their specific functions, in order to achieve the design of new compounds with predetermined pharmacological properties.     

A molecular dynamics simulation study of an aminoglycoside/A-site RNA complex: conformational and hydration patterns

Aminoglycoside antibiotics interfere with the translation mechanism by binding to the tRNA decoding site of the 16S ribosomal RNA. Crystallographic structures of aminoglycosides bound to A-site systems clarified many static aspects of RNA-ligand interactions. To gain some insight on the dynamic aspects of recognition phenomena, we conducted molecular dynamics simulations of the aminoglycoside paromomycin bound to a eubacterial ribosomal decoding A-site oligonucleotide. Results from 25 ns of simulation time revealed that: (i) the neamine part of the antibiotic represents the main anchor for binding, (ii) additional sugar rings provide limited and fragile contacts, (iii) long-resident water molecules present at the drug/RNA interface are involved in the recognition phenomena. The combination of MD simulations together with systematic structural information offers striking insights into the molecular recognition processes underlying RNA/aminoglycoside binding. Important methodological considerations related to the use of medium resolution starting structures and associated sampling problems are thoroughly discussed.     

Characterization of the ColE1 mobilization region and its protein products

Summary A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. Themob region is genetically complex: twomob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis ofmob. Fourmob genes essential for mobilization by R64drd11 were thus identified. Fragments ofmob were subcloned under control of the P_tac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of themob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essentialmob genes are conserved is also presented.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

Integrity of H1 helix in prion protein revealed by molecular dynamic simulations to be especially vulnerable to changes in the relative orientation of H1 and its S1 flank

In the template-assistance model, normal prion protein (PrPC), the pathogenic cause of prion diseases such as Creutzfeldt-Jakob in human, bovine spongiform encephalopathy in cow, and scrapie in sheep, converts to infectious prion (PrPSc) through an autocatalytic process triggered by a transient interaction between PrPC and PrPSc. Conventional studies suggest the S1-H1-S2 region in PrPC to be the template of S1-S2 β-sheet in PrPSc, and the conformational conversion of PrPC into PrPSc may involve an unfolding of H1 in PrPC and its refolding into the β-sheet in PrPSc. Here we conduct a series of simulation experiments to test the idea of transient interaction of the template-assistance model. We find that the integrity of H1 in PrPC is vulnerable to a transient interaction that alters the native dihedral angles at residue Asn¹⁴³, which connects the S1 flank to H1, but not to interactions that alter the internal structure of the S1 flank, nor to those that alter the relative orientation between H1 and the S2 flank.     

Computational studies of pandemic 1918 and 2009 H1N1 hemagglutinins bound to avian and human receptor analogs

The purpose of this work was to study the binding properties of two pandemic influenza A virus 1918 H1N1 (SC1918) and 2009 H1N1 (CA09) hemagglutinin (HA) with avian and human receptors. The quantum chemical calculations have been performed to analyze the interactions of 130 loop, 190 helix, 220 loop region, and conserved residues 95,145,153–155, of pandemic viruses’ HA with sialo-trisaccharide receptor of avian and human using density functional theory. The HA’s residues Tyr 95, Ala 138, Gln 191, Arg 220, and Asp 225 from the above regions have stronger interaction with avian receptor. The residues Thr 136, Trp 153, His 183, and Asp 190 of HA are important and play a significant role to bind with human receptor. The residues Tyr 95, Ala 138, Lys 145, Trp 153, Gln 192, and Gln 226 of HA of CA09 virus have found more interaction energies with human than avian receptors. Due to mutations in the active residues of HA of CA09 virus comparing with SC1918, the binding capabilities of HA with human have been increased. The molecular dynamics simulation was made to understand the different dynamical properties of HA and molecular interactions between HA of these two viruses with sialo-trisaccharide receptors of avian and human receptors. The interaction energy of HA of CA09 virus with human receptor decreases due to the human receptor far away from conserved residue region of HA protein. This reveals that the conserved residues particularly Lys 145 play major contribution to interaction with human receptor in HA of CA09 virus.     

Binding mode prediction and inhibitor design of anti-influenza virus diketo acids targeting metalloenzyme RNA polymerase by molecular docking

Influenza is a yearly seasonal threat and major cause of mortality, particularly in children and the elderly. Although neuraminidase inhibitors and M2 protein blockers are used for medication, drug resistance has gradually emerged. Thus, the development of effective anti-influenza drugs targeting different constituent proteins of the virus is urgently desired. In this light, we carried out molecular docking to predict the binding modes of anti-influenza diketo acid inhibitors in the active site of the PAN subunit of the metalloenzyme RNA polymerase of influenza virus. The calculations suggested that the dianionic forms of the diketo acids should chelate the dinuclear manganese center as dinucleating ligands and sequester it. They also indicated that the diketo acid derivatives with larger hydrophobic substituents should block a hydrophobic cavity in the active site more tightly. These assumptions could adequately explain the enzyme inhibition by these compounds. Furthermore, we designed potential inhibitors by lead optimization of a diketo acid inhibitor from the thermodynamic points of view. Molecular docking results showed that the newly designed diketo acid derivatives might inhibit the metalloenzyme RNA polymerase more strongly than the lead inhibitor.     

Modeling of Transition State by Molecular Dynamics. Prediction of Catalytic Efficiency of the Mutants of Mandelate Racemase

Abstract

Modeling of transition state by molecular dynamic method often requires modification of the force field parameters to describe energy profile accurately. In this work, we avoided the modification by modeling a series of mutants at binding-related site. In predicting the catalytic efficiency (k _cat /K _m ) of the mutants of mandelate racemase (MR), the prediction performance of three energy subsets was investigated. It was indicated that the interaction-energy subset exhibited better prediction performance than whole-system subset and binding-site subset in both quantity and trend. When prediction error (PE) criterion was equal to 5%, 10 out of 12 samples were predicted correctly within interaction-energy subset, which demonstrated a great application potential of this method in prediction of enzyme catalytic efficiency and enzyme rational design.     

Synthesis,molecular modelling studies and biological evaluation of new oxoeicosanoid receptor 1 agonists

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC₅₀) and efficacy (E_max) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles.