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1.
ACS Omega ; 8(29): 25850-25860, 2023 Jul 25.
Article in English | MEDLINE | ID: mdl-37521650

ABSTRACT

In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds.

2.
Bioorg Med Chem ; 33: 116019, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33486159

ABSTRACT

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of patients with bladder cancer harboring genetic alterations in FGFR3. We identified pyrimidine derivative 20b, which induced tumor regression following oral administration to a bladder cancer xenograft mouse model. Compound 20b was discovered by optimizing lead compound 1, which we reported previously. Specifically, reducing the molecular size of the substituent at the 4-position and replacing the linker of the 5-position in the pyrimidine scaffold resulted in an increase in systemic exposure. Furthermore, introduction of two fluorine atoms into the 3,5-dimethoxyphenyl ring enhanced FGFR3 inhibitory activity. Molecular dynamics (MD) simulation of 20b suggested that the fluorine atom interacts with the main chain NH moiety of Asp635 via a hydrogen bond.


Subject(s)
Antineoplastic Agents/pharmacology , Pyrimidines/pharmacology , Receptor, Fibroblast Growth Factor, Type 3/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Dynamics Simulation , Molecular Structure , NIH 3T3 Cells , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Pyrimidines/administration & dosage , Pyrimidines/chemistry , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Solubility , Structure-Activity Relationship , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology
3.
Bioorg Med Chem ; 28(10): 115453, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32278710

ABSTRACT

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of bladder cancer. We identified 1,3,5-triazine derivative 18b and pyrimidine derivative 40a as novel structures with potent and highly selective FGFR3 inhibitory activity over vascular endothelial growth factor receptor 2 (VEGFR2) using a structure-based drug design (SBDD) approach. X-ray crystal structure analysis suggests that interactions between 18b and amino acid residues located in the solvent region (Lys476 and Met488), and between 40a and Met529 located in the back pocket of FGFR3 may underlie the potent FGFR3 inhibitory activity and high kinase selectivity over VEGFR2.


Subject(s)
Drug Design , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Receptor, Fibroblast Growth Factor, Type 3/antagonists & inhibitors , Triazines/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Cell Line , Dose-Response Relationship, Drug , Humans , Molecular Structure , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Structure-Activity Relationship , Triazines/chemistry , Vascular Endothelial Growth Factor Receptor-2/metabolism
5.
J Med Chem ; 61(11): 5047-5053, 2018 06 14.
Article in English | MEDLINE | ID: mdl-29771525

ABSTRACT

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.


Subject(s)
Enzyme Inhibitors/pharmacology , Intramolecular Transferases/antagonists & inhibitors , Trypanosoma cruzi/enzymology , Drug Discovery , Humans , Inhibitory Concentration 50 , Intramolecular Transferases/chemistry , Nuclear Magnetic Resonance, Biomolecular , Trypanosoma cruzi/drug effects
6.
Sci Rep ; 7(1): 6666, 2017 07 27.
Article in English | MEDLINE | ID: mdl-28751689

ABSTRACT

Chagas disease results from infection by Trypanosoma cruzi and is a neglected tropical disease (NTD). Although some treatment drugs are available, their use is associated with severe problems, including adverse effects and limited effectiveness during the chronic disease phase. To develop a novel anti-Chagas drug, we virtually screened 4.8 million small molecules against spermidine synthase (SpdSyn) as the target protein using our super computer "TSUBAME2.5" and conducted in vitro enzyme assays to determine the half-maximal inhibitory concentration values. We identified four hit compounds that inhibit T. cruzi SpdSyn (TcSpdSyn) by in silico and in vitro screening. We also determined the TcSpdSyn-hit compound complex structure using X-ray crystallography, which shows that the hit compound binds to the putrescine-binding site and interacts with Asp171 through a salt bridge.


Subject(s)
Chagas Disease/enzymology , Enzyme Inhibitors/pharmacology , Spermidine Synthase/antagonists & inhibitors , Trypanosoma cruzi/enzymology , Binding Sites , Chagas Disease/drug therapy , Computer Simulation , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/therapeutic use , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism , Spermidine Synthase/metabolism , Trypanosoma cruzi/drug effects
7.
J Med Chem ; 59(5): 2261-6, 2016 Mar 10.
Article in English | MEDLINE | ID: mdl-26881725

ABSTRACT

Although NMR in fragment-based drug discovery is utilized almost exclusively to evaluate physical binding between molecules, it should be also a powerful tool for biochemical assay, evaluating inhibitory effect of compounds on enzymatic activity. Time-dependent spectral change in real-time monitoring or inhibitor concentration-dependent spectral change after constant-time reaction was processed by factor analysis, by which reaction rate or IC50 value was obtained. Applications to spermidine synthase of Trypanosoma cruzi, which causes Chagas disease, are described.


Subject(s)
Cyclohexylamines/pharmacology , Drug Discovery , Nuclear Magnetic Resonance, Biomolecular , Spermidine Synthase/antagonists & inhibitors , Trypanosoma cruzi/enzymology , Chagas Disease/drug therapy , Cyclohexylamines/chemical synthesis , Cyclohexylamines/chemistry , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Molecular Structure , Spermidine Synthase/metabolism , Structure-Activity Relationship , Time Factors
8.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 9): 1879-89, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26327378

ABSTRACT

Trypanosoma cruzi causes Chagas disease, a severe disease affecting 8-10 million people in Latin America. While nifurtimox and benznidazole are used to treat this disease, their efficacy is limited and adverse effects are observed. New therapeutic targets and novel drugs are therefore urgently required. Enzymes in the polyamine-trypanothione pathway are promising targets for the treatment of Chagas disease. Spermidine synthase is a key enzyme in this pathway that catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. Fragment-based drug discovery was therefore conducted to identify novel, potent inhibitors of spermidine synthase from T. cruzi (TcSpdSyn). Here, crystal structures of TcSpdSyn in complex with dcSAM, trans-4-methylcyclohexylamine and hit compounds from fragment screening are reported. The structure of dcSAM complexed with TcSpdSyn indicates that dcSAM stabilizes the conformation of the `gatekeeping' loop to form the putrescine-binding pocket. The structures of fragments bound to TcSpdSyn revealed two fragment-binding sites: the putrescine-binding pocket and the dimer interface. The putrescine-binding pocket was extended by an induced-fit mechanism. The crystal structures indicate that the conformation of the dimer interface is required to stabilize the gatekeeping loop and that fragments binding to this interface inhibit TcSpdSyn by disrupting its conformation. These results suggest that utilizing the dynamic structural changes in TcSpdSyn that occur upon inhibitor binding will facilitate the development of more selective and potent inhibitors.


Subject(s)
Spermidine Synthase/chemistry , Trypanosoma cruzi/enzymology , Allosteric Regulation , Animals , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydrogen Bonding , Models, Molecular , Protein Conformation , Spermidine Synthase/antagonists & inhibitors
9.
J Biol Chem ; 284(4): 2435-47, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-19017645

ABSTRACT

Heat shock factor 2 (HSF2) is a member of a vertebrate transcription factor family for genes of heat shock proteins and is involved in the regulation of development and cellular differentiation. The DNA binding property of HSF2 is modulated by the post-translational modification of a specific lysine residue in its DNA binding domain by small ubiquitin-like modifier (SUMO), but the consequences of SUMOylation and its underlying molecular mechanism remain unclear. Here we show the inhibitory effect of SUMOylation on the interaction between HSF2 and DNA based on biochemical analysis using isolated recombinant HSF2. NMR study of the SUMOylated DNA binding domain of HSF2 indicates that the SUMO moiety is flexible with respect to the DNA binding domain and has neither a noncovalent interface with nor a structural effect on the domain. Combined with data from double electron-electron resonance and paramagnetic NMR relaxation enhancement experiments, these results suggest that SUMO attachment negatively modulates the formation of the protein-DNA complex through a randomly distributed steric interference.


Subject(s)
DNA/metabolism , Heat-Shock Proteins/metabolism , SUMO-1 Protein/metabolism , Transcription Factors/metabolism , Base Sequence , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Humans , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , SUMO-1 Protein/chemistry , Transcription Factors/chemistry , Transcription Factors/genetics
10.
Biochim Biophys Acta ; 1768(12): 3001-11, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17996724

ABSTRACT

PACAP (pituitary adenylate cyclase-activating polypeptide) is a member of the VIP/secretin/glucagon family, which includes the ligands of class II G-protein coupled receptors. Since the recognition of PACAP by the receptor may involve the binding of PACAP to membranes, its membrane-bound structure should be important. We have carried out structural analysis of uniformly 13C,15N labeled PACAP27 and its C-terminal truncated form PACAP(1-21)NH2 (PACAP21) bound to membranes with high resolution solid-state NMR. Phosphatidylcholine bilayers and phosphatidylcholine/phosphatidylglycerol bilayers were used for PACAP27 and PACAP21, respectively. Most backbone signals were assigned for PACAP27 and PACAP21. TALOS analysis revealed that both peptides take on extended conformations on the membranes. Dilution of PACAP21 did not change the conformation of the major part. Selective polarization transfer experiment confirmed that PACAP27 is interacting with the membranes. It was concluded that the interaction of PACAP with the membrane surface causes their extended conformation. PACAP27 is reported to take an alpha-helical conformation in dodecylphosphocholine micelles and membrane-binding peptides usually take similar conformations in micelles and in membranes. Therefore, the property of PACAP27 changing its conformation in response to its environment is unique. Its conformational flexibility may be associated with its wide variety of functions.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Pituitary Adenylate Cyclase-Activating Polypeptide/chemistry , Amino Acid Sequence , Carbon Isotopes , Cell Membrane/chemistry , Cell Membrane/metabolism , Molecular Sequence Data , Nitrogen Isotopes , Phospholipids/chemistry , Phospholipids/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism
11.
FEBS Lett ; 580(1): 23-6, 2006 Jan 09.
Article in English | MEDLINE | ID: mdl-16364317

ABSTRACT

We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.


Subject(s)
Receptor, Muscarinic M2/chemistry , Animals , Circular Dichroism , Escherichia coli , Gene Expression , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Receptor, Muscarinic M2/genetics , Receptor, Muscarinic M2/isolation & purification
12.
Protein Eng Des Sel ; 17(4): 305-14, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15166312

ABSTRACT

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.


Subject(s)
Genetic Vectors , Peptides/genetics , Amino Acid Sequence , Base Sequence , DNA , Factor Xa/metabolism , Isotope Labeling , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Open Reading Frames , Peptides/chemistry , Peptides/isolation & purification
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