Synthesis and Evaluation of Habiterpenol Analogs.

Habiterpenol is a G2 checkpoint inhibitor isolated from the culture broth of Phytohabitans sp. 3787_5. Here, we report the synthesis of new habiterpenol analogs through the total synthesis process of habiterpenol and evaluating the analogs for G2 checkpoint inhibitory activity. We investigated two different synthetic approaches for total synthesis, with intramolecular conjugate addition and Ti(III)-mediated radical cyclization as key reactions. Although the former was unsuccessful, the latter reaction facilitated stereoselective total synthesis and determination of the absolute configuration of habiterpenol. The extension of these chemistries to a structure-activity relationship (SAR) study gave new habiterpenol analogs, which could not be derived from natural habiterpenol and only be synthesized by applying the total synthesis. Therefore, this study provides important insights into SAR studies of habiterpenol.

Identification of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.

Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is overexpressed in many cancers. Numerous EZH2 inhibitors have been developed as anticancer agents, but recent studies have also focused on protein-protein interaction (PPI) between embryonic ectoderm development (EED) and EZH2 as a novel drug discovery target. Because EED indirectly enhances EZH2 enzymatic activity, EED-EZH2 PPI inhibitors suppress the methyltransferase activity and inhibit cancer growth. By contrast to the numerous promising EZH2 inhibitors, there are a paucity of EED-EZH2 PPI inhibitors reported in the literature. Here, we aimed to discover novel EED-EZH2 PPI inhibitors by first identifying possible binders of EED using an in-house knowledge-based in silico fragment mapping method. Next, 3D pharmacophore models were constructed from the arrangement pattern of the potential binders mapped onto the EED surface. In all, 16 compounds were selected by 3D pharmacophore-based virtual screening followed by docking-based virtual screening. In vitro evaluation revealed that five of these compounds exhibited inhibitory activities. This study has provided structural insights into the discovery and the molecular design of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.

In silico fragment-mapping method: a new tool for fragment-based/structure-based drug discovery.

Here, we propose an in silico fragment-mapping method as a potential tool for fragment-based/structure-based drug discovery (FBDD/SBDD). For this method, we created a database named Canonical Subsite-Fragment DataBase (CSFDB) and developed a knowledge-based fragment-mapping program, Fsubsite. CSFDB consists of various pairs of subsite-fragments derived from X-ray crystal structures of known protein-ligand complexes. Using three-dimensional similarity-matching between subsites on one protein and another, Fsubsite compares the surface of a target protein with all subsites in CSFDB. When a local topography similar to the subsite is found on the surface, Fsubsite places a fragment combined with the subsite in CSFDB on the target protein. For validation purposes, we applied the method to the apo-structure of cyclin-dependent kinase 2 (CDK2) and identified four compounds containing three mapped fragments that existed in the list of known inhibitors of CDK2. Next, the utility of our fragment-mapping method for fragment-growing was examined on the complex structure of tRNA-guanine transglycosylase with a small ligand. Fsubsite mapped appropriate fragments on the same position as the binding ligand or in the vicinity of the ligand. Finally, a 3D-pharmacophore model was constructed from the fragments mapped on the apo-structure of heat shock protein 90-α (HSP90α). Then, 3D pharmacophore-based virtual screening was carried out using a commercially available compound database. The resultant hit compounds were very similar to a known ligand of HSP90α. As a result of these findings, this in silico fragment-mapping method seems to be a useful tool for computational FBDD and SBDD.

Validation of molecular force field parameters for peptides including isomerized amino acids.

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age-related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l-α-amino acids, the accuracy of the computational results for d-amino acids and ß-amino acids was comparable to those for l-α-amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.

Linear Discriminant Analysis for the in Silico Discovery of Mechanism-Based Reversible Covalent Inhibitors of a Serine Protease: Application of Hydration Thermodynamics Analysis and Semi-empirical Molecular Orbital Calculation.

We recently reported that the Gibbs free energy of hydrolytic water molecules (ΔGwat) in acyl-trypsin intermediates calculated by hydration thermodynamics analysis could be a useful metric for estimating the catalytic rate constants (kcat) of mechanism-based reversible covalent inhibitors. For thorough evaluation, the proposed method was tested with an increased number of covalent ligands that have no corresponding crystal structures. After modeling acyl-trypsin intermediate structures using flexible molecular superposition, ΔGwat values were calculated according to the proposed method. The orbital energies of antibonding π* molecular orbitals (MOs) of carbonyl C=O in covalently modified catalytic serine (Eorb) were also calculated by semi-empirical MO calculations. Then, linear discriminant analysis (LDA) was performed to build a model that can discriminate covalent inhibitor candidates from substrate-like ligands using ΔGwat and Eorb. The model was built using a training set (10 compounds) and then validated by a test set (4 compounds). As a result, the training set and test set ligands were perfectly discriminated by the model. Hydrolysis was slower when (1) the hydrolytic water molecule has lower ΔGwat; (2) the covalent ligand presents higher Eorb (higher reaction barrier). Results also showed that the entropic term of hydrolytic water molecule (-TΔSwat) could be used for estimating kcat and for covalent inhibitor optimization; when the rotational freedom of the hydrolytic water molecule is limited, the chance for favorable interaction with the electrophilic acyl group would also be limited. The method proposed in this study would be useful for screening and optimizing the mechanism-based reversible covalent inhibitors.

Design of a New α-1-C-Alkyl-DAB Derivative Acting as a Pharmacological Chaperone for ß-Glucocerebrosidase Using Ligand Docking and Molecular Dynamics Simulation.

Some point mutations in ß-glucocerebrosidase cause either improper folding or instability of this protein, resulting in Gaucher disease. Pharmacological chaperones bind to the mutant enzyme and stabilize this enzyme; thus, pharmacological chaperone therapy was proposed as a potential treatment for Gaucher disease. The binding affinities of α-1-C-alkyl 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives, which act as pharmacological chaperones for ß-glucocerebrosidase, abruptly increased upon elongation of their alkyl chain. In this study, the primary causes of such an increase in binding affinity were analyzed using protein⁻ligand docking and molecular dynamics simulations. We found that the activity cliff between α-1-C-heptyl-DAB and α-1-C-octyl-DAB was due to the shape and size of the hydrophobic binding site accommodating the alkyl chains, and that the interaction with this hydrophobic site controlled the binding affinity of the ligands well. Furthermore, based on the aromatic/hydrophobic properties of the binding site, a 7-(tetralin-2-yl)-heptyl-DAB compound was designed and synthesized. This compound had significantly enhanced activity. The design strategy in consideration of aromatic interactions in the hydrophobic pocket was useful for generating effective pharmacological chaperones for the treatment of Gaucher disease.

Gibbs Free Energy of Hydrolytic Water Molecule in Acyl-Enzyme Intermediates of a Serine Protease: A Potential Application for Computer-Aided Discovery of Mechanism-Based Reversible Covalent Inhibitors.

In order to predict the potencies of mechanism-based reversible covalent inhibitors, the relationships between calculated Gibbs free energy of hydrolytic water molecule in acyl-trypsin intermediates and experimentally measured catalytic rate constants (kcat) were investigated. After obtaining representative solution structures by molecular dynamics (MD) simulations, hydration thermodynamics analyses using WaterMap™ were conducted. Consequently, we found for the first time that when Gibbs free energy of the hydrolytic water molecule was lower, logarithms of kcat were also lower. The hydrolytic water molecule with favorable Gibbs free energy may hydrolyze acylated serine slowly. Gibbs free energy of hydrolytic water molecule might be a useful descriptor for computer-aided discovery of mechanism-based reversible covalent inhibitors of hydrolytic enzymes.

Synthesis of a novel universal opioid receptor agonist with the 1,3,5-trioxazatriquinane skeleton and its pharmacologies.

We designed and synthesized of 1,3,5-trioxazatriquinanes with o- or p-hydroxyphenyl rings as analogs of the κ opioid receptor agonist SYK-146 with m-hydroxyphenyl groups. Although almost all tested compounds did not bind to the opioid receptors, only 17b (SYK-524) with two o-hydroxyphenyl rings showed moderate or potent binding affinities and exhibited agonistic activities for the three opioid receptor types. Because the basicity of the nitrogen atom in the 1,3,5-trioxazatriquinane structure was predicted to be very low due to the electron withdrawing effect of the three oxygen atoms, SYK-524 was a novel non-morphinan and nonpeptidic opioid universal agonist lacking a basic nitrogen atom.

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy.

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

Brazilian propolis-derived components inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes.

BACKGROUND: Previous reports suggest that Brazilian propolis has multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about the molecular mechanisms by which these compounds inhibit the downregulation of adiponectin. METHODS: The effect of various Brazilian propolis-derived components on inhibition of tumor necrosis factor-α (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes and molecular mechanism was investigated. RESULTS AND CONCLUSIONS: Pretreatment with either artepillin C (C3) or its derivative (C4) significantly inhibited TNF-α-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Interestingly, C3 strongly activated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. Treatment of adipocytes with C3 resulted in the upregulation of adiponectin and fatty acid-binding protein 4 expression, but C4 did not significantly induce PPARγ transactivation. C4 did, however, inhibit the TNF-α-induced c-Jun-NH(2)-terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on hPPARγ with C3 and JNK1 with C4 clearly supported our experimental results. These data demonstrate that 1) both C3 and C4 significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes, 2) C3 functions as a PPARγ agonist, and its inhibition of the effect of TNF-α is due to this PPARγ transactivation, and 3) C4 is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin. GENERAL SIGNIFICANCE: Brazilian propolis-derived components (C3 and C4) can significantly inhibit TNF-α-mediated downregulation of adiponectin in adipocytes, although they do so via different mechanisms.

Essential structure of opioid κ receptor agonist nalfurafine for binding to κ receptor 1: synthesis of decahydroisoquinoline derivatives and their pharmacologies.

On the basis of the three-dimensional pharmacophore model of opioid κ agonists, we simplified the structure of nalfurafine (selective κ agonist) to find the essential structural moieties for binding the opioid receptors, especially κ receptor type. As a result, we found that the trans-fused decahydroisoquinoline derivatives without a phenol ring bound the opioid receptor in micromolar order and that both the amide side chain and the nitrogen substituted by the cyclopropylmethyl group were indispensable moieties for eliciting the κ selectivity. The simple decahydroisoquinoline without amide side chain also bound the opioid receptor without receptor type selectivity, suggesting that the message-address concept would be applicable to even these simple derivatives. These findings that the simple decahydroisoquinoline derivatives showed the affinities for the opioid receptors, especially some of the compounds showed κ selectivity, are the first example in the opioid field.

3D-pharmacophore identification for kappa-opioid agonists using ligand-based drug-design techniques.

A selective kappa-opioid receptor (KOR) agonist might act as a powerful analgesic without the side effects of micro-opioid receptor-selective drugs such as morphine. The eight classes of known KOR agonists have different chemical structures, making it difficult to construct a pharmacophore model that takes them all into account. Here, we summarize previous efforts to identify the pharmacophore for kappa-opioid agonists and propose a new three-dimensional pharmacophore model that encompasses the kappa-activities of all classes. This utilizes conformational sampling of agonists by high-temperature molecular dynamics and pharmacophore extraction through a series of molecular superpositions.

Drug design and synthesis of a novel kappa opioid receptor agonist with an oxabicyclo[2.2.2]octane skeleton and its pharmacology.

A conformational analysis of kappa opioid receptor agonists, TRK-820 and U-50,488H indicated an active conformation of TRK-820 in which the C-ring was in the boat form with the 14-OH interacting with the amide nitrogen. Based on the obtained active conformation of TRK-820, we designed and synthesized a novel kappa agonist KNT-63 with oxabicyclo[2.2.2]octane skeleton. KNT-63 showed profound antinociceptive effects via the kappa receptor which were as potent as that of TRK-820.

Identification of the three-dimensional pharmacophore of kappa-opioid receptor agonists.

A selective kappa-opioid receptor agonist might act as a powerful analgesic without the side effects of mu-opioid receptor-selective drugs such as morphine. The eight classes of known kappa-opioid receptor agonists have different chemical structures, making it difficult to construct a pharmacophore model that takes them all into account. Here we propose a new three-dimensional pharmacophore model that encompasses the kappa-activities of all classes, which utilizes conformational sampling of agonists by high-temperature molecular dynamics and pharmacophore extraction through a series of molecular superpositions.

NMR spectroscopy and computational analysis of interaction between Serratia marcescens chitinase B and a dipeptide derived from natural-product cyclopentapeptide chitinase inhibitor argifin.

The dipeptide N-acetyl-Arg{N(omega)-(N-methylcarbamoyl)}-N-methyl-Phe(2), which is a part of the natural-product cyclopentapeptide chitinase inhibitor argifin (1), inhibits chitinase B from Serratia marcescens (SmChiB) with a half-maximal inhibitory concentration (IC(50)) of 3.7 microM. Despite the relatively small size of 2, its inhibitory activity is comparable with that of 1 (IC(50)=6.4 microM). To elucidate the basis for this interesting phenomenon, we investigated the interaction between 2 and SmChiB using a combination of nuclear magnetic resonance spectroscopy and computational methods. The transferred nuclear Overhauser effect (TRNOE) experiment obtained structural information on the SmChiB-bound conformation of 2. The binding mode of 2 and SmChiB was modeled by the novel molecular-docking approach proposed in our laboratory, which can explicitly consider water-mediated hydrogen-bonding interactions in protein-ligand interfaces. The SmChiB-bound conformation of 2 in the resulting model satisfied all proton-proton distance constraints derived from the TRNOE experiment, indicating that our model structure of the 2-SmChiB complex is reasonable. A molecular dynamics (MD) simulation examined the stability of the resultant complex structure and suggested that 2 binds to SmChiB in a similar fashion to the binding mode observed for N(omega)-(N-methylcarbamoyl)-Arg(1) and N-methyl-Phe(2) of 1 in the crystal structure of the argifin-SmChiB complex. Finally, the binding free energies of 1 and 2 with SmChiB were estimated by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method using the MD trajectory. The MM-PBSA calculation suggested that both 1 and 2 bind to SmChiB with similar affinities, which is consistent with their experimental IC(50) values. Energetic analysis revealed that the van der Waals interaction of 2 with SmChiB is much less than that of 1, but is completely compensated by the more favorable contribution of solute entropy and the total electrostatic component. The improved total electrostatic component was derived from more favorable electrostatic interactions. Therefore, we conclude that dipeptide 2 was also better optimized against SmChiB than 1 in an electrostatic point of view.

Evaluation of the searching abilities of HBOP and HBSITE for binding pocket detection.

We evaluated the pocket-searching abilities of the computer programs HBOP and HBSITE, in which hydrophobic potentials calculated on grid points generated around a protein function as an indicator of the pocket-like property-using a test set of 458 experimentally observed structures of protein-ligand complexes. The results were compared with those obtained using the alternative algorithms PASS and SiteID, which only consider geometric properties, and Q-SiteFinder, which considers not only geometry but also physicochemical properties. The comparison revealed that HBOP and HBSITE could detect experimentally observed ligand-binding pockets for more test complexes than PASS and SiteID. In addition, the success rate of HBOP for detecting binding pockets was higher than that of Q-SiteFinder, and that of HBSITE was comparable with that of Q-SiteFinder. Results of tests for ligand-unbound state proteins indicated that HBSITE was more appropriate than Q-SiteFinder for pocket searches of ligand-unbound proteins, and HBSITE was more robust than Q-SiteFinder against structural differences between ligand-bound and -unbound state proteins.

Structure-based virtual screening for novel chymase inhibitors by in silico fragment mapping.

The term chymase refers to a family of chymotrypsin-like serine proteases stored within the secretory granules of mast cells. Recently, a variety of small molecule inhibitors for chymase have been developed with a primary focus on the treatment of cardiovascular diseases. Despite the expected therapeutic benefit of these chymase inhibitors, they have not been used clinically. Here, we attempted to identify new chymase inhibitors using a multistep structure-based virtual screening protocol combined with our knowledge-based in silico fragment mapping technique. The mapping procedure identified fragments with novel modes of interaction at the oxyanion hole of chymase. Next, we constructed a three-dimensional (3D) pharmacophore model and retrieved eight candidate chymase inhibitors from a commercial database that included approximately five million compounds. This selection was achieved using a multistep virtual screening protocol, which combined a 3D pharmacophore-based search, docking calculations, and analyses of binding free energy. One of the eight compounds exhibited concentration-dependent chymase inhibitory activity, which could be further optimized to develop more potent chymase inhibitors.

[Identifying Inhibitors of USP7-HDM2 Protein-Protein Interaction (PPI) by the in Silico Fragment-mapping Method].

Proteolysis mediated by the ubiquitin-proteome system plays an important role in cancer. Recently, a deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7) has attracted attention as a key regulator of the p53-human double minute 2 (HDM2) pathway in cancer cells. Although some USP7 enzyme inhibitors have been identified, issues related to activity and selectivity prevent their therapeutic application. In this study, we aimed to search for novel USP7-HDM2 protein-protein interaction (PPI) inhibitors that do not affect the USP7 enzyme activity. Using the fragment-mapping program Fsubsite and the canonical subsite-fragment database (CSFDB) developed in our laboratory, we mapped a variety of fragments onto USP7 protein and constructed 3D-pharmacophore models based on the arrangement patterns of the mapped fragments. Finally, we performed 3D pharmacophore-based virtual screening of a commercial compound database and successfully selected promising USP7-HDM2 PPI inhibitor candidates.

In silico multi-filter screening approaches for developing novel beta-secretase inhibitors.

A large database of chemical structures was screened for potential inhibitors of beta-secretase was carried out using in silico multi-filter techniques. Substructure screening, computer-aided ligand docking, binding free energy calculations, and partial interaction energy analyses were performed successively to identify chemical compounds which could serve as different scaffolds from known beta-secretase inhibitors for future drug design. We showed that our in silico multi-filter screening retrieved all known inhibitors from the compound database investigated, which suggests that the other compounds identified as inhibitors by this computerized screening process are potential beta-secretase inhibitors.

Precise prediction of activators for the human constitutive androstane receptor using structure-based three-dimensional quantitative structure-activity relationship methods.

The constitutive androstane receptor (CAR, NR1I3) regulates the expression of numerous drug-metabolizing enzymes and transporters. The upregulation of various enzymes, including CYP2B6, by CAR activators is a critical problem leading to clinically severe drug-drug interactions (DDIs). To date, however, few effective computational approaches for identifying CAR activators exist. In this study, we aimed to develop three-dimensional quantitative structure-activity relationship (3D-QSAR) models to predict the CAR activating potency of compounds emerging in the drug-discovery process. Models were constructed using comparative molecular field analysis (CoMFA) based on the molecular alignments of ligands binding to CAR, which were obtained from ensemble ligand-docking using 28 compounds as a training set. The CoMFA model, modified by adding a lipophilic parameter with calculated logD7.4 (S+logD7.4), demonstrated statistically good predictive ability (r2 = 0.99, q2 = 0.74). We also confirmed the excellent predictability of the 3D-QSAR model for CAR activation (r2pred = 0.71) using seven compounds as a test set for external validation. Collectively, our results indicate that the 3D-QSAR model developed in this study provides precise prediction of CAR activating potency and, thus, should be useful for selecting drug candidates with minimized DDI risk related to enzyme-induction in the early drug-discovery stage.