Ohgi Takahashi

Verruculides A and B, two new protein tyrosine phosphatase 1B inhibitors from an Indonesian ascidian-derived Penicillium verruculosum.

Two new merosesquiterpenes, verruculides A (1) and B (2), were isolated from a culture broth of the Indonesian ascidian-derived Penicillium verruculosum TPU1311, together with three known congeners, chrodrimanins A (3), B (4), and H (5). The structures of 1 and 2 were assigned on the basis of their spectroscopic data (1D and 2D NMR, HRMS, UV, CD, and IR). Compound 2 had a linear sesquiterpene moiety and was considered to be the derivative of the biosynthetic precursor for 1 and 3-5. Compounds 1, 3, and 5 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 8.4, 8.5, and 14.9 μM, respectively. Compound 2 showed 40% inhibition at 23.1 μM, while 4 was not active at 20.7 μM.

Modeling the Enolization of Succinimide Derivatives, a Key Step of Racemization of Aspartic Acid Residues: Importance of a Two‐H2O Mechanism

Racemization of aspartic acid residues in peptides and proteins is assumed to proceed via succinimide intermediates. An enolization of the succinimide intermediate is required for the racemization to occur. In this study, we modeled the enolization step by density‐functional theory (DFT) calculations (B3LYP/6‐31+G**), using two model compounds, N‐methylsuccinimide (1) and its formylamino derivarive 2. Three mechanisms were investigated for 1, i.e., the direct mechanism without active participation of H2O molecules, and one‐H2O and two‐H2O mechanisms, in which one or two H2O molecules actively participate in the reaction. We found that the two‐H2O mechanism was the most favorable with an activation barrier of 37 kcal mol−1. In the two‐H2O mechanism, a concerted bond reorganization involving a triple H‐atom transfer occurred in an eight‐membered cyclic structure formed between the imide and two H2O molecules. For 2, we investigated only the two‐H2O mechanism and found that the activation barrier was lowered to 31 kcal mol−1 due to an H‐bond between the CO O‐atom of the formylamino group (‘the neighboring residue’) and one of the H2O molecules. Our results suggest that, in proteins, the Asp racemization is severely controlled by the accessibility of H2O molecules to the reaction site of the succinimide intermediate.

Romidepsin (FK228) and its analogs directly inhibit phosphatidylinositol 3‐kinase activity and potently induce apoptosis as histone deacetylase/phosphatidylinositol 3‐kinase dual inhibitors

Activation of phosphatidylinositol 3‐kinase (PI3K) signaling is involved in carcinogenesis and cancer progression. The PI3K inhibitors are considered candidate drugs for cancer treatment. Here, we describe a drug screening system for novel PI3K inhibitors using Saccharomyces cerevisiae strains with deleterious mutations in the ATP‐binding cassette transporter genes, because wild‐type S. cerevisiae uses drug efflux pumps for reducing intracellular drug concentrations. By screening the chemical library of the Screening Committee of Anticancer Drugs, we identified the histone deacetylase (HDAC) inhibitor romidepsin (FK228) and its novel analogs. In vitro PI3K activity assays confirmed that these compounds directly inhibit PI3K activity at μM‐range concentrations. FK‐A5 analog was the most potent inhibitor. Western blotting revealed that these compounds inhibit phosphorylation of protein kinase B and downstream signaling components. Molecular modeling of the PI3K–FK228 complex indicated that FK228 binds to the ATP‐binding pocket of PI3K. At μM‐range concentrations, FK228 and FK‐A5 show potent cytotoxicity, inducing apoptosis even in HDAC inhibitor‐resistant cells. Furthermore, HDAC/PI3K dual inhibition by FK228 and FK‐A5 at μM‐range concentrations potentiates the apoptosis induction, mimicking the effect of combining specific HDAC and PI3K inhibitors. In this study, we showed that FK228 and its analogs directly inhibit PI3K activity and induce apoptosis at μM‐range concentrations, similar to HDAC/PI3K dual inhibition. In future, optimizing the potency of FK228 and its analogs against PI3K may contribute to the development of novel HDAC/PI3K dual inhibitors for cancer treatment.

Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai.

Three new N-methyladenine-containing diterpenes, 2-oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G (10), and ageline B (11). The structures of 1-3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5-11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 μg/disc, respectively. All compounds were inactive (IC50 > 10 μM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 15 μM.

The relationship between local liquid density and force applied on a tip of atomic force microscope: A theoretical analysis for simple liquids

The density of a liquid is not uniform when placed on a solid. The structured liquid pushes or pulls a probe employed in atomic force microscopy, as demonstrated in a number of experimental studies. In the present study, the relation between the force on a probe and the local density of a liquid is derived based on the statistical mechanics of simple liquids. When the probe is identical to a solvent molecule, the strength of the force is shown to be proportional to the vertical gradient of ln(ρDS) with the local liquids density on a solid surface being ρDS. The intrinsic liquids density on a solid is numerically calculated and compared with the density reconstructed from the force on a probe that is identical or not identical to the solvent molecule.

Euryspongins A-C, three new unique sesquiterpenes from a marine sponge Euryspongia sp.

Three new unique sesquiterpenes, euryspongins A-C (1-3), were isolated from a marine sponge Euryspongia sp. collected at Iriomote Island, Okinawa, Japan. Compound 1 possessed a bicyclic furanosesquiterpene structure with six- and eight-membered rings, whereas compounds 2 and 3 had an α,β-unsaturated-γ-lactone ring instead of the furan ring in 1. Only five natural products in this class have been reported, and compounds 1-3 are the sixth-eighth examples of natural products. Compounds 1-3 had no inhibition effect against PTP1B, an important target enzyme for the treatment of diabetes, while the dehydro derivative of 1 [dehydroeuryspongin A (4)] exhibited inhibitory activity (IC(50)=3.6 μM).

Two new protein tyrosine phosphatase 1B inhibitors, hyattellactones A and B, from the Indonesian marine sponge Hyattella sp.

Two unique sesterterpenes, hyattellactones A (1) and B (2), together with two known sesterterpenes, phyllofolactones F (3) and G (4), were isolated from the Indonesian marine sponge Hyattella sp. The structures of the two new compounds, 1 and 2 were assigned based on their spectroscopic data. Hyattellactone A (1) was a scalarane sesterterpene with an α,β-unsaturated-γ-lactone ring and C-ethyl group, while B (2) was an epimer of 1 at the C-24 position. Compounds 1 and 3 inhibited PTP1B activity with IC50 values of 7.45 and 7.47μM, respectively. On the other hand, compounds 2 and 4 (24S-isomers of 1 and 3, respectively) showed much reduced activity than the 24R-isomers.

Strongylophorines, new protein tyrosine phosphatase 1B inhibitors, from the marine sponge Strongylophora strongilata collected at Iriomote Island.

A new meroditerpene, 26-O-ethylstrongylophorine-14 (1), was isolated from the Okinawan marine sponge Strongylophora strongilata together with six known strongylophorines: 26-O-methylstrongylophorine-16 (2) and strongylophorines-2 (3), -3 (4), -8 (5), -15 (6), and -17 (7). The structure of 1 was assigned on the basis of its spectroscopic data. Compound 1 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 8.7 μM, while known compounds 2-8 gave IC50 values of 8.5, >24.4, 9.0, 21.2, 11.9, and 14.8 μM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7 μM (IC50). The inhibitory activities of strongylophorines possessing the acetal moiety at C-26 (1, 2, and 6) were stronger than those of the lactone derivatives (3 and 5). This is the first study to demonstrate that meroditerpenes inhibit PTP1B activity.

Evaluation of Influence of Single Nucleotide Polymorphisms in Cytochrome P450 2B6 on Substrate Recognition Using Computational Docking and Molecular Dynamics Simulation

In this study, we investigated the influence of single nucleotide polymorphisms on the conformation of mutated cytochrome P450 (CYP) 2B6 proteins using molecular dynamics (MD) simulation. Some of these mutations influence drug metabolism activities, leading to individual variations in drug efficacy and pharmacokinetics. Using computational docking, we predicted the structure of the complex between the antimalarial agent artemether and CYP2B6 whose conformations were obtained by MD simulation. The simulation demonstrated that the entire structure of the protein changes even when a single residue is mutated. Moreover, the structural flexibility is affected by the mutations and it may influence the enzyme activity. The results suggest that some of the inactive mutants cannot recognize artemether due to structural changes caused by the mutation.

Roles of Intramolecular and Intermolecular Hydrogen Bonding in a Three-Water-Assisted Mechanism of Succinimide Formation from Aspartic Acid Residues

Aspartic acid (Asp) residues in peptides and proteins are prone to isomerization to the β-form and racemization via a five-membered succinimide intermediate. These nonenzymatic reactions have relevance to aging and age-related diseases. In this paper, we report a three water molecule-assisted, six-step mechanism for the formation of succinimide from Asp residues found by density functional theory calculations. The first two steps constitute a stepwise iminolization of the C-terminal amide group. This iminolization involves a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds formed by the C-terminal amide group, the side-chain carboxyl group, and the three water molecules. After a conformational change (which breaks the intramolecular hydrogen bond involving the iminol nitrogen) and a reorganization of water molecules, the iminol nitrogen nucleophilically attacks the carboxyl carbon of the Asp side chain to form a five-membered ring. This cyclization is accompanied by a triple proton transfer involving two water molecules, so that a gem-diol tetrahedral intermediate is formed. The last step is dehydration of the gem-diol group catalyzed by one water molecule, and this is the rate-determining step. The calculated overall activation barrier (26.7 kcal mol−1) agrees well with an experimental activation energy.