Discovery of TP0628103: A Highly Potent and Selective MMP-7 Inhibitor with Reduced OATP-Mediated Clearance Designed by Shifting Isoelectric Points.

Matrix metalloproteinase-7 (MMP-7) has been shown to play an important role in pathophysiological processes such as cancer and fibrosis. We previously discovered selective MMP-7 inhibitors by molecular hybridization and structure-based drug design. However, the systemic clearance (CLtot) of the biologically active lead compound was very high. Because our studies revealed that hepatic uptake by organic anion transporting polypeptide (OATP) was responsible for the high CLtot, we found a novel approach to reducing their uptake based on isoelectric point (IP) values as an indicator for substrate recognition by OATP1B1/1B3. Our "IP shift strategy" to adjust the IP values culminated in the discovery of TP0628103 (18), which is characterized by reduced in vitro OATP-mediated hepatic uptake and in vivo CLtot. Our in vitro-in vivo extrapolation of OATP-mediated clearance and the "IP shift strategy" provide crucial insights for a new medicinal chemistry approach to reducing the systemic clearance of OATP1B1/1B3 substrates.

Discovery of novel indole derivatives as potent and selective inhibitors of proMMP-9 activation.

Matrix metalloproteinase-9 (MMP-9) is a secreted zinc-dependent endopeptidase that degrades the extracellular matrix and basement membrane of neurons, and then contributes to synaptic plasticity by remodeling the extracellular matrix. Inhibition of MMP-9 activity has therapeutic potential for neurodegenerative diseases such as fragile X syndrome. This paper reports the molecular design, synthesis, and in vitro studies of novel indole derivatives as inhibitors of proMMP-9 activation. High-throughput screening (HTS) of our internal compound library and subsequent merging of hit compounds 1 and 2 provided compound 4 as a bona-fide lead. X-ray structure-based design and subsequent lead optimization led to the discovery of compound 33, a highly potent and selective inhibitor of proMMP-9 activation.

Structure-Based Optimization and Biological Evaluation of Potent and Selective MMP-7 Inhibitors for Kidney Fibrosis.

Matrix metalloproteinase-7 (MMP-7) has been shown to play important roles in pathophysiological processes involved in the development/progression of diseases such as cancer and fibrosis. We discovered selective MMP-7 inhibitors composed of arylsulfonamide, carboxylate, and short peptides by a molecular hybridization approach. These compounds interacted with MMP-7 via multiple hydrogen bonds in the cocrystal structures. To obtain compounds for in vivo evaluation, we attempted structural optimization, particularly targeting Tyr167 at the S3 subsite through structure-based drug design, and identified compound 15 as showing improved MMP-7 potency and MMP subtype selectivity. A novel π-π stacking interaction with Tyr167 was achieved when 4-pyridylalanine was introduced as the P3 residue. Compound 15 suppressed the progression of kidney fibrosis in a dose-dependent manner in a mouse model of unilateral ureteral obstruction. Thus, we demonstrated, for the first time, that potent and selective MMP-7 inhibitors could prevent the progression of kidney fibrosis.

Discovery of Highly Potent and Selective Matrix Metalloproteinase-7 Inhibitors by Hybridizing the S1' Subsite Binder with Short Peptides.

Matrix metalloproteinase-7 (MMP-7) has emerged as a protein playing important roles in both physiological and pathophysiological processes. Despite the growing interest in MMP-7 as a potential therapeutic target for diseases including cancer and fibrosis, potent and selective MMP-7 inhibitors have yet to be identified. Compound 1, previously reported by Edman and co-workers, binds to the S1' subsite of MMP-7, exhibiting moderate inhibitory activity and selectivity. To achieve both higher inhibitory activity and selectivity, we conceived hybridizing 1 with short peptides. The initially designed compound 6, which was a hybrid molecule between 1 and a tripeptide (Ala-Leu-Met) derived from an MMP-2-inhibitory peptide (APP-IP), showed enhanced MMP-7-inhibitory activity. Subsequent optimization of the peptide moiety led to the development of compound 18 with remarkable potency for MMP-7 and selectivity over other MMP subtypes.

Adjuvant capecitabine plus oxaliplatin after D2 gastrectomy in Japanese patients with gastric cancer: a phase II study.

BACKGROUND: Adjuvant chemotherapy with XELOX (capecitabine plus oxaliplatin) has been shown to be beneficial following resection of gastric cancer in South Korean, Chinese, and Taiwanese patients. This phase II study (J-CLASSIC-PII) was undertaken to evaluate the feasibility of XELOX in Japanese patients with resected gastric cancer. METHODS: Patients with stage II or III gastric cancer who underwent curative D2 gastrectomy received adjuvant XELOX (eight 3-week cycles of oral capecitabine, 1000 mg/m2 twice daily on days 1-14, plus intravenous oxaliplatin 130 mg/m2 on day 1). The primary endpoint was dose intensity. Secondary endpoints were safety, proportion of patients completing treatment, and 1-year disease-free survival (DFS) rate. RESULTS: One hundred patients were enrolled, 76 of whom completed the study as planned. The mean dose intensity was 67.2 % (95 % CI, 61.9-72.5 %) for capecitabine and 73.4 % (95 % CI, 68.4-78.4 %) for oxaliplatin, which were higher than the predefined age-adjusted threshold values of 63.4 % and 69.4 %, respectively, and the study therefore met its primary endpoint. The 1-year DFS rate was 86 % (95 % CI, 77-91 %). No new safety signals were identified. CONCLUSIONS: The feasibility of adjuvant XELOX in Japanese patients with resected gastric cancer is similar to that observed in South Korean, Chinese, and Taiwanese patients in the Capecitabine and Oxaliplatin Adjuvant Study in Stomach Cancer (CLASSIC) study. Based on findings from this study and the CLASSIC study, the XELOX regimen can be considered an adjuvant treatment option for Japanese gastric cancer patients who have undergone curative resection.

Anionic liposome template synthesis of raspberry-like hollow silica particle under ambient conditions with basic catalyst.

Hollow silica particle was obtained with a vesicle template synthesis in water under ambient conditions in the presence of ammonia. Biomimetic vesicles, liposomes were used, which consisted of a zwitterionic phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and a tiny amount of charged amphiphiles, hexadecylamine (HDA) or dicetylphosphate (DCP). Aggregation of silica occurred for DPPC or cationic DPPC/HDA liposome, whereas well-dispersed hollow silica particle could be obtained for anionic DPPC/DCP liposome. The hollow particle synthesized with the anionic liposome had single-layered and raspberry-like structures. Electrostatic repulsion between anionic vesicles maintained stable dispersion of the as-synthesized particles during the reaction. Formation of the raspberry-like morphology is explained by silica particle precipitation selectively induced around the liposomes under basic conditions due to affinity of silica precursors for the liposomes. Synthesis of well-dispersed hollow silica particle with a raspberry-like morphology is the first report in vesicle template syntheses.

Clinical outcome in Japanese elderly patients with non-valvular atrial fibrillation taking warfarin: a single-center observational study.

BACKGROUND: Although a lower target prothrombin time-international normalized ratio (PT-INR) with warfarin therapy is recommended in Japan for atrial fibrillation (AF) patients ≥70 years of age, few studies have provided supporting data. The current study aimed to evaluate the clinical outcome in elderly Japanese patients with non-valvular AF who were taking warfarin. METHODS: We conducted a cohort study of 845 consecutive non-valvular AF patients ≥70 years of age who were taking warfarin (median age, 74 years; 30.5% women) with a median follow-up period of 27 months (4-69 months). Of these patients, 29.7% had a history of stoke/transient ischemic attack (TIA), and 73.1% of the patients had a CHADS(2) score ≥2. The occurrence of thromboembolic events, including ischemic stroke, TIA and other systemic embolisms, and major bleeding events were validated through a review of medical records. RESULTS: The incidence of thromboembolic and major bleeding events were 3.8 and 2.1% per year, respectively. A higher incidence of both events was observed in patients with a CHADS(2) score ≥3. The multivariate analysis showed that prior stroke/TIA (odds ratio 1.7, 95% CI 1.0-2.7) and diabetes (odds ratio 1.7, 95% CI 1.0-2.8) were independent risks of thromoembolic events. A HAS-BLED score ≥3 represented a risk for major bleeding (hazard ratio 2.8, 95% CI 1.7-4.6). A PT-INR of 1.5-2.5 indicated a low incidence of thromboembolic and major bleeding events in patients with a CHADS(2) score ≥2. CONCLUSIONS: Our results demonstrate that a target PT-INR of 2.0 and a range of 1.5-2.5 may be safe for elderly Japanese patients with non-valvular AF.

Selenite reduction by the thioredoxin system: kinetics and identification of protein-bound selenide.

Selenite (SeO(3)(2-)) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/autoradiography using (75)Se-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).

A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique.

Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P(2) at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-delta1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P(2) distribution quantitatively. By using this method, we found that PI(4,5)P(2) is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P(2) was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P(2) level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P(2) level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P(2) level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P(2) pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.

Radical scavenging activity of 'Nikogori' gelatin gel food made from head, bone, skin, tail and scales of fishes measured using the chemiluminescence method.

Head, bone, skin and tail mixed parts of yellowtail and bream, scales of bream and head of salmon which have nutritional values and are usually discarded were made into 'Nikogori' gelatin gel. They are not only a good source of protein but are also useful for elderly people with swallowing problems. Soy sauce, a traditional seasoning in Japan, was added to enhance the taste of 'Nikogori'. The rheological properties were examined by a rheometer and the peroxyl radical scavenging activity was measured by the chemiluminescence method. Sensory evaluation was also conducted employing 20 faculty members and students of the Laboratory of Cookery Science, Tokyo Kasei University, Japan to assess its acceptance. It was found that 'Nikogori' has peroxyl radical scavenging activity. Moreover, addition of soy sauce to 'Nikogori' enhanced the peroxyl radical scavenging activity. The rheological properties of 'Nikogori' and of the soy sauce added conformed to the standard set by the Ministry of Health, Labour and Welfare of Japan for elderly people with swallowing problems. From the data of the antioxidative activity and the sensory evaluation, the soy sauce-added 'Nikogori' was preferrable to that of the non-added one.

Cholesterol depletion induces autophagy.

Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5mM methyl-beta-cyclodextrin or 10-20microg/ml nystatin for 1h, or metabolically by 20microM mevastatin and 200microM mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50microm in length. The increase of LC3-II by methyl-beta-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft.

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.

Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane.

Lipid droplets (LDs) are organelles that store neutral lipids, but their regulatory mechanism is not well understood. In the present study, we identified Rab18 as an LD component of HepG2 cells by proteomic analysis, and confirmed its localization by immunohistochemistry and western blotting. Wild-type and dominant-active Rab18 localized to LDs but the dominant-negative form did not. Endogenous Rab18 coexisted with adipocyte differentiation-related protein (ADRP) in LDs, but the labeling intensity of the two proteins showed clear reciprocity. Consistent with this observation, overexpression of Rab18 induced a decrease in the amounts of ADRP in LDs in HepG2 and BALB/c 3T3 cells. Furthermore, Rab18 overexpression caused close apposition of LDs to membrane cisternae connected to the rough ER. Two other procedures that decrease ADRP, i.e. RNA interference and brefeldin A treatment, induced the same morphological change, indicating that decrease in ADRP was the cause of the LD-ER apposition. In accordance with similar structures found between ER and other organelles, we propose that the ER membrane apposed to LDs should be named the LD-associated membrane, or LAM. The present results suggested that Rab18 regulates LAM formation, which is likely to be involved in mobilizing lipid esters stored in LDs.

19F and 1H MRI detection of amyloid beta plaques in vivo.

Formation of senile plaques composed of amyloid beta peptide, a pathological hallmark of Alzheimer disease, in human brains precedes disease onset by many years. Noninvasive detection of such plaques could be critical in presymptomatic diagnosis and could contribute to early preventive treatment strategies. Using amyloid precursor protein (APP) transgenic mice as a model of amyloid beta amyloidosis, we demonstrate here that an intravenously administered (19)F-containing amyloidophilic compound labels brain plaques and allows them to be visualized in living mice by magnetic resonance imaging (MRI) using (19)F and (1)H. Our findings provide a new direction for specific noninvasive amyloid imaging without the danger of exposure to radiation. This approach could be used in longitudinal studies in mouse models of Alzheimer disease to search for biomarkers associated with amyloid beta pathology as well as to track disease course after treatment with candidate medications.

Fluoro-substituted and 13C-labeled styrylbenzene derivatives for detecting brain amyloid plaques.

Two styrylbenzene derivatives, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB) and (E,E)-1-bromo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene-alpha,alpha'-(13)C(2) ([(13)C]BSB), were synthesized for use as a histochemical stain to detect amyloid plaques of Alzheimer's disease (AD) brain sections. An analysis of fluorescence spectra demonstrated that FSB shows approximately twofold fluorescence intensity relative to the conventional styrylbenzene derivative, (E,E)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB). Moreover, FSB was found to stain amyloid plaques and neurofibrillary tangles of AD brains with greater fluorescence intensity and a lower level of background signals compared to BSB. These finding indicate that FSB can be an excellent fluorescent compound to label human amyloid lesions with high sensitivity and specificity. Because of the possession of a nuclide with a quantized angular momentum, both FSB and [(13)C]BSB are also potential contrast agents for magnetic resonance imaging to locate AD pathologies in vivo.

Convergent total syntheses of gambierol and 16-epi-gambierol and their biological activities.

The convergent total syntheses of gambierol (1) and 16-epi-gambierol (2) have been achieved. The ABC and FGH ring segments 4 and 5 were prepared from known compounds 6 and 13, respectively, by linear manners. The fragments prepared were connected by our own synthetic strategy including the intramolecular allylation of alpha-acetoxy ether followed by ring-closing metathesis to furnish the octacyclic ether 3. The diiodoalkene 45, prepared from 3, was converted to the Z-iodoalkene 50 via a novel and stereoselective hydrogenolysis followed by deprotection. Construction of the triene side chain was performed by the modified Stille coupling of 50 with the Z-vinylic stannane 41 to afford 1. The similar transformations were carried out on the epimeric octacycle 34 to give 2, which showed no toxicity against mice at the concentration of 14 mg/kg.

Total synthesis of gambierol.

The convergent total synthesis of gambierol (1) is described. The octacyclic ether framework of 1 was constructed via the intramolecular allylation of alpha-chloroacetoxy ether followed by ring-closing metathesis. A modified Stille coupling was successfully applied to the synthesis of the triene side chain.

The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition.

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.

Concise synthesis of cyclic ethers via the palladium-catalyzed coupling of ketene acetal triflates and organozinc reagents. Application to the iterative synthesis of polycyclic ethers.

The reaction of the ketene acetal triflates 9a-e and a zinc homoenolate 10 in the presence of a catalytic amount of Pd(PPh(3))(4) gave the enol ethers 11a-e in good yields. The products were converted to the corresponding cyclic ethers 14a and 14b by hydroboration and lactonization. The present methodology allowed us to synthesize the DE and GH ring segment of gambierol in a concise manner. Iterative syntheses of the polycyclic ethers 26 and 32 are also described.