Enhanced Suppression of a Protein-Protein Interaction in Cells Using Small-Molecule Covalent Inhibitors Based on an N-Acyl-N-alkyl Sulfonamide Warhead.

Protein-protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.

Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors.

Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.

Total Synthesis of (+)-Goniodenin.

Goniodenin is a lipophilic polyketide originating from plant sources and which possesses a potent cytotoxic activity against cancer cell lines. The first total synthesis of (+)-goniodenin has been achieved in 23 steps from (R)-glycidol. The synthetic sequence featured a cross metathesis for the formation of the C8-C9 bond and installation of the terminal γ-butenolactone ring unit by the alkylation of α-phenylthio-γ-butyrolactone with the corresponding C3-O-triflate. The stereogenic center at C18 carbon was created by Hiyama-Fujita reduction of the corresponding ketone with high diastereoselectivity.

Integrated cross-coupling strategy for an α-carboline-based Aurora B kinase inhibitor.

An efficient and practical synthetic process for an α-carboline-based Aurora B kinase inhibitor was achieved using an integrated Pd-catalyzed cross-coupling strategy. The process features a mild and efficient method for construction of the α-carboline core by employing a Pd-catalyzed sequence of Buchwald-Hartwig amination and intramolecular direct C-H arylation at the ortho position of an unsubstituted aniline moiety, which is a key functionality for further derivatization with a Suzuki coupling via Sandmeyer iodination. The process has eliminated expensive starting materials and column chromatography purifications and enabled considerable enhancement of the total yield from 11% to 48%.

A case of adrenal metastasis of hepatocellular carcinoma diagnosed by endoscopic ultrasound-guided fine-needle aspiration.

An 82-year-old man had been treated for lung adenocarcinoma and hepatocellular carcinoma (HCC). Contrast-enhanced computed tomography examination showed swelling of the left adrenal gland, suggesting metastasis of lung adenocarcinoma, HCC, or primary adrenal tumor. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) was performed for the pathological diagnosis, and adrenal metastasis of HCC was diagnosed. No notable complications due to EUS-FNA were found. There have been reports of adrenal metastasis due to various cancers, but there are few reports that can confirm the diagnosis of adrenal metastasis of HCC using EUS-FNA. Adrenal metastasis of HCC is not a rare condition, but it may be difficult to diagnose in the case of multiple cancer complications. We experienced a case in which EUS-FNA was useful for the diagnosis of adrenal metastasis of HCC.

Neurofibromatosis Type 1 with a Giant Diffuse Plexiform Neurofibroma Invading the Liver.

Plexiform neurofibromas (PNs) occur in approximately 50% of patients with neurofibromatosis type 1 (NF1). PNs are rare in the abdominal cavity and especially rare in hepatobiliary lesions. A 31-year-old man with NF1 had a tumor extending along the celiac artery, superior mesenteric artery, and intrahepatic portal vein. We diagnosed him with diffuse PN based on liver tumor biopsy findings and the tumor form. Because the tumor had invaded along the intrahepatic portal vein, surgical resection was deemed difficult, and the patient was followed up with imaging studies. The patient remained asymptomatic without tumor growth.

In Situ Construction of Protein-Based Semisynthetic Biosensors.

Chemically constructed biosensors consisting of a protein scaffold and an artificial small molecule have recently been recognized as attractive analytical tools for the specific detection and real-time monitoring of various biological substances or events in cells. Conventionally, such semisynthetic biosensors have been prepared in test tubes and then introduced into cells using invasive methods. With the impressive advances seen in bioorthogonal protein conjugation methodologies, however, it is now becoming feasible to directly construct semisynthetic biosensors in living cells, providing unprecedented tools for life-science research. We discuss here recent efforts regarding the in situ construction of protein-based semisynthetic biosensors and highlight their uses in the visualization and quantification of biomolecules and events in multimolecular and crowded cellular systems.

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide.

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M-1 s-1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Flonicamid, a novel insecticide with a rapid inhibitory effect on aphid feeding.

Flonicamid (IKI220; N-cyanomethyl-4-trifluoromethylnicotinamide), a pyridinecarboxamide compound, is a novel systemic insecticide with selective activity against hemipterous pests, such as aphids and whiteflies, and thysanopterous pests. The purpose of this study is to clarify the biological properties of flonicamid against aphids. Flonicamid is very active against aphids, regardless of differences in species, stages and morphs. This compound inhibited the feeding behaviour of aphids within 0.5 h of treatment without noticeable poisoning symptoms such as convulsion, and this antifeeding activity was not recoverable until death. The nymphs born from adults exposed to flonicamid for 3 h showed high mortality. The effect of flonicamid on the feeding activity of an individual aphid was studied using electronic monitoring of insect feeding behaviour (EMIF). Although the treated aphid attached the head of its proboscis to the leaf surface, salivation and sap feeding were strongly inhibited. These results suggest that the main insecticidal mechanism of flonicamid is starvation based on the inhibition of stylet penetration to plant tissues.

Construction of Iterative Tetrahydrofuran Ring Units and Total Synthesis of (+)-Goniocin.

Cytotoxic acetogenin (+)-goniocin has been synthesized in 17 steps from (R)-O-tritylglycidol. The core structure of the contiguous C22-C10 threo-trans-threo-trans-threo-trans-tris-tetrahydrofuran (THF) ring involving an iterative THF-ring unit was synthesized. An iterative THF ring unit was constructed from an alkenyl-substituted THF ring in four steps including a Pd(II)-catalyzed ring-closing reaction and cross-metathesis. This method is general and allows the preparation of both trans-threo-trans- and trans-threo-cis-THF ring units flexibly.

Synthesis of planar-chiral paracyclophanes via samarium(II)-catalyzed intramolecular pinacol coupling.

[reaction: see text] A series of [n]paracyclophanediols (n = 8-12) was synthesized by samarium-catalyzed pinacol coupling for their ansa-bridge formation. Enantiomerically pure [n]paracyclophane esters were derived from the diols in a several steps via chiral resolution (for n = 10) or via crystallization-induced asymmetric transformation (for n = 11) by using amino alcohol auxiliaries and their selective cleavages.

Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma.

Suppression of apoptosis is important for carcinogenesis and tumor growth. Recent studies revealed that survivin not only inhibited apoptosis but also accelerated cancer cell proliferative activity. To investigate the prognostic role of expression of the antiapoptosis gene, survivin, in hepatocellular carcinoma (HCC), the authors analyzed the correlation between the expression pattern of survivin messenger RNA (mRNA) and clinicopathologic findings of patients. Tissues were obtained by surgical resection of livers from 51 patients with HCC and 6 patients without HCC. Expression of survivin mRNA was evaluated using reverse transcription-polymerase chain reaction in 51 tumors, 51 adjacent histologically noncancerous livers, and 6 normal livers. Survivin protein expression was evaluated using Western blotting, and apoptotic cancer cells were detected by immunostaining with polyclonal rabbit anti-single-stranded DNA. Survivin mRNA expression was detected in 21 of 51 (41%) tumors, 2 of 51 (4%) noncancerous livers, and none of the 6 normal livers. Survivin mRNA expression did not correlate with tumor size or stage of HCC. Percentage of apoptotic cancer cells of 30 survivin mRNA-negative tumors (5.2 +/- 3.4%) was significantly higher than that of 21 survivin mRNA-positive tumors (2.2 +/- 2.3%, P = 0.0019). The disease-free 5-year survival rate of 21 patients positive for survivin mRNA (19%) was significantly poorer than that of 30 patients negative for survivin mRNA (39%, P = 0.0148). Survivin mRNA was detected in 57% (17/30) patients with HCC recurrence but in only 19% (4/21) of patients without recurrence (P = 0.0072). These results indicated that survivin mRNA expression could be used as an independent prognostic factor for patients with HCC after hepatectomy.

Expression of the murine double minute gene 2 oncoprotein in esophageal squamous cell carcinoma as a novel marker for lack of response to chemoradiotreatment.

The protein product of the murine double minute gene 2 (MDM2) negatively controls the work of the p53 protein and the retinoblastoma protein (pRB). Recent studies have found that MDM2 expression correlates with chemoresistance of tumor cells. In the present study, the correlation between MDM2 expression and chemoradioresistance was evaluated in patients with esophageal squamous cell carcinoma (ESCC). The immunoreactivities of p53, pRB, and MDM2 were evaluated in 148 surgically resected ESCC by using monoclonal antibodies. The disease-free survival of 107 surviving patients who underwent curative surgery was compared among groups with positive and negative expressions of p53, pRB, and MDM2. High immunoreactivities of p53, pRB, and MDM2 were detected in 47.3%, 64.2%, and 32.4% of cases, respectively. In 107 patients undergoing curative surgery, pRB losses and MDM2 overexpression were found to be poor prognostic factors by univariate analysis. In multivariate analysis, only MDM2 expression was determined to be a prognostic factor independent of the tumor stage. Moreover, we found that MDM2 expression correlates with short survival of patients undergoing postoperative adjuvant chemoradiotherapy. In patients without adjuvant therapy, however, the MDM2 status did not correlate with patient survival. The present results indicate that MDM2 expression may be not only a prognostic marker for patients with ESCC, but also a novel marker for predicting a lack of response to chemoradiotreatment.

Palladium-catalyzed fluorocarbonylation using N-formylsaccharin as CO source: general access to carboxylic acid derivatives.

N-formylsaccharin, an easily accessible crystalline compound, has been employed as an efficient CO source in Pd-catalyzed fluorocarbonylation of aryl halides to afford the corresponding acyl fluorides in high yields. The reactions use a near-stoichiometric amount of the CO source (1.2 equiv) and tolerate diverse functional groups. The acyl fluorides obtained could be readily transformed into various carboxylic acid derivatives such as carboxylic acid, esters, thioesters, and amides in a one-pot procedure.

Trichlorophenyl formate: highly reactive and easily accessible crystalline CO surrogate for palladium-catalyzed carbonylation of aryl/alkenyl halides and triflates.

The high utility of 2,4,6-trichlorophenyl formate, a highly reactive and easily accessible crystalline CO surrogate, is demonstrated. The decarbonylation with NEt(3) to generate CO proceeded rapidly at rt, thereby allowing external-CO-free Pd-catalyzed carbonylation of aryl/alkenyl halides and triflates. The high reactivity of the CO surrogate enabled carbonylation at rt and significantly reduced the quantities of formate to near-stoichiometric levels. The obtained trichlorophenyl esters can be readily converted to a variety of carboxylic acid derivatives in high yields.

Palladium-catalyzed carbonylation of aryl, alkenyl, and allyl halides with phenyl formate.

Highly efficient palladium-catalyzed carbonylation of aryl, alkenyl, and allyl halides with phenyl formate is reported. This procedure does not use carbon monoxide and affords one-carbon-elongated carboxylic acid phenyl esters in excellent yields. The reaction proceeds smoothly under mild conditions and tolerates a wide range of functional groups including aldehyde, ether, ketone, ester, and cyano groups. Furthermore, a variety of heteroaromatic bromides can be converted to the corresponding phenyl esters in high yields.

Remarkable improvement achieved by imidazole derivatives in ruthenium-catalyzed hydroesterification of alkenes using formates.

Imidazole derivatives are revealed to be effective ligands in the Ru-catalyzed hydroesterification of alkenes using formates, affording one-carbon-elongated esters in high yields. Further, intramolecular hydroesterification was successfully performed to give lactones for the first time. Imidazole derivatives can contribute to promote the reaction as well as to suppress the undesired decarbonylation of formate. Toxic CO gas, a directing group, and large excess alkenes are not required.

Ternary complex formation of Ino2p-Ino4p transcription factors and Apl2p adaptin beta subunit in yeast.

Yeast Ino2p-Ino4p heterodimeric complex is well known as a transcriptional activator for the genes regulated by inositol and choline, such as the INO1 gene. Apl2p is a large subunit of the yeast adaptin complex, an adaptor complex required for the clathrin coat to bind to the membrane. We found that Ino2p, Ino4p, and Apl2p form a ternary complex. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation. Ino2p and Ino4p bind to Apl2p in the same region of Apl2p, viz., at the middle part and the C-terminal part. Ino2p and Ino4p bind to Apl2p independently, but more strongly when both are present. Furthermore, a disruption of APL2 together with INO2 or INO4 rendered yeast cells sensitive to oxidative stress. INO2-APL2 double disruptants also showed growth inability in non-fermentable carbon sources, such as glycerol. These results indicate a genetic interaction between APL2, INO2 and INO4 and uncovere novel functions of the Ino2p-Ino4p-Apl2p complex in yeast.