Synthesis of NG-061 and its analogs, and their biological evaluation as an enhancer of nerve growth factor

A novel potentiator of nerve growth factor (NGF), NG-061, which had been isolated from the fermentation broth of Penicillium minioluteum F-4627, was synthesized from methoxybenzoquinone and phenylacetylhydrazine in a single step. A series of acyl hydrazone derivatives were also synthesized and their potentiator activity of neurotrophic effect of NGF on neurite outgrowth was evaluated by assay with a rat pheochromocytoma cell line PC12.     

Illicium Sesquiterpenes: Divergent Synthetic Strategy and Neurotrophic Activity Studies

Majucin‐type sesquiterpenes from Illicium sp., such as jiadifenolide ( 2 ), jiadifenin ( 3 ), and (1R,10S)‐2‐oxo‐3,4‐dehydroxyneomajucin ( 4 , ODNM), possess a complex caged chemical architecture and remarkable neurotrophic activities. As such, they represent attractive small‐molecule leads against various neurodegenerative diseases. We present an efficient, enantioselective, and unified synthesis of 2 , 3 , and 4 and designed analogues that diverge from tetracyclic key intermediate 7 . The synthesis of 7 is highlighted by the use of an enantioselective Robinson annulation reaction (construction of the AB rings), a Pd‐mediated carbomethoxylation reaction (construction of the C ring), and a one‐pot oxidative reaction cascade (construction of the D ring). Evaluation of the neurotrophic activity of these compounds led to the identification of several highly potent small molecules that significantly enhanced the activity of nerve growth factor (NGF) in PC‐12 cells. Moreover, efforts to define the common pharmacophoric motif suggest that substitution at the C‐10 center significantly affects bioactivity, while the hemiketal moiety of 2 and 3 and the C‐1 substitution might not be critical to the neurotrophic activity.     

Discovery of a potent and highly β1 specific proteasome inhibitor from a focused library of urea-containing peptide vinyl sulfones and peptide epoxyketones

Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the proteasome. Compounds 13 and 15 are β5 selective and their potency exceeds that of syringolin A. In contrast, 5 may well be the most potent β1 selective compound active in living cells reported to date.     

Ubiquitin Associates with the N‐Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI‐MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF_1–14, which is a short model peptide encompassing the first 14 N‐terminal residues of NGF, binds the copper‐binding regions of Ub (K_D=8.6 10^?5 m ). Moreover, the peptide undergoes a random coil–polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF_1–14/Ub interactions. Further experiments performed with the full‐length NGF confirmed the existence of a copper(II)‐dependent association between Ub and NGF and indicated that the N‐terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full‐length protein.     

The total synthesis of a ganglioside Hp-s1 analogue possessing neuritogenic activity by chemoselective activation glycosylation

The total synthesis of ganglioside 2, an analogue of the ganglioside Hp-s1 (1) which displays neuritogenic activity toward the rat pheochromocytoma cell line PC-12 cell in the presence of nerve growth factor (NGF) with an effect (34.0%) greater than that of the mammalian ganglioside GM 1 (25.4%), was accomplished by applying a chemoselective-activation glycosylation strategy. Moreover, we also demonstrate that the synthesized ganglioside 2 exhibited neuritogenic activity toward the human neuroblastoma cell line SH-SY5Y without the presence of NGF.     

Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells**

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 ( 12 ) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.     

A Set of Activity‐Based Probes to Visualize Human (Immuno)proteasome Activities

Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, β1c, β2c, and β5c. Lymphoid tissues also express the immunoproteasome subunits β1i, β2i, and β5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity‐based probes that enables simultaneous gel‐based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for β1c, β2c, β5c, and β2i, to compare the active‐site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of β5i and β1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.     

Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases

BACKGROUND: The 26S proteasome contains six highly related ATPases of the AAA family. We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome. Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM. Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed. RESULTS: We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function. Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant. Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome. In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism. CONCLUSIONS: These results describe a useful technique for the selective inactivation of AAA proteins. In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation.     

The inorganic perspective of nerve growth factor: interactions of Cu2+ and Zn2+ with the N-terminus fragment of nerve growth factor encompassing the recognition domain of the TrkA receptor

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity.     

Selective small molecule peptidomimetic ligands of TrkC and TrkA receptors afford discrete or complete neurotrophic activities

We designed a minilibrary of 55 small molecule peptidomimetics based on beta-turns of the neurotrophin growth factor polypeptides neurotrophin-3 (NT-3) and nerve growth factor (NGF). Direct binding, binding competition, and biological screens identified agonistic ligands of the ectodomain of the neurotrophin receptors TrkC and TrkA. Agonism is intrinsic to the peptidomimetic ligand (in the absence of neurotrophins), and/or can also be detected as potentiation of neurotrophin action. Remarkably, some peptidomimetics afford both neurotrophic activities of cell survival and neuronal differentiation, while others afford discrete signals leading to either survival or differentiation. The high rate of hits identified suggests that focused minilibraries may be desirable for developing bioactive ligands of cell surface receptors. Small, selective, proteolytically stable ligands with defined biological activity may have therapeutic potential.     

Encapsulation of proteins in poly(l-lactide-co-caprolactone) fibers by emulsion electrospinning

This study was aimed at investigating emulsion electrospinning to prepare biodegradable fibrous mats with encapsulation of human-nerve growth factor (NGF). One of the best methods for fabricating a bio-functional tissue engineering scaffold is to load bioactive agent into the scaffold. In this work, the feasibility of incorporating NGF into poly(l-lactide-co-caprolactone) fibers by emulsion electrospinning has been studied. The release behavior of encapsulated bovine serum albumin (BSA) was investigated. The bioactivity of NGF released from fibrous mats was verified by testing the neurite outgrowth of rat pheochromocytoma cells (PC12). Furthermore, the process of fiber forming during emulsion electrospinning was discussed. The results demonstrate that emulsion electrospun fibers can successfully encapsulate proteins and release them in a sustained manner. The bioactivity of NGF released from emulsion electrospun fibers was confirmed by PC12 bioassays.     

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.     

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

Background: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate,     

p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.     

Glucokinase-activating sesquinlignans from the rhizomes of Acorus tatarinowii Schott

Three novel sesquinlignans, tatanans A (1), B (2), and C (3), have been isolated from the rhizomes of Acorus tatarinowii Schott. Their structures were established by spectroscopic techniques and single-crystal X-ray analysis. Tatanans A-C potently increase GK enzymatic activity with EC(1.5) values in the range of 0.16-1.85 μM. The potent GK activity and unique structural features of tatanans make them promising leads for therapeutic development of antihyperglycemic drugs.     

SPR imaging biosensor for the 20S proteasome: sensor development and application to measurement of proteasomes in human blood plasma

The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome’s catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng·mL-1 on average for control, and 42300 ng·mL-1 on average for leukemia patients).

Electrophoretic analysis of phosphorylation of the yeast 20S proteasome

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.     

Towards subunit-specific proteasome inhibitors: synthesis and evaluation of peptide alpha',beta'-epoxyketones

BACKGROUND: The proteasome is a large multicatalytic protease complex (700 kDa) involved in a number of highly regulated processes. It has three major catalytic activities: a chymotrypsin-like activity, a trypsin-like activity and a post-glutamyl peptide hydrolyzing (PGPH) activity. To be useful as molecular probes, which could help dissect the cellular functions of the proteasome, inhibitors should be specific for the proteasome, active in vivo and selectively block only one of the three catalytic activities. To date, few inhibitors fulfill these requirements so we set out to make novel proteasome inhibitors that incorporate these characteristics. RESULTS: A panel of amino-terminally acetylated peptide alpha',beta'-epoxyketones with leucine in P1 and various aliphatic or aromatic amino acids in P2-P4 were prepared and evaluated. Most compounds selectively inhibited the chymotrypsin-like activity, while only weakly inhibiting the trypsin-like and PGPH activities. After optimization, one inhibitor, Ac-hFLFL-epoxide, was found to be more potent and selective for the inhibition of the chymotrypsin-like activity than several previously described inhibitors. This inhibitor also exhibited strong in vivo anti-inflammatory activity. CONCLUSIONS: Optimization of amino-terminally acetylated peptide alpha',beta'-epoxyketones furnished a potent proteasome inhibitor, Ac-hFLFL-epoxide, that has an excellent selectivity for the chymotrypsin-like activity. The inhibitor also proved to be a potent antiproliferative and anti-inflammatory agent. The strong in vivo and in vitro activities suggest that this class of proteasome inhibitors could be both molecular probes and therapeutic agents.     

The ubiquitin proteasome system in Huntington's disease and the spinocerebellar ataxias

Huntington's disease and several of the spinocerebellar ataxias are caused by the abnormal expansion of a CAG repeat within the coding region of the disease gene. This results in the production of a mutant protein with an abnormally expanded polyglutamine tract. Although these disorders have a clear monogenic cause, each polyglutamine expansion mutation is likely to cause the dysfunction of many pathways and processes within the cell. It has been proposed that the ubiquitin proteasome system is impaired in polyglutamine expansion disorders and that this contributes to pathology. However, this is controversial with some groups demonstrating decreased proteasome activity in polyglutamine expansion disorders, some showing no change in activity and others demonstrating an increase in proteasome activity. It remains unknown whether the ubiquitin proteasome system is a feasible therapeutic target in these disorders. Here we review the conflicting results obtained from different assays performed in a variety of different systems. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).