Chemical proteomics on ligand protein

2009 - 2010The emerging field of mass spectrometry-based chemical proteomics provides a powerful instrument in the target discovery of bioactive small-molecules, such as drugs or natural products[1]. The identification of their macromolecular targets is required for a comprehensive understanding of their bio-pharmacological role and for unraveling their mechanism of action[1, 2]. Indeed, the target discovery of bioactive molecules endowed with intriguing pharmacological profiles is one of the main issues in the field of pharmaceutical sciences, since this is necessary for a rational development of potential drugs. Nevertheless, several bioactive compounds have been mainly evaluated for their pharmacological effects, whereas the exact mechanism of action at molecular level still remains unknown[3, 4]. Moreover, a complementary point of view about the effect of a small bioactive molecule on a cellular system can be given by label-based quantitative proteomic analysis[5]. Indeed, the identification of biologically relevant changes in the expression of proteins in a cell, after a treatment with a bioactive compound, could help to understand the exact mechanism of action of such active compound. Here, we report the application of chemical proteomics to the analysis of the cellular interactome of three marine bioactive metabolites, all showing an intriguing anti-inflammatory pharmacological profile, and the application of quantitative chemical proteomics to the platelets activation mechanism by collagen. In more detail, the chemical proteomic approach was applied to Petrosaspongiolide M (PM)[6-8], Bolinaquinone (BLQ)[9-11] and Perthamide C (PRT)[12] target discovery. Thus, these molecules were immobilized onto agarose beads through an α,ω-diamino polyethylene glycol spacer. The modified beads were then used as baits for fishing the potential partners of the bioactive compounds in macrophages cell lysate. The application of such technique allowed us to identify 20S proteasome, clathrin and endoplasmin (GRP94) as main partners of PM, BLQ and PRT, respectively. Then, in vitro and in cell fluorescence assays were developed to assess the effect of PM onto the 20S proteasome enzymatic system, allowing us to measure the inhibition potency of this sesterterpene on the different proteolytic sites of the proteasome machinery. The BLQ ability to modulate clathrin mediated endocytosis has been assessed through cytofluorimetric and microscopy analysis, suggesting a new application of BLQ as biotechnological tool in the modulation of trafficking pathways. SPR technology has been employed to prove the ability of PRT to interact with GRP94 and Hsp90, opening the way to further investigations on the role of PRT in the modulation of heat shock protein functions. Finally, we report the application of quantitative chemical proteomics to discover the effect of collagen on platelet activation. Since cAMP and cGMP plays a key role in platelet activation[13], we combined quantitative chemical proteomics approach with the specific enrichment of cAMP/cGMP signaling nodes[14], to investigate how PKA but also cGMP-dependent protein kinases (PKG) spatially reorganizes in activated human platelets. [edited by author]IX n.s

Modulation of Tau Protein Fibrillization by Oleocanthal

Among the phenolic compounds extracted from extra virgin olive oil, oleocanthal (1) has attracted considerable attention in the modulation of many human diseases, such as inflammation and Alzheimer’s disease (AD). Indeed, 1 is capable of altering the fibrillization of tau protein, which is one of the key factors at the basis of neurodegenerative diseases, and of covalently reacting with lysine ε- amino groups of the tau fragment K18 in an unspecific fashion. In the present study, an investigation of the recognition process and the reaction profile between 1 and the wild-type tau protein has been conducted by a circular dichroism, surface plasmon resonance, fluorescence, and mass spectrometry combined approach. As a result, 1 has been found to interact with tau-441, inducing stable conformational modifications of the protein secondary structure and also interfering with tau aggregation. These findings provide experimental support for the potential reduced risk of AD and related neurodegenerative diseases associated with olive oil consumption and may offer a new chemical scaffold for the development of AD-modulating agents

Through-the-Annulus Threading of the Larger Calix[8]arene Macrocycle

A complete study of the through-the-annulus threading of the larger calix[8]arene macrocycle with di-n-alkylammonium cations has been performed in the presence of the “superweak” TFPB counterion. Thus, it was found that such threading occurs only upon partial preorganization of the calix[8]arene macroring by intramolecular bridging. In particular, 1,5-bridged calix[8]arenes with a meta- or para-xylylene bridge (2 and 3) gave pseudo[2]rotaxanes in which one dialkylammonium axle (4a−4e+) was threaded into one of the two subcavities of the calix[8]-wheel. Conformational studies by using chemical shift surface maps and DFT calculations evidenced a 3/4-cone geometry for these subcavities. Higher pseudorotaxane Kass values were obtained for calix[8]-wheels 2 and 3, with respect to calix[6]-host 1a, due to the cooperative effect of their two subcavities. Dynamic NMR studies on calix[8]-pseudorotaxanes evidenced a direct correlation between Kass (and ΔGass) values and energy barriers for calix inversion due to the effectiveness of thread templation. In accordance with DFT calculations, an endo-alkyl preference, over the endo-benzyl one, was observed by threading calix[8]-wheel 3 with the directional n-butylbenzylammonium axle 4d+

New insights on the interaction mechanism between tau protein and oleocanthal, an extra-virgin olive-oil bioactive component.

Oleocanthal (OLC) is a phenolic component of extra-virgin olive oil, recently supposed to be involved in the modulation of some human diseases, such as inflammation and Alzheimer. In particular, OLC has been shown to abrogate fibrillization of tau protein, one of the main causes of Alzheimer neurodegeneration. A recent interpretation of this mechanism has been attempted on the basis of OLC reactivity with the fibrillogenic tau hexapeptide VQIVYK and SDS-PAGE of OLC/tau incubation mixtures, suggesting that covalent modification events modulate tau fibrillization. In this paper we report a detailed mass spectrometric investigation of the OLC reactive profile with both tau protein fibrillogenic fragment K18 and propylamine in biomimetic conditions. We show that K18 is prone to be covalently modified by OLC through Schiff base formation between the 3-amino group of lysine residues and OLC aldehyde carbonyls. Moreover, as expected from its de-structured conformation, K18 shows a non-selective modification profile, reacting with several lysine residues to give cyclic pyridinium-like stable adducts. These data give new insights on the mechanism of inhibition of tau fibrillization mediated by OLC

Chemical Proteomics Reveals Bolinaquinone as a Clathrin-Mediated Endocytosis Inhibitor

Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Herein we report the mode of action of perthamide C, a natural cyclopeptide isolated from the marine sponge Theonella swinhoei. Through an emerging mass spectrometry-based chemical proteomics approach, Heat Shock Protein 90 and Glucose Regulated Protein 94 were identified as key targets of perthamide C and this evidence has been validated using surface plasmon resonance. The ability of perthamide C to influence heat shock protein-mediated cell apoptosis revealed that this marine metabolite could be a good candidate for the development of a lead compound with therapeutic applications based on apoptosis modulation