Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of ...context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.
Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water ...soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90α. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2‘-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2‘-halide is rotated approximately 180° away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2‘-halo derivatives of the 3‘,4‘,5‘-trimethoxy series where Cl > Br > I compared to the 4‘,5‘-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.
The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a ...transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.
Molecular chaperone heat shock protein 90 (Hsp90) is an important target in cancer and neurodegenerative diseases, and has rapidly become the focus of several drug discovery efforts. Among small ...molecule Hsp90 inhibitors with clinical applicability are derivatives of 8-arylmethyl-9H-purin-6-amine class. Here we report the use of microwave-assisted chemistry for the successful one-pot delivery of 8-arylmethyl-9H-purin-6-amines. We discuss the applicability as well as the limitations of this method towards the creation of a large chemical diversity in the 8-arylmethyl-9H-purin-6-amine series.
To elucidate the mechanism and origins of chemo- and enantioselectivities of the reaction between aliphatic aldehydes and hydrazones catalyzed by triazolium-derived NHC, density functional theory ...computations have been performed. According to our calculated results, the whole catalytic cycle for the formation of dihydropyridazinones proceeds via the initial nucleophilic addition of NHC to an aliphatic aldehyde, followed by the concerted intramolecular proton transfer and C–Cl bond cleavage. Subsequent deprotonation generates an enolate intermediate. The enolate intermediate then undergoes 1,4-addition to hydrazone to construct a new carbon–carbon bond. The following ring-closure would lead to a six-membered ring intermediate, which, upon the release of NHC, affords the final product dihydropyridazinone. The computation results reveal that intramolecular proton transfer is significantly promoted by the Brønsted acid DIPEA·H+. The carbon–carbon bond formation step could determine not only the chemoselectivity but also the stereoselectivity and lead to the S-isomer product. It was found that the stereoselectivity arises from a combination of weak interactions, including C–H···O, C–H···N, C–H···π, and LP···π. NHC could enhance the nucleophilicity of the aliphatic aldehyde and facilitate further reaction with hydrazone. This work could be beneficial for the development of new catalytic strategies in the future.
An approach toward visual detection and chemical utilization of NO2/N2O4 is proposed, which employs simple calix4arenes. Exposure of tetra-O-alkylated calix4arenes 1 and 2, possessing either a cone ...or a 1,3-alternate conformation, to NO2/N2O4, both in chloroform solution and in the solid state, results in deeply colored calixarene−nitrosonium (NO+) complexes. In the presence of a Lewis acid, such as SnCl4, stable calixarene-NO+ complexes 7 and 8 were isolated in a quantitative yield and characterized by UV−vis, FTIR, high-resolution 1H NMR spectroscopy and elemental analysis. NO+ is found encapsulated within the calixarene cavity, and stable charge-transfer complexes result with K ass > 106 M-1 (CDCl3). The NO+ encapsulation was also demonstrated in titration experiments with calixarenes 1, 2, and 5 and commercially available NO+SbF6 - salt in chloroform. The complexation process is reversible, and the complexes dissociate upon addition of water and alcohol, recovering the parent calixarenes. Attachment of functionalized calix4arenes to silica gel was demonstrated, which afforded a solid material 15 capable of visual detection and entrapment of NO2/N2O4. Calixarene-NO+ complexes can be utilized for the NO+ transfer processes and nitrosation reactions. The NO+ guest transfer between two calixarene containers 2 and 5 was achieved and studied by UV−vis and 1H NMR spectroscopy. Chemical fixation of NO2/N2O4 was demonstrated through their quantitative transformation into the calixarene-NO+ complex and its use as a nitrosonium transfer agent in the synthesis of N-nitrosoamides. These results may lead toward novel nitrogen oxides storing materials.
Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the ...stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.
The 70
kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of ...proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.
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