A Selective Chemical Probe for Coenzyme A-Requiring Enzymes

Department of Chemistry, University of Illinois, Urbana-Champaign, Urbana, Illinois, United States
Angewandte Chemie International Edition (Impact Factor: 11.26). 10/2007; 46(40):7621-4. DOI: 10.1002/anie.200702485
Source: PubMed


Transferasen markiert: Eine auf dem Coenzym A (CoA) basierende Affinitätssonde mit einer Sulfoxycarbamat-Funktion erkennt eine Reihe von Acetyltransferasen selektiv gegenüber anderen Enzymen und Proteinen. Es verbleibt eine Desthiobiotin-Markierung, die für Western-Blots und die massenspektrometrische Charakterisierung genutzt werden kann (siehe Bild; Nu=Nucleophil).

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    • "Based on enzyme kinetics a ping-pong mechanism was proposed [23]. However, it was demonstrated that an electrophilic acetyl-CoA affinity labellingbased probe did not target a residue that is critical for catalysis, arguing against a ping-pong mechanism [24], but a ternary complex mechanism could not be confirmed either. Despite the fact that p300 contains a cysteine in the binding pocket no mutagenesis studies have yet been performed to investigate whether this cysteine is important for HAT activity. "
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    ABSTRACT: Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT.
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    • "As all cysteines of HSP90 are located within its middle domain, small molecules that react with those cysteines may represent a new class of inhibitors distinct from those that target either the N-terminal ATP-binding pocket or the C-terminal domain of the chaperone. It was previously shown that cysteine residues of coenzyme A (CoA)-utilising enzymes can be selectively targeted by a mildly electrophilic sulphoxythiocarbamate-CoA analogue with the formation of stable thiocarbamate adducts (Hwang et al, 2007). "
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    ABSTRACT: Background: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. Methods: Combining chemical synthesis, biological evaluation, and structure–activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. Results: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. Conclusion: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.
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    ABSTRACT: An erratum for this article appears in the January, 2004 issue of Movement Disorders (Mov Disord 2004;19:119). Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). The loss of these neurons is associated with a glial response composed mainly of activated microglial cells and, to a lesser extent, of reactive astrocytes. This glial response may be the source of trophic factors and can protect against reactive oxygen species and glutamate. Alternatively, this glial response can also mediate a variety of deleterious events related to the production of pro-oxidant reactive species, and pro-inflammatory prostaglandin and cytokines. We discuss the potential protective and deleterious effects of glial cells in the SNpc of PD and examine how those factors may contribute to the pathogenesis of this disease.
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