Rapid Identification of a Protein Binding Partner for the Marine Natural Product Kahalalide F by Using Reverse Chemical Proteomics

Piggott, Andrew M.; Karuso, Peter

doi:10.1002/cbic.200700608

Cited by 41 publications

(25 citation statements)

References 48 publications

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“…Further studies revealed hNopp140 as molecular target of doxorubicin,169 and Bcl‐2, NSC‐1, and NFX1 as targets of paclitaxel (Table 1, entry 53),170 Ca 2+ /Calmodulin as target of the curcumin derivative HBC (Table 1, entry 54),171 UQCRB in mitochondrial complex III as target protein for terpestacin (Table 1, entry 55)172 and TACC3 as novel target for bisphenol A (Table 1, entry 56) 173. Piggott and Caruso could identify the human ribosomal protein S25 as the molecular target for the anticancer drug kahalalide F (Table 1, entry 57) using three different T7 phage‐displayed human disease cDNA libraries 174. By fingerprinting the selected clones with the frequent base cutter Hin f1 many clones could be screened very quickly, thereby facilitating the analysis of a far greater number of phages without the need for sequencing.…”

Section: Approaches To Target Identificationmentioning

confidence: 99%

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

et al. 2013

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Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins.

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Section: Approaches To Target Identificationmentioning

confidence: 99%

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

et al. 2013

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“…Protein-To explore the molecular mechanisms underlying inhibition of angiogenesis by terpestacin, phage display biopanning, an approach that has proven to be a powerful means to identify cellular binding proteins of bioactive small molecules, was applied to identify terpestacin binding proteins (15,19). Terpestacin was biotinylated at either the C-24 (BT1) or C-17 (BT2) position hydroxyl group to produce an affinity ligand for the isolation of its binding proteins (Fig.…”

Section: Identification Of Human Uqcrb As a Terpestacin-bindingmentioning

confidence: 99%

Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing

Jung

Shim

Lee

et al. 2010

Journal of Biological Chemistry

110

102

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Cellular oxygen sensing is required for hypoxia-inducible factor-1␣ stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.Progression of many solid tumors requires angiogenesis (1). Mitochondrial function has been linked to angiogenesis, because mitochondria are the primary sites of oxygen consumption, and angiogenesis is an oxygen concentration-sensitive process (2). Reports suggest that reactive oxygen species (ROS) 3 generation at mitochondrial Complex III is necessary and sufficient to trigger HIF-1␣ stabilization during hypoxia, and cells lacking mitochondrial DNA and electron transport activity ( cells) fail to exhibit increased ROS or up-regulation of HIF-1␣ target genes during hypoxia (3-5). Inhibitors of Complex III such as myxothiazol and stigmatellin also block mitochondrial ROS generation and inhibit the stabilization and transcriptional activity of HIF-1␣ during hypoxia. These findings suggest that the generation of ROS from mitochondrial Complex III is a critical event in the signaling of cellular hypoxia (6, 7). However, details regarding which of the components of Complex III contribute to this signaling remain to be described.Biological screening tools are useful for identifying naturally occurring small molecules capable of inducing a specific phenotype change (8, 9). We performed a large scale screen of microbial extracts in an attempt to identify small molecules that could inhibit the angiogenic response to pro-angiogenic stimuli, such as hypoxia, in endothelial cells. We identified terpestacin as a small molecule with unique bicyclo sesterterpene structure capable of inhibiting the angiogenic response at concentrations below the toxic threshold (10). Terpestacin strongly inhibits the functional response to hypoxia of human umbilical vein endothelial cells in vitro and angiogenesis within the embryonic chick chorioallantoic membrane in vivo. In addition to this anti-angiogenic activity, terpestacin has previously been reported to inhibit syncytium formation during human immunodeficiency virus infection and has been chemically synthesized (11-13). However, neither the molecular target of this compound nor the cellular mechanism of its anti-angiogenic activity has been identified.In the present study we identified the binding protein of terpestacin and...

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“…It shows antitumor activities [346] both on transformed cell lines and on tumor specimens derived from a variety of solid human tumors [347,348]. Although the molecular bases of the tumoricidal activity of Kahalalide F are not fully established, the permeabilization of the plasma membrane leading to lyses [351], alterations in lysosome morphology [352,353], and inhibition of the ErbB3 signaling pathways [354][355][356] are involved in the mechanisms of this activity. In June 2009, PharmaMar licensed kahalalide F to Medimetriks Pharmaceuticals for uses other than oncology and neurology [350].…”

Section: Dolabella Auriculariamentioning

confidence: 99%

Plant and Marine Sources

Amedei

Niccolai

2014

Natural Products Analysis

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Rapid Identification of a Protein Binding Partner for the Marine Natural Product Kahalalide F by Using Reverse Chemical Proteomics

Cited by 41 publications

References 48 publications

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack

Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing

Plant and Marine Sources

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