Chemical genetics strategies for identification of molecular targets

Botubol-Ares, José Manuel; Durán‐Peña, María Jesús; Hernández-Galán, Rosario; Collado, Isidro González

doi:10.1007/s11101-013-9312-6

Cited by 8 publications

(8 citation statements)

References 133 publications

(198 reference statements)

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“…However, when the target is identified, it may lead to major breakthroughs in cell biology (Pucheault, 2008 ). Gratefully, current technologies render this task increasingly easier (Ares et al, 2013 ).…”

Section: Rapalogues: Temsirolimus® and Everolimus®mentioning

confidence: 99%

Cancer wars: natural products strike back

et al. 2014

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Natural products have historically been a mainstay source of anticancer drugs, but in the 90's they fell out of favor in pharmaceutical companies with the emergence of targeted therapies, which rely on antibodies or small synthetic molecules identified by high throughput screening. Although targeted therapies greatly improved the treatment of a few cancers, the benefit has remained disappointing for many solid tumors, which revitalized the interest in natural products. With the approval of rapamycin in 2007, 12 novel natural product derivatives have been brought to market. The present review describes the discovery and development of these new anticancer drugs and highlights the peculiarities of natural product and new trends in this exciting field of drug discovery.

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Section: Rapalogues: Temsirolimus® and Everolimus®mentioning

confidence: 99%

Cancer wars: natural products strike back

et al. 2014

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“…Current methods for target identification of small molecules are described by various recent reviews (Ares et al, 2013;Ziegler et al, 2013). Futamura compared the advantages and disadvantages of the methods (Futamura et al, 2013).…”

Section: In Vitro Target Identification Approachesmentioning

confidence: 99%

Target Identification and Polypharmacology of Nutraceuticals

Dormán¹,

Flachner²,

Hajdú³

et al. 2016

Nutraceuticals

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“…Whilst cytotoxicity is often perceived as an end-point, addressing the phenotypic response can be carried out through two processes, namely reverse chemical genetics or forward chemical genetics screening, respectively. 38,39,40,41 Developments made in the fields of biochemistry and molecular biology makes screening a library of compounds against specifically selected proteins a readily accessible method for identifying new bioactive small molecules. This is known as reverse chemical genetics or target-based screening.…”

Section: Target Identification and Mechanism Of Actionmentioning

confidence: 99%

“…When a small bioactive molecule is identified, it is then introduced into a cell or organism in attempts to study its mechanism of action. 38 However, although reverse chemical genetics may result in the identification of a highly specific bioactive small molecule, targeting a specific protein may prevent identification of potential off-target effects that can arise in more complex systems as well as the activation of multiple signalling pathways. 42 Additionally, targeting a specific protein involved in the pathophysiology of disease can ultimately impede the progress of drug development and discovery as it does not lead to the identification of new targets.…”

Section: Target Identification and Mechanism Of Actionmentioning

confidence: 99%

“…Affinity purification to identify bioactive small molecules and their targets has proved to be the most direct method for target identification in which whole cell extracts are passed through the affinity matrices and a protein target are isolated. 44 For example, the identification of calcineurin as a common target for both immunosuppressant's FK506 and cyclosporin A 39,38,45 was achieved by subjecting a whole cell lysate to a FK506 and cyclospoirn A affi-gel 10 affinity matrix. Affinity purification is considered to be the oldest, yet most commonly used method for target identification.…”

Section: Affinity-based Identificationmentioning

confidence: 99%

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Identifying the Mechanism of Action of Bioactive 1,2-Cyclopropyl Carbohydrates

Lassueur¹

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<p>Cyclopropanes and carbohydrates have long been used in the field of drug development. Previous work has shown that 1,2-cyclopropyl carbohydrates display bioactivity in both HeLa cancer cell lines¹ and in yeast² with a tentatively proposed mechanism of inhibition occurring through an enzymatic cyclopropane ring opening reaction and subsequent formation of a covalent bond with a target enzyme.² A small library of 1,2-cyclopropyl carbohydrate derivatives were synthesised based on known pharmacophores to examine further the potential mechanism of inhibition of such compounds and confirm the occurrence of enzyme-catalysed cyclopropane ring-opening reactions. Initial synthetic efforts were focused on the synthesis of the 1,2-dichlorocyclopropyl carbohydrate 23, which, through the optimisation of an essential C-6 detritylation reaction, was achieved in moderate yields of 32% over 7 steps. Following this, the ethoxycarbonyl substituted 1,2-cyclopropyl carbohydrate 54 was synthesised over 7 steps in a 22% yield through a rhodium acetate-catalysed addition of ethyl diazoacetate (49) to the glucal substrate 40. It was envisioned that if enzymatic cyclopropane ring-opening was occurring to form a C-7 carbanion, this would in turn be stabilised through the potential enolate formation of 54. Use of N,N-ditosylhydrazine in the synthesis of propargyl diazoacetate (58) followed by a rhodium acetate-catalysed cyclopropanation of 58 with substrate 40 resulted in the successful synthesis of 61 over 7 steps in a total yield of 9%. The incorporation of the propargyl substituent in 61 was introduced as a molecular probe in an attempt to isolate the target protein through an affinity purification procedure. The bioactivity of the propargyl derivative 61 was consistent with the synthesised compounds 23 and 54. It was proposed that these compounds undergo an enzymatic cyclopropane ring opening reaction accompanied with a clear diastereoselective preference for the α-stereoisomer of the cyclopropane ring, consistent with a target-based activation of the compounds. Chemical genetic analysis of the resulting bioactive compounds was undertaken using a deletion mutant array of Saccharomyces cerevisiae to elucidate a potential mechanism of action. Analysis of the results showed that, of the 4800 homozygous deletion strains tested in the high-throughput screens, a total of 122 strains were found following validation to sensitise and 68 to give resistance against 23 and 54. These sensitive and resistant mutants were subjected to a validation assay. Following validation, genes whose deletion led to sensitivity or resistance were then subjected to gene ontology term enrichment analysis which showed enrichment in the cytosolic ribosome, SNARE complex and SNAP receptor activity for resistant strains and enrichment in endoplasmic reticulum and endomembrane systems was found for the sensitive strain. Genes whose deletion sensitised to both compounds showed strong enrichment in cellular protein localisation, intra-golgi vesicale-mediated transport and the endomembrane system. Target identification and isolation were attempted through an affinity purification procedure using compound 61 and an azide-modified agarose resin. However, this was without success, either through inaccessibility of the alkyne of the target probe or because the target resides in the membrane-associated fraction which was discarded prior to treatment with the probe. This study suggests that the 1,2-cyclopropyl carbohydrates synthesised function through a cyclopropane ring-opening reaction, assisted by an enzymatic nucleophile. Chemical genetic analysis showed that the target of these compounds is involved in protein transport and protein localisation most likely relating to the vesicle tethering. Although many aspects of this work still need further investigation, either through the synthesis of new 1,2-cyclopropyl carbohydrates to increase bioactivity and better understand the enzymatic target, or through further biological procedures to better understand the mechanism of action, the use of 1,2-cyclopropyl carbohydrates as a potential pharmaceuticals or probes of protein trafficking shows much promise.</p>

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Chemical genetics strategies for identification of molecular targets

Cited by 8 publications

References 133 publications

Cancer wars: natural products strike back

Cancer wars: natural products strike back

Target Identification and Polypharmacology of Nutraceuticals

Identifying the Mechanism of Action of Bioactive 1,2-Cyclopropyl Carbohydrates

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