The cellular thermal shift assay for evaluating drug target interactions in cells

Jafari, Rozbeh; Almqvist, Helena; Axelsson, Hanna; Ignatushchenko, Marina; Lundbäck, Thomas; Nordlund, P.; Molina, Daniel Martinez

doi:10.1038/nprot.2014.138

Cited by 1,002 publications

(1,039 citation statements)

References 30 publications

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“…To further confirm our conclusion, we carried out a thermal shift assay (Molina et al 2013;Jafari et al 2014) for the p{dper(WT)} and p{dper(OP1)} extracts. This assay quantifies changes in thermal denaturation and aggregation temperature of a protein as a result of treatment by increasing temperatures, and such changes indicate structural changes of a protein.…”

Section: Codon Optimization Results In Altered Per Structurementioning

confidence: 68%

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

Murphy

Zhou

et al. 2016

Genes Dev.

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Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding.

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Section: Codon Optimization Results In Altered Per Structurementioning

confidence: 68%

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

Murphy

Zhou

et al. 2016

Genes Dev.

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“…In this study, we used Sypro Orange thermal shift on purified protein. With the recent development of cellular thermal shift assay, 29,30 it is certainly possible, using the principles discussed in this work, to apply cellular thermal shift assay on cells to screen for triclosan-resistant ENR/ FabI mutants of species requiring enoyl-ACP-linked substrate. This method may also be used to select effective triclosan analogues that, together with NAD(P) + , bind to the active site of ENR/FabI to inhibit the enzymatic activity, and thus inhibit cell growth.…”

Section: Discussionmentioning

confidence: 99%

An Efficient and Economical Assay to Screen for Triclosan Binding to FabI

et al. 2016

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Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosanresistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP + to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

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“…The supernatants were analyzed by Western blot, and the density of AlkB protein bands was plotted using GraphPad Prism 5.0 TM . All performances were repeated in triplicate (39,40).…”

Section: Methodsmentioning

confidence: 99%

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

Huang

Liu

et al. 2016

Journal of Biological Chemistry

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The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)-and 2-oxoglutarate-dependent dioxygenases that efficiently repair N 1 -methyladenine and N 3 -methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N 1 -methyladenine and N 3 -methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo. In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.The nucleic acids in living cells are subject to modification by both endogenous and environmental agents (1). Direct-acting chemicals constantly damage nucleic acids and generate various methyl lesions with mutagenic and/or cytotoxic consequences (2, 3). O 6 -Methylguanine (O 6 mG) 2 and N 3 -methyladenine lesions have the highest potential for methylating damage by an SN1 agent such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), which block replication and are thought to be toxic (4, 5). For the most part, the SN2 agent such as methyl methane sulfonate (MMS) produces N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) lesions in single-stranded DNA (ssDNA). Accumulation of these adducts can lead to cell death (6, 7). Organisms have evolved several mechanisms to efficiently remove various methyl lesions, including suicidal methyltransferases, DNA glycosylases, and the AlkB family dioxygenases (see Fig. 1A) (8, 9). To date, AlkB repair appears to be the major natural defense mechanism with the power to restore the canonical base structure in vivo. Escherichia coli AlkB and its human homologues, ALKBH2 and ALKBH3, utilize iron(II) and 2-oxoglutarate (2OG) to achieve oxidative demethylation of m 1 A and m 3 C (see Fig. 1B) (10 -12). The lack of AlkB repair results in increased sensitivity to MMS, elevated level of mutations, and reduced cell proliferation (13-16). Furthermore, the accumulation of m 1 A and m 3 C lesions could also occur on RNA. Research suggests that the oxidative demethylation in mRNA and tRNA acts as a part of AlkB or ALKBH3 repair to protect cells against MMS (17, 18). The scope of substrates for AlkB repair has been largely extended to all simple N-alkyl lesions at the WatsonCrick base-pairing interface on the four bases (19), thus indicating the importance of oxidative demethylation for cell survival. In addition, human enzymes have been broadly linked to cancer. The housekeeping ...

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The cellular thermal shift assay for evaluating drug target interactions in cells

Cited by 1,002 publications

References 30 publications

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

An Efficient and Economical Assay to Screen for Triclosan Binding to FabI

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

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