Experimental Methods Used for Identifying Small-Molecule Inhibitors of Protein-Protein Interaction

Zhou, Mi; Li, Qing; Kong, Wenna; Wang, Renxiao

doi:10.1007/978-981-13-0773-7_5

Cited by 2 publications

(2 citation statements)

References 177 publications

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“…Fortunately, A29 did not clog the open capillary, thus waiting and flushing A29 out was sufficient to resolve the problem. For the reference measurements with TSA, a melting temperature cut-off of Δ T m > 1.5 °C was selected, which corresponds to the 5-fold sum of the mean and standard deviation of the negative control A24 . Likewise, the SPR cut-off was set to the 3-fold of the highest observed mean normalized signal of A24 .…”

Section: Results and Discussionmentioning

confidence: 99%

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

et al. 2021

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Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

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Section: Results and Discussionmentioning

confidence: 99%

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

et al. 2021

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“…At an in vitro level (with the exception of Campesterol), these phytochemical compounds have been reported to stabilize and accumulate p53 in the nuclei of different cancer cell lines, possibly through the disruption of mortalin-p53 complexes ( Wang et al, 2014 ; Nigam et al, 2015 ; Bhargava et al, 2018 ; Garg et al, 2019 ; Pham et al, 2019 ). However, common biochemical methodologies, for instance, affinity chromatography approaches (pull-down and co-immunoprecipitation assays) and fluorescence or bioluminescence resonance energy transfer approaches (FRET or BRET), have not been implemented to mechanistically confirm the potentiality of these molecules as mortalin-p53 interaction inhibitors ( Zhou et al, 2018 ). Additionally, based on in silico analyses that were further validated by in vitro co-immunoprecipitation assays, Caffeic Acid Phenethyl Ester (CAPE) ( Wadhwa et al, 2016 ; Sari et al, 2020 ) and Withanone ( Grover et al, 2012 ) have been previously reported as naturally occurring molecules with the capability to abrogate mortalin-p53’s interaction.…”

Section: Compounds That Abrogate the Interaction Between Mortalin And...mentioning

confidence: 99%

Abrogating the Interaction Between p53 and Mortalin (Grp75/HSPA9/mtHsp70) for Cancer Therapy: The Story so far

Elwakeel

2022

Front. Cell Dev. Biol.

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p53 is a transcription factor that activates the expression of a set of genes that serve as a critical barrier to oncogenesis. Inactivation of p53 is the most common characteristic in sporadic human cancers. Mortalin is a differentially sub-cellularly localized member of the heat shock protein 70 family of chaperones that has essential mitochondrial and extra-mitochondrial functions. Elevated mortalin levels in multiple cancerous tissues and tumor-derived cell lines emphasized its key role in oncogenesis. One of mortalin’s major oncogenic roles is the inactivation of p53. Mortalin binds to p53 sequestering it in the cytoplasm. Hence, p53 cannot freely shuttle to the nucleus to perform its tumor suppressor functions as a transcription factor. This protein-protein interaction was reported to be cancer-specific, hence, a selective druggable target for a rationalistic cancer therapeutic strategy. In this review article, the chronological identification of mortalin-p53 interactions is summarized, the challenges and general strategies for targeting protein-protein interactions are briefly discussed, and information about compounds that have been reported to abrogate mortalin-p53 interaction is provided. Finally, the reasons why the disruption of this druggable interaction has not yet been applied clinically are discussed.

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Experimental Methods Used for Identifying Small-Molecule Inhibitors of Protein-Protein Interaction

Cited by 2 publications

References 177 publications

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

Abrogating the Interaction Between p53 and Mortalin (Grp75/HSPA9/mtHsp70) for Cancer Therapy: The Story so far

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