A Proteomic Platform to Identify Off-Target Proteins Associated with Therapeutic Modalities that Induce Protein Degradation or Gene Silencing

Abstract Novel modalities such as Proteolysis Targeting Chimera (PROTAC) and RNA interference (RNAi) have the ability to inadvertently alter the abundance of endogenous proteins. Currently available in vitro secondary pharmacology assays, which evaluate off-target binding or activity of small molecules, do not fully assess the off-target effects of PROTAC and are not applicable to RNAi. To address this gap, we developed a proteomics-based platform to comprehensively evaluate abundance of off-target proteins. The first part of the study involves selecting a panel of off-target proteins and a platform of cell lines using evidence from genetics and pharmacology. This process yielded 2,813 proteins, forming the basis of a panel that we refer to as the “selected off-target proteome” (SOTP). An iterative algorithm was then used to identify appropriate cell lines. Four human cell lines out of 932 were selected that, collectively, expressed ~ 80% of the SOTP based on transcriptome data. Second, we used mass spectrometry to quantify the intracellular and extracellular proteins of interest in the 4 selected cell lines. Among over 10,000 quantifiable proteins identified, 1,828 were part of the predefined SOTP. The SOTP was designed to be easily modified or expanded, owing rationale selection process developed and the label free LC-MS/MS approach chosen. This versatility inherent to our platform is essential to design fit-for-purpose studies that can address the dynamic questions faced in investigative toxicology.

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A proteomic platform to identify off-target proteins associated with therapeutic modalities that induce protein degradation or gene silencing

Scientific Reports ◽

10.1038/s41598-021-95354-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xin Liu ◽

Ye Zhang ◽

Lucas D. Ward ◽

Qinghong Yan ◽

Tanggis Bohnuud ◽

...

Keyword(s):

Cell Lines ◽

Selection Process ◽

Extracellular Proteins ◽

Label Free ◽

Target Proteins ◽

Therapeutic Modalities ◽

Endogenous Proteins ◽

Or Gene ◽

Target Binding

AbstractNovel modalities such as PROTAC and RNAi have the ability to inadvertently alter the abundance of endogenous proteins. Currently available in vitro secondary pharmacology assays, which evaluate off-target binding or activity of small molecules, do not fully assess the off-target effects of PROTAC and are not applicable to RNAi. To address this gap, we developed a proteomics-based platform to comprehensively evaluate the abundance of off-target proteins. First, we selected off-target proteins using genetics and pharmacology evidence. This process yielded 2813 proteins, which we refer to as the “selected off-target proteome” (SOTP). An iterative algorithm was then used to identify four human cell lines out of 932. The 4 cell lines collectively expressed ~ 80% of the SOTP based on transcriptome data. Second, we used mass spectrometry to quantify the intracellular and extracellular proteins from the selected cell lines. Among over 10,000 quantifiable proteins identified, 1828 were part of the predefined SOTP. The SOTP was designed to be easily modified or expanded, owing to the rational selection process developed and the label free LC–MS/MS approach chosen. This versatility inherent to our platform is essential to design fit-for-purpose studies that can address the dynamic questions faced in investigative toxicology.

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A Proteomic Platform to Identify Off-Target Proteins Associated with Therapeutic Modalities that Induce Protein Degradation or Gene Silencing

10.1101/2020.11.18.389148 ◽

2020 ◽

Author(s):

Xin Liu ◽

Ye Zhang ◽

Lucas D. Ward ◽

Qinghong Yan ◽

Tanggis Bohnuud ◽

...

Keyword(s):

Cell Lines ◽

Selection Process ◽

Extracellular Proteins ◽

Label Free ◽

Target Proteins ◽

Therapeutic Modalities ◽

Fit For Purpose ◽

Or Gene ◽

Target Binding

ABSTRACTNovel modalities such as Proteolysis Targeting Chimera (PROTAC) and RNA interference (RNAi) have a mechanism of action-based potential to alter the abundance of off-target proteins. The current in vitro secondary pharmacology assays, which evaluate off-target binding or activity of small molecules, do not fully assess the off-target effects of PROTAC and are not applicable to RNAi. To address this gap, we developed a proteomics-based platform to comprehensively evaluated abundance of off-target proteins. The first part of the manuscript describes the rationale and process through which the off-target proteins and cell lines were selected. The off-target proteins were selected from the entire human proteome based on genetics and pharmacology data (Deaton et al., 2018). The selection yielded 2,813 proteins, forming the nexus of a panel that we refer to as the “selected off-target proteome” (SOTP). An algorithm was then used to identify appropriate cell lines. Four human cell lines out of 932 were selected that, collectively, expressed ~ 80% of the SOTP based on transcriptome data. The second part of the manuscript describes the LC-MS/MS experimentation to quantify the intracellular and extracellular proteins of interest in the 4 selected cell lines. Among over 10,000 quantifiable proteins identified, 1,828 were part of the predefined SOTP. The SOTP was designed to be easily modified or expanded, owing rationale selection process developed and the label free LC-MS/MS approach chosen. This versatility inherent to our platform is essential to design fit-for-purpose studies that can address the dynamic questions faced in investigative toxicology.

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Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines

Particle and Fibre Toxicology ◽

10.1186/1743-8977-10-50 ◽

2013 ◽

Vol 10 (1) ◽

pp. 50 ◽

Cited By ~ 65

Author(s):

Melissa A Vetten ◽

Nonhlanhla Tlotleng ◽

Delia Tanner Rascher ◽

Amanda Skepu ◽

Frankline K Keter ◽

...

Keyword(s):

Gold Nanoparticles ◽

Cell Lines ◽

In Vitro Toxicity ◽

Label Free

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An Aptamer-Functionalized Microfluidic Platform for Biomolecular Purification and Sensing

ASME 2009 7th International Conference on Nanochannels, Microchannels and Minichannels ◽

10.1115/icnmm2009-82142 ◽

2009 ◽

Author(s):

Thai Huu Nguyen ◽

Qiao Lin

Keyword(s):

Small Molecules ◽

Label Free ◽

Temperature Dependent ◽

Synthetic Process ◽

Microfluidic Platform ◽

Thermally Induced ◽

Complex Samples ◽

Target Binding ◽

External Stimuli

Aptamers are oligonucleotides (DNA or RNA) that bind to chemical and biological analyte targets via affinity interactions. Through an in vitro synthetic process, aptamers can be developed for an extremely broad spectrum of analytes, such as small molecules, proteins, cells, viruses, and bacteria. Target recognition by aptamers is highly selective, as affinity interactions result in secondary aptamer conformational structures that specifically fit the target. The aptamer-target binding is also reversible and depends strongly on external stimuli such as pH and temperature. The specificity and stimuli-responsiveness of aptamers are highly attractive to biological purification and sensing, which generally involve isolating minute quantities of targets from complex samples with non-specific molecules and impurities present at orders-of-magnitude higher concentrations. We present an aptamer-functionalized microfluidic platform that by design exploits the specificity and temperature-dependent reversibility of aptamers to enable biomolecular purification and sensing. Using the specificity of aptamers, we demonstrate highly selective capture and enrichment of biomolecules. Employing thermally induced, reversible disruption of aptamer-target binding, we accomplish isocratic elution of the captured analytes and regeneration of the aptamer surfaces, thereby eliminating the use of potentially harsh reagents. Using integrated microfluidic control, the eluted analytes are detected in a label-free fashion by mass spectrometric methods.

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Detecting Drug-Target Binding in Cells using Fluorescence Activated Cell Sorting Coupled with Mass Spectrometry Analysis

10.1101/121988 ◽

2017 ◽

Author(s):

Kris Wilson ◽

Scott P Webster ◽

John P Iredale ◽

Xiaozhong Zheng ◽

Natalie Z Homer ◽

...

Keyword(s):

Drug Target ◽

Drug Targets ◽

Mass Spectrometry Analysis ◽

Protein Targets ◽

Target Proteins ◽

Spectrometry Analysis ◽

Cellular Permeability ◽

Target Binding ◽

Hit Validation

AbstractThe assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity - the Toxicity-Affinity-Permeability-Selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

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Antisense Oligonucleotide in LNA-Gapmer Design Targeting TGFBR2—A Key Single Gene Target for Safe and Effective Inhibition of TGFβ Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms21061952 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1952

Author(s):

Sabrina Kuespert ◽

Rosmarie Heydn ◽

Sebastian Peters ◽

Eva Wirkert ◽

Anne-Louise Meyer ◽

...

Keyword(s):

Cell Lines ◽

Selection Process ◽

Single Gene ◽

Locked Nucleic Acid ◽

Drug Candidate ◽

Tgfβ Signaling ◽

Gene Target ◽

Neuronal Stem Cell

Antisense Oligonucleotides (ASOs) are an emerging drug class in gene modification. In our study we developed a safe, stable, and effective ASO drug candidate in locked nucleic acid (LNA)-gapmer design, targeting TGFβ receptor II (TGFBR2) mRNA. Discovery was performed as a process using state-of-the-art library development and screening. We intended to identify a drug candidate optimized for clinical development, therefore human specificity and gymnotic delivery were favored by design. A staggered process was implemented spanning in-silico-design, in-vitro transfection, and in-vitro gymnotic delivery of small batch syntheses. Primary in-vitro and in-vivo toxicity studies and modification of pre-lead candidates were also part of this selection process. The resulting lead compound NVP-13 unites human specificity and highest efficacy with lowest toxicity. We particularly focused at attenuation of TGFβ signaling, addressing both safety and efficacy. Hence, developing a treatment to potentially recondition numerous pathological processes mediated by elevated TGFβ signaling, we have chosen to create our data in human lung cell lines and human neuronal stem cell lines, each representative for prospective drug developments in pulmonary fibrosis and neurodegeneration. We show that TGFBR2 mRNA as a single gene target for NVP-13 responds well, and that it bears great potential to be safe and efficient in TGFβ signaling related disorders.

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