Label-Free Proteome Profiling as a Quantitative Target Identification Technique for Bioactive Small Molecules

Kyung Tae Hong†,‡ and Jun-Seok Lee*,†,‡
†Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Hwarangro 14 gil 5, Seunbguk-gu, Seoul 02792, Republic of Korea
‡Bio-Med Program, KIST-School UST, Hwarangro 14 gil 5, Seungbuk-gu, Seoul 02792, Republic of Korea

Chemical probes are unique tools for investigating the function of proteins or a new mode of action of a medicine. Many bioactive chemical probes were discovered from either a diversity-oriented chemical library screening or derivatization of natural compounds. Inspired by Paul Ehrlich’s magic bullet concept, the reductionist approach has been dominated in the chemical biology field, and researchers sought a target that was involved in the mechanism of action causing the phenotype. In conventional chemical genetics research, identifying the target protein of the bioactive
which demonstrate the robustness of this method. More importantly, TPP results also revealed the new potential endogenous target of staurosporine, other than kinases, such as coproporphyrinogen III oxidase and ferrochelatase. Though it was not clear about the mechanism of thermal perturbations at this moment, this clue would be apparently beneficial to further investigate the off-target effects of staurosporine. Another interesting finding from the TPP experiment was reported more recently from Helleday’s group. They discovered MTH1 enzyme sanitize oxidized dNTP precursors
compound is the most challenging task based on a
generated by dysfunctional
redox regulation, thus avoidingphenotype-based approach. Several strategies have been developed to unveil the target protein of small molecules, including immunoprecipitation (IP) using the affinity matrix, chemical labeling method using the electrophilic tag, and in situ photo-cross-linking method.1

However, a majority of conven- tional strategies require chemical modification of the hit compounds, which require tedious SAR studies to load on to the solid resin for IP or direct covalent labeling to the target. With the advances of high-resolution mass spectrometry techniques and in-depth understanding of protein stability, label-free target identification methods have emerged recently.2 Most label-free target deconvolution methods rely on the changes in protein stability upon compound binding events. The distinctions are made based on how each technique induces protein stability disruption using various perturbations,such as thermal, limited proteolysis, or oxidation stress.

Thermal proteome profiling (TPP) is a measurement of an intact protein state under thermal perturbation.3 Under thermal stress, proteins generally unfold and spontaneously expose their hydrophobic domains that induce aggregation. As unfolded protein aggregates are easily removed by precip- itations, unfolded proteins are recovered and quantitatively monitored. Initially, thermal profiling has been applied to individual purified protein conditions, and then the principle was exploited extensively into the mixture conditions in lysate and even live cell samples. Compared to the conventional target identification methods, the cellular thermal shift assay (CETSA) has excellent merits in that the assay is working in live cell or tissue without any chemical modification process since the major challenge of drug efficacy monitoring is that drug binding cannot be measured in a live cell environment.
In 2014, Savitski and co-workers reported a comprehensive
incorporation of a false oxidized nucleotide into the DNA that otherwise yields lethal DNA mutation and cell death. To evaluate MTH1 as a general target for cancer treatment, they searched inhibitors from the compound library and discovered 2-aminopyrimidine scaffold compounds, TH287 and TH588. Next, they further optimized TH588 into TH1579 (Kar- onudib) to enhance potency and oral delivery, followed by unbiased target validation using TPP. A total of 6405 complete melting curve data were obtained, and TH1579 successfully targeted the MTH1 with the most prominent thermal shift of almost 12 °C. Interestingly, they could also find 9 additional proteins that showed a consistent melting temperature shift, and two of them (deoxycytidine kinase, monoglyceride lipase) exhibited a notably distinct shift pattern from the cell extract and intact cell sample. Discovery of these new off-targets gave us a better understanding of the safety aspect of drug candidates, and it is the significant merit of TPP over conventional labeling-based methods. Besides, this result showed that the target engagement of bioactive compounds could significantly differ depending on the intact cellular environment (Figure 1).

The drug affinity responsive target stability (DARTS) is a measurement of ligand binding stabilization induced proteol- ysis resistance of the target. A low concentration of a protease (e.g., Pronase, thermolysin) is used to cleave exposed regions of proteins, and drug targets are relatively protected from the proteolysis. Though this technique inherently limits the assay condition only in cell extract conditions, it does not require modification of drug/bioactive compounds, and the experi- ment is relatively straightforward. Since DARTS was first introduced in 2009, various research groups have actively exploited this technique to unveil target proteins of bioactivecomparison between TPP and conventional “kinobead” profiling results of staurosporine in K562 cells. TPP comprised an almost comparable number of kinases (175) compared to the traditional method (229) even in live conditions, and 49 of them showed a reproducible thermal shift of greater than 1 °C,
Special Issue: Future of Biochemistry: The Asia-Pacific Issue
Received: October 31, 2019

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Image© XXXX American Chemical Society A DOI: 10.1021/acs.biochem.9b00975

Representative label-free proteome profiling for target identification.molecules, including syrosingopine (antihypertensive drug), MSL (autophagy enhancer), V-9302 (antagonist of a glutamine transporter), and MCC950 (inhibitor for NLRP3 pathway).4

The stability of proteins from rates of oxidation (SPROX) is a measurement of thermodynamic stability change under the exposure of chemical denaturants (e.g., urea) by oxidation of the unfolded or exposed domain containing methionine residues. Pioneered by the Fitzgerald group in 2008, SPROX provides a structural folding state quantitatively based on the mass intensity of oxidized or nonoxidized methionine. Thus, it is possible to monitor the drug binding effect on the domain level. On the other hand, the SPROX method applies only in the cell extract sample. Recently, SPROX has been applied to unveil the distinction between the targets of tamoxifen and N- desmethyl tamoxifen.5
As we have observed from the recent literature, more and more chemical biology research has adopted label-free profiling methods for the target deconvolution. Though there are still unique merits of a conventional cross-linking strategy for particular aims such as imaging or intracellular translocation tracking for the target, comprehensive phenotypic analysis requires proteome-wide functional scrutiny, and unbiased label-free profiling methods are valuable in this regard. How would label-free profiling be advanced in the future? We foresee that advances in the dynamic range of a mass spectrometer and more choices of the multiplicity of the isobaric tag will significantly enrich the contents of label-free profiling results. In such situation, we expect greatly diverse applications of label-free profiling methods will be applied in chemical biology research soon.

⦁ AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
ORCID
ImageJun-Seok Lee: 0000-0003-3641-1728
Notes

■The authors declare no competing financial interest.

ACKNOWLEDGMENTS

■This work is supported by the Bio & Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT & Future Planning (2018M3A9H4079286).

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