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. 2020 Feb 5;11(4):535–540. doi: 10.1021/acsmedchemlett.9b00658

Quantitative Proteomics Reveals Cellular Off-Targets of a DDR1 Inhibitor

Jiaqian Xu †,‡, Zhang Zhang †, Ligen Lin §, Hongyan Sun ‡, Lorenzo V White †, Ke Ding †,*, Zhengqiu Li †,*
PMCID: PMC7153277  PMID: 32292561

Abstract

graphic file with name ml9b00658_0005.jpg

Target identification of small molecules is a great challenge but an essential step in drug discovery. Here, a quantitative proteomics approach has been used to characterize the cellular targets of DR, a DDR1 inhibitor. By taking advantage of competitive affinity-based protein profiling coupled with bioimaging, Cathepsin D (CTSD) was found to be the principle off-target of DR in human cancer cells. Further findings suggest the potential of DR as a novel CTSD inhibitor for breast cancer treatment. In addition, a trans-cyclooctene (TCO) containing probe was developed to track the binding between DR and its target proteins in living systems and could be a useful tool for DDR1 detection.

Keywords: DDR1, bioimaging, chemoproteomics, target identification, photoaffinity labeling

Target-based drug discovery is the prevalent paradigm for delivery of therapeutic agents into the clinic,1 but widespread concerns exist regarding its safety and potential side effects due to off-target binding.2 As a result, comprehensive knowledge of the full target spectrum of lead compounds can provide a molecular basis with which to evaluate the possible side effects. It can guide subsequent structural optimization and may offer novel therapeutic applications.3 Over the past decades, affinity-based protein profiling (AfBP) coupled with bioimaging has been widely used in the investigation of genuine drug target engagement in native cellular environments.4−8 To facilitate the synthesis of high-quality probes, a suite of bioorthogonal-handle containing linkers were developed for reversible and irreversible inhibitors, respectively, which enable simultaneous proteome profiling and bioimaging studies to improve the accuracy in target identification.9−13

Discoidin domain receptors (DDRs), such as DDR1 and DDR2, are members of the transmembrane receptor tyrosine kinase (RTK) superfamily, which are involved in fundamental cellular processes including morphogenesis, differentiation, proliferation, adhesion, migration, and invasion.14,15 Increasing evidence suggests that the known functional abnormality of DDR1 is associated with a variety of inflammatory diseases, such as atherosclerosis, osteoarthritis, and organ fibrosis, and thus DDR1 is considered to be a novel drug target for treatment of inflammation. Recently, a tetrahydroisoquinoline-7-carboxamide derivative DR, capable of exhibiting potent inhibitory activity against DDR1 (IC50 = 38.3 nM), was reported by our group (Figure 1A).16,17 Further structural optimization yielded two representative compounds 2-2ar and 7ae, which tightly bind to the DDR1 protein with Kd values of 4.7 and 2.2 nM and demonstrated inhibition of DDR1 with IC50 values of 9.4 and 6.6 nM, respectively. In addition, these compounds were clearly less potent against most of the 403 wild-type kinases, showing excellent target specificity.16,17 However, these results were in vitro based, and their cellular targets other than the kinome remain obscure. In an attempt to identify the cellular on/off-targets of DR in situ, competitive affinity-based proteome profiling coupled with bioimaging was carried out, with the aim of guiding subsequent structural optimization and assessing potential side effects. In view of the fact that DDR1 is a novel drug target for which no imaging probe has been reported to date, a bioorthogonal probe containing a trans-cyclooctene (TCO) handle was developed for imaging DDR1 in a living system. This provides potential applications in diagnosis and therapy of DDR1-related diseases.

Figure 1.

Figure 1

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(A) Structures of parent inhibitor DR and probes DR-1 and DR-2 for proteome profiling; DR-3 is designed for bioimaging in live cells. (B) Co-crystal structure of DR with DDR1 (PDB code: 5FDP). (C) IC50 values of DR and probes DR-1/DR-2/DR-3 against DDR1.

We began our study with the chemical design and synthesis of photoreactive probes DR-1 and DR-2 and a bioorthogonal probe, DR-3, based on the cocrystal structure of DR and DDR1 (Figure 1B), which suggests that the modification of the piperazine group should not compromise the bioactivity. Thus, a minimalist photo-cross-linker L1/2 or a trans-cyclooctene (TCO) was incorporated into amenable sites of DR to afford the probes DR-1, DR-2, and DR-3, respectively (Figure 1A). The conjugation of different photo-cross-linkers L1/2 in DR-1 and DR-2 is for comparison in proteome profiling and bioimaging. The TCO-containing probe is designed for a rapid, copper-free, TCO-tetrazine ligation reaction, allowing DDR1 imaging in a living system. All probes were fully characterized before their use in subsequent biological evaluations.

With these probes in hand, we first evaluated the biological activities of these probes using standard in vitro DDR1 kinase inhibition assays with DR as a positive control. As shown in Figure 1C, all probes DR-1, DR-2, and DR-3 displayed moderate inhibitory activity against DDR1 with IC50 values of 258.6 nM, 576.6 nM, and 397.4 nM, respectively. They are less potent than the parent molecule DR, suggesting that the introduction of a minimalist photo-cross-linker and trans-cyclooctene (TCO) might have a slightly detrimental effect on the binding of the probe to target proteins. However, the probes were found to be suitable for proteome profiling and bioimaging studies in subsequent experiments.

Subsequently, proteome profiling and bioimaging experiments were carried out to assess their labeling performances both in situ and in vitro in complex proteome. The H1299 cell line was selected as a biological model which significantly overexpresses DDR1 kinase and has been used for DDR1 inhibitor development.16 After incubation of DR-1 and DR-2 with live cells or cell lysates for 1–3 h, the samples were then irradiated for 20 min with UV light at 365 nm to allow formation of stable linkages between the probes and target proteins. After conjugation with TAMRA-N3, the probe-labeled proteomes were then separated by SDS-PAGE and visualized by in-gel fluorescence scanning. As shown in Figure 2A, notable different labeling bands could be observed between the experiments from in vitro and in situ proteome profiling, indicating that the probes interact with different sets of targets in the two biological environments, while identical labeling bands were visible between probes DR-1 and DR-2 treated samples. Two obvious fluorescent bands under in situ environment at around 110 kDa and 33 kDa could be observed (* marked bands, Figure 2A). These bands became much weaker or disappeared entirely upon pretreatment with excess of the parent inhibitors (10 × DR), indicating that they were specific targets of DR. To determine whether the known target DDR1 was the labeling band of approximately 110 kDa, the probe-labeled proteomes in live cells were further clicked with biotin-N3 followed by affinity enrichment with streptavidin beads. The enriched samples were further validated by Western blotting with a DDR1 antibody (Figure 2B), which demonstrated that both probes DR-1 and DR-2 successfully label the known target DDR1 in the native cellular environment.

Figure 2.

Figure 2

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(A) Proteome reactivity profiles of live H1299 cells and cell lysates with DR-1/DR-2. (B) Validation of DDR1 by pull-down/Western blotting in live H1299 cells. (C) Confocal fluorescence imaging of H1299 cells with probes DR-1/DR-2 in the presence or absence of excess DR. Nu = nucleus, DIC = differential interference contrast. Scale bar = 10 μm.

To visualize the subcellular localization of probe labeled targets, bioimaging experiments were then carried out. After probe incubation, UV-cross-linking, fixation, and permeabilization, cells were conjugated with TAMRA-N3 and were then imaged. Strong fluorescence signals could be observed mainly in the cytoplasm and cell membrane in the probe-treated samples, while a sharp decrease in the presence of excessive parent inhibitors was observed and no fluorescence signal was detected in negative control samples (Figures 2C and S1). Given that DDR1 has been considered to be a transmembrane protein, the difference in locations between DDR1 and results of bioimaging could be attributed to the existence of off-targets. Collectively, the results of competitive protein profiling and bioimaging prove that the probes are capable of efficiently capturing the intended cellular targets of DR in situ.

Since both probes produce identical labeling bands and DR-1 shows higher inhibition activity, large-scale pull-down/LC-MS/MS experiments were carried out with DR-1 to identify the potential cellular targets of DR. Stable isotope labeling by amino acids (SILAC) labeled H1299 cells were treated with DR-1 for 2–4 h, and this was followed by UV-irradiation. The probe-labeled proteomes were then clicked with biotin-N3, enriched and analyzed by LC-MS/MS upon digestion. Control experiments with excessive parent inhibitors or DMSO were carried out concurrently to distinguish genuine targets from nonspecific labeling. Aiming to minimize false positives, the proteomics experiments were carried out in biological triplicates and the identified protein hits were further refined using SILAC ratios. The obtained protein hits were analyzed by corresponding volcano plots as the log2- of the competitive ratio (DR-1/DR-1 + DR) against the statistical significance (−log10p-value). Protein hits with both log2(-competitive ratio) and −log10p-value higher than 1.0 were considered to be significant hits. Based on these criteria, 3 proteins hits, CTSD (Cathepsin D), GPR107 (Protein GPR107), and COMT (Catechol O-methyltransferase), met this requirement (Figure 3A). These protein hits were also detected in enrichment experiments (DR-1/DMSO), indicating their high reliability (Figure 3B). The DDR1 was not shown in the protein list, which could be accounted for by low solubility of DDR1, a membrane-located protein, in lysis buffer.3 The top hit, Cathepsin D (CTSD), plays crucial roles in metabolic degradation of intracellular proteins and is also involved in antigen processor activation and regulation of programmed cell death. GPR107 was proposed as a receptor for neuronostatin and may play an important role in the central control of cardiovascular function.18 It was reported that catechol-O-methyltransferase (COMT) is a key enzyme for inactivation and metabolism of the catecholamine neurotransmitters and catechol hormones, which might be a promising therapy target for parkinson’s disease treatment.19 These protein hits could be valuable clues for the understanding of the mechanism of drug action and potential side effects.

Figure 3.

Figure 3

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Volcano plots of enriched proteins in SILAC experiments with DR-1 in the presence of parent inhibitor DR (A) or DMSO (B). Red dots depict selected targets that were both significantly enriched in two experiments. (C) Target validation of CTSD by pull-down/Western blotting in the presence or absence of excess DR. (D) Thermal shift binding assay to evaluate the binding of DR with CTSD in live H1299 cells, the top protein hit identified by DR-1. (E) Inhibition of DR against CTSD. (F) Docking experiments to predict the binding mode of DR with CTSD. (G) Live H1299 cells were pretreated with lysosome-neutralizing agent NH4Cl followed by CTSD validation with pull-down/Western blotting. (H) IC50 values of DR against several breast cancer lines: MCF-7, MDA-MB-231, and SK-BR-3.

Human Cathepsin D (CTSD) is translated as a precursor proenzyme which, after cleavage of an N-terminal peptide, produces an inactive pro-enzyme, Pro CTSD. Pro CTSD is then converted to a single-chain intermediate and finally matures into a heavy chain and light chain noncovalently bonded complex. The molecular weights of pro-, single chain, heavy chain, and light chain CTSD are ∼53, 48, 33, and 14 kDa, respectively.20 Pull-down/Western blotting experiments with an anti-CTSD antibody were further carried out (Figure 3C), which proved that DR-1 primarily labels the heavy chain of mature CTSD and coincides well with the labeling band at ∼30 kDa visualized by in-gel fluorescence scanning (Figure 2A, asterisked bands). It was found that mature CTSD is mainly located in the lysosome, which is also consistent with the imaging results (Figure 2C). In addition, cellular thermal-shift assay (CETSA) was performed to compare the thermal stability of mature CTSD heavy chain between DR- and DMSO-treated live H1299 cells. CTSD was stabilized upon DR treatment (Figure 3D), indicating that DR physically engages in binding with CTSD under in situ environment. In vitro based enzymatic assay demonstrated that DR can display moderate inhibition toward CTSD protein (Figure 3E). Docking experiments were then carried out to evaluate the interaction of DR with CTSD at the atomic level. As shown in Figure 3F, DR could form two hydrogen bond interactions with Ser235 and Thr234 in the CTSD heavy chain, and these contribute greatly to the relatively low binding free energy predicted by AutoDock vina. Given that DR is a basic molecule, we tested whether acid lysosomal accumulation might be the reason for the in situ reactivity of DR against CTSD. After pretreatment of H1299 cells with lysosomal neutralizing agents, 10 mM ammonium chloride for 30 min, pull-down/Western blotting with DR-1 was carried out. As shown in Figure 3G, consistent with previous studies,20,21 labeling of CTSD with DR-1 was blocked by pretreatment with lysosomal neutralizing agents, indicating that DR can accumulate in the lysosome and interact with CTSD. Previous studies have shown that CTSD is a well established therapeutic target for breast cancer treatment, but no drug with very few scaffolds has yet been approved for general use.22,23 Accordingly, we further evaluated the potential therapeutic utility of DR in breast cancer. As shown in Figure 3H, DR exhibited micromolar antiproliferative IC50 values against MCF-7, MDA-MB-231, and SK-BR-3 breast cancer cell lines, which significantly overexpress CTSD. This provides a novel molecular scaffold for development of CTSD inhibitors.

Considering that DDR1 is a novel target for the treatment of inflammation related diseases and to the best of our knowledge, no imaging probe has been reported to date, we further evaluated the imaging performances with the TCO-containing probe, DR-3. After incubation of live H1299 cells with DR-3 for 4 h, followed by fixation, permeabilization, and conjugation with tetrazine-TAMRA, the cells were washed with fresh medium and then analyzed by microscopy. As shown in Figure 4, the fluorescence signals generated from probe-treated cells were to some extent colocalized with the fluorescence signals from DDR1. More fluorescence could be observed in the cytosol and could be accounted for by the off-target binding. Little fluorescence was detected in the negative control samples. Collectively, these imaging results suggest that the probe DR-3 can be used for the detection of DDR1 and drug uptake or distribution.

Figure 4.

Figure 4

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Cellular imaging of H1299 cells with DR-3. Immunofluorescence (IF) staining using anti-DDR1 antibody.

In summary, two photoaffinity probes based on a potent DDR1 kinase inhibitor have been developed. These probes can be used to perform proteome profiling and bioimaging studies with cancer cells. CTSD was successfully identified by chemoproteomics as a cellular off-target of DR and further validated by Western blotting and CETSA assays. Further findings suggest potential therapeutic applications of DR as a lead compound in treatment of breast cancer by targeting CTSD, facilitating the use of AfBP in an acceleration of drug repurposing.24 The bioorthogonal probe targeting DDR1 could be a useful tool in diagnosis and therapy of DDR1-related diseases.

Acknowledgments

Funding was provided by National Key R&D Program of China (2019YFC1711000), National Natural Science Foundation of China (21877050), Science and Technology Program of Guangdong Province (2017A050506028, 2019B151502025), Science and Technology Program of Guangzhou (201704030060, 201805010007). We thank Prof. Shao Q. Yao (NUS, Singapore) for the invaluable suggestions on this work.

Glossary

Abbreviations

AfBP

affinity-based protein profiling

DDR

ciscoidin domain receptors

RTKs

receptor tyrosine kinases

TCO

trans-cycloocetene

IC50

the half maximal (50%) inhibitory concentration of a substance

CTSD

Cathepsin D

CETSA

cellular thermal shift assay

SILAC

stable isotope labeling by amino acids

TAMRA

carboxytetramethylrhodamine

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00658.

  • Synthetic procedures and compound characterization, biological methods, and 1H NMR and 13C NMR spectra of probes (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml9b00658_si_001.pdf (1.3MB, pdf)

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Supplementary Materials

ml9b00658_si_001.pdf (1.3MB, pdf)

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