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. 2024 May 10;15(6):906–916. doi: 10.1021/acsmedchemlett.4c00104

Development of Biochemical and Cellular Probes to Study RIPK1 Target Engagement

Xin Yu †, Hanfeng Lin †, Feng Li ‡, Jin Wang †,§,*, Dong Lu †,*
PMCID: PMC11181498  PMID: 38894934

Abstract

graphic file with name ml4c00104_0009.jpg

RIPK1 inhibitors have emerged as promising candidates for treating diverse diseases, including inflammatory diseases, autoimmune disorders, Alzheimer’s disease, and cancer. However, the previously reported binding assays have limited sensitivity and stability, impeding high-throughput screening and robust characterization of the RIPK1 inhibitors. To address this challenge, we introduced two probes, T2-BDP-FL and T3-BDP-FL, derived from distinct RIPK1 inhibitors with different binding modes to establish time-resolved fluorescence resonance energy transfer (TR-FRET) displacement assays. Employing our TR-FRET displacement assays, we quantified the biochemical binding affinities of a series of RIPK1 inhibitors with diverse structural and binding modes for human RIPK1. Consistent results were obtained with these two probes in the TR-FRET displacement assay. Furthermore, we developed a RIPK1 fluorescent probe, T2-BDP589, for the NanoBRET assay. This assay enabled the characterization of RIPK1 target engagement by various RIPK1 inhibitors for both human and mouse RIPK1 in live cells. Our developed fluorescent probe displacement assays offer a sensitive and high-throughput approach to identify RIPK1 inhibitors based on both biochemical and cellular activities.

Keywords: RIPK1, Fluorescent probe, TR-FRET, NanoBRET, Target engagement

Receptor-interacting protein kinase 1 (RIPK1) stands as a pivotal master regulator governing programmed cell death, cell survival, and pro-inflammatory processes.1−5 Extensive investigations have illuminated the critical role of RIPK1 in the pathogenesis of diverse disorders, encompassing ischemia-reperfusion injury,6,7 neurodegenerative diseases,8−10 inflammatory ailments,6,11,12 infectious disease,13−15 and tumor metastasis.16−19

In recent years, a number of RIPK1 inhibitors have emerged, categorized into Type I, Type II, Type III, and other classes based on their binding modes (Figure 1).20−28 Encouragingly, a subset of these inhibitors is currently undergoing clinical evaluation for a spectrum of conditions, including inflammatory diseases,28 autoimmune disorders,29 Alzheimer’s disease,28 and cancer.30,31 This rapid advancement in RIPK1 inhibitor development holds immense promise for the therapeutic modulation of disease processes driven by RIPK1 dysregulation.

Figure 1.

Figure 1

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Structures of representative RIPK1 inhibitors.

To evaluate RIPK1 inhibitor activities, a range of in vitro assays have emerged, including cellular necroptosis assays,19,20,30,32 ADP-Glo kinase assays,20,23,30 the cellular thermal shift assay (CETSA),33 and fluorescence polarization (FP).22,23 Among these, both the cellular necroptosis assay and CETSA lack direct quantification of target engagement, failing to assess engagement under equilibrium conditions or provide quantitative data on target affinity or occupancy in live cells. The ADP-Glo kinase assay, while effective, necessitates substantial enzyme quantities for generating robust signals, which imposes constraints on the lower limit of potency resolution.34 Additionally, these methodologies are labor-intensive and are susceptible to notable false negatives and false positives,35 thereby constraining their utility within high-throughput screening settings. FP assays typically demand a high protein concentration to attain satisfactory signal-to-noise ratios.36,37 The potency resolution of FP assays is bounded by the binding dissociation constant (Kd) value of the tracer, thereby complicating the accurate determination of ligand potencies that bind more tightly to the receptor than the tracer.37 Moreover, FP assays are vulnerable to interference from autofluorescent substances or light scattering from precipitates, yielding potential false positive outcomes.38

In parallel with FP assays, the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay emerges as another homogeneous fluorescence-based method extensively harnessed for High-Throughput Screening (HTS).36,37,39−41 The TR-FRET assay operates through a distance-dependent transfer of excited-state energy from a donor fluorophore to an acceptor fluorophore, governed by dipole–dipole interactions, once the fluorophore pair with spectral overlap is brought into close proximity.37,39

Illustratively, as depicted in Figure 2, the targeted protein tagged with the 6xHis tag is noncovalently labeled with the Tb-anti-His antibody, serving as the donor fluorophore. Upon the binding of a binder-derived BODIPY probe to the protein, the resulting proximity of the two fluorophores triggers energy transfer from terbium (Tb) to the fluorescence acceptor, thereby emitting fluorescence at 520 nm from the acceptor BODIPY-FL. Conversely, when the BODIPY probe is displaced from the protein by an inhibitor, the distance between Tb and the unbound fluorescent probe in solution prevents energy transfer. Consequently, the binding affinity of the test inhibitor to the protein can be ascertained by measuring fluorescence intensities at 520 versus 495 nm. These homogeneous fluorescence-based assays provide a rapid and convenient means for quantifying the inhibitory activities of novel compounds.

Figure 2.

Figure 2

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Illustration of the mechanism of TR-FRET and Nano-BRET target engagement assay.

Specifically, the TR-FRET assay boasts several notable advantages over the FP assay. The time-resolved fluorescence measurement yields minimal background noise and a high signal-to-noise ratio.37 The luminescence lifetime of terbium (Tb) spans the millisecond range, distinctly longer than the nanosecond range of background fluorescence from sample components such as organic compounds, proteins, and cells.42 This substantial difference in emission lifetime permits a time delay between donor excitation and fluorescence measurement, effectively allowing interfering signals from short-lived emissions to decay before the measurement. Furthermore, the proximity-based approach involving a donor–acceptor pair contributes to a reduced incidence of false positives or negatives, often encountered in FP assays.43 Moreover, the TR-FRET assay operates effectively at low protein and tracer concentrations, enabling discrimination of inhibitors over a broader range of affinities, down to the picomolar inhibition constant (Ki) range.37,44

Concurrently, NanoBRET, a subtype of Bioluminescence Resonance Energy Transfer (BRET), provides a remarkably sensitive and quantitative means of assessing target engagement within living cells, encompassing both equilibrium and nonequilibrium conditions.45−47 In essence, this technique involves the assembly of reporter complexes within viable cells, where a nanoLuc-tagged target protein attains dynamic equilibrium with a cell-permeable fluorescent probe. Upon compound binding, a competitive displacement of the probe occurs, leading to an attenuated energy transfer within live cells (Figure 2).39,47

Here, we developed both TR-FRET and NanoBRET displacement assays to evaluate biochemical and intracellular RIPK1 target engagement, respectively. In the TR-FRET displacement assay, we designed and developed two probes derived from a Type II RIPK1 inhibitor and a Type III RIPK1 inhibitor. The NanoBRET displacement assay was established using a BRET probe, T2-BDP589. A series of RIPK1 inhibitors with distinct structural and binding models were tested for both human RIPK1 and mouse RIPK1 in both the TR-FRET and NanoBRET assays. Our established TR-FRET displacement assay and NanoBRET displacement assay were more sensitive than the previously reported FP assay and ADP-Glo assay, enabling the characterization of inhibitors with a picomolar potency range. Overall, these assays provide a sensitive and high-throughput approach to identify RIPK1 inhibitors based on both biochemical and cellular activities.

Design of TR-FRET and NanoBRET Probes

Type II and Type III inhibitors constitute the prevailing classes of RIPK1 inhibitors. In our endeavor to investigate the impact of the probe’s binding position for the TR-TRET assay, we intend to develop two TR-FRET probes derived from a Type II and Type III RIPK1 inhibitor, respectively. Compound 22b arises from a screening hit against RIPK1 and subsequent structural optimization (Figure 1).26 Computational modeling has confirmed the Type II binding mode of compound 22b, interacting with a DLG-out inactive form of RIPK1. The scaffold 1H-pyrazolo[3,4-d]pyrimidin-4-amine established two hydrogen bonds with residues Glu93 and Met95 in the hinge region, while the carbonyl of compound 22b formed a hydrogen bond with Asp156 (Figure 3a). Importantly, the ethyl group of the scaffold extended toward the solvent region, serving as an exit vector for linking with the BODIPY dye (Figure 3c). Guided by the elucidated cocrystal structure of the Type III RIPK1 inhibitor GSK547 bound to RIPK1 (Figure 3b),31 we devised a distinct probe. Capitalizing on a similar conceptual framework and leveraging a solvent-exposed vector, we extended linkers from the parent molecule GSK547 to the BODIPY dye, yielding the Type III probe denoted as T3-BDP-FL (Figure 3c).

Figure 3.

Figure 3

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Design of fluorescent probes derived from different types of RIPK1 inhibitors. (A) 2D view of the predicted binding mode of compound 22b in the binding pocket of human RIPK1 (PDB: 4NEU). (B) 2D view of the crystal structure of GSK547 bound to the kinase domain of RIPK1 (PDB: 6HHO). (C) Design of TR-FRET and NanoBRET probes derived from Type II and Type III inhibitors.

Chemistry

The route employed to synthesize Type II inhibitor-based probes is outlined in Scheme 1. Amide 1 was synthesized by condensation with 2-(3-(trifluoromethoxy)phenyl)acetic acid. Simultaneously, 5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine was reacted with tert-butyl(3-bromopropyl)carbamate to produce intermediate 2. Suzuki cross coupling reaction of intermediate 1 and 2 followed by deprotection to afford amine 3, which were transformed to the respective probes by condensation with respective BODIPY dyes.

Scheme 1. Synthesis of Type II Inhibitor-Based Probes.

Scheme 1

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Reagents and conditions: (a) 2-(3-(trifluoromethoxy)phenyl)acetic acid, HATU, TEA, DMF, 65 °C, 5 h, 80%; (b) tert-butyl(3-bromopropyl)carbamate, K2CO3, DMF, 50 °C, overnight, 72%; (c) (i) Pd(dppf)2Cl2·CH2Cl2, K2CO3, dioxane/H2O = 5/1, 90 °C, overnight; (ii) 3 N HCl in methanol, DCM, 0 °C to rt, 3 h, 36%; (d) BDP FL NHS ester or BDP 576/589 NHS ester, DIPEA, DMF, rt, 0.5 h, 50–70%.

Alternatively, the Suzuki cross coupling reaction of 4,6-dichloropyrimidine gave carboxylic ester 4. Intermediate 5 was prepared according to reported procedures in the literature.20 Nucleophilic substitution of 4 with 5 followed by hydrolysis gave carboxylic acid 6. Finally, carboxylic acid 6 was coupled with BDP FL NHS amine to afford target probe T3-BDP-FL (Scheme 2).

Scheme 2. Synthesis of Type III Inhibitor-Based Probe.

Scheme 2

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Reagents and conditions: (a) (4-(methoxycarbonyl)phenyl)boronic acid, Pd(dppf)2Cl2·CH2Cl2, K2CO3, 1,4-dioxane/H2O = 5/1, 90 °C, overnight, 81%; (b) (i) 4, DIPEA, ACN, 80 °C, overnight; (ii) LiOH·H2O, THF/H2O = 10/1, rt, overnight, 55%; (c) BDP FL NHS ester, DIPEA, DMF, rt, 0.5 h, 57%.

Optimization of RIPK1 Enzyme Concentration for the TR-FRET Assay

To obtain the optimal signal-to-noise ratio, a simultaneous titration of the RIPK1 enzyme and probe was performed. The probe was 1-to-5 serially diluted (from 0.32 to 5000 nM) in the presence of 0.33 nM Tb-anti-His, along with three different concentrations of RIPK1 enzyme groups (0.33, 1, and 3.3 nM). The group without the RIPK1 enzyme was selected as the background. After incubation for 4 h at room temperature, the TR-FRET signals were measured.

As shown in Figure 4a, the signal increased when the T2-BDP-FL concentration increased. In addition, the signal also increased when the concentration of human RIPK1 enzyme (hRIPK1) increased at the same concentration of T2-BDP-FL. The maximum signal-to-noise ratio for three different concentrations of hRIPK1 was 3.1, 8.9, and 19.5, respectively (Figure 4c). We also tested the signal-to-noise ratio of T3-BDP-FL for hRIPK1. T3-BDP-FL generally exhibited higher fluorescence intensities when bound to hRIPK1 in comparison with T2-BDP-FL (Figure 4b). The maximum signal-to-noise ratios for the three different concentrations of hRIPK1 were 4.2, 14.2, and 32.6, respectively (Figure 4d).

Figure 4.

Figure 4

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Determination of the RIPK1 enzyme concentration and incubation time in the TR-FRET assay. Dose–response effect of T2-BDP-FL (A) or T3-BDP-FL (B) in the presence of 0.33, 1.0, or 3.3 nM RIPK1 enzyme. Signal-to-noise ratio of T2-BDP-FL (C) or T3-BDP-FL (D) in the presence of 0.33, 1.0, or 3.3 nM RIPK1 enzyme. Dose–response curves of T2-BDP-FL (E) or T3-BDP-FL (F) for the hRIPK1 enzyme at the indicated incubation times. (G) DMSO tolerance of the T2-BDP-FL mediated TR-FRET assay.

To ensure a robust signal-to-noise ratio while minimizing protein use, 1 nM RIPK1 enzymes and 0.33 nM Tb-anti-His were selected for further assay development.

Optimization of Incubation Time for the TR-FRET Assay

To ascertain the equilibration time and assess assay stability, we monitored TR-FRET signals for both T2-BDP-FL and T3-BDP-FL at time points of 40, 80, 120, 160, 200, 240, and 280 min, maintaining a constant presence of 1 nM RIPK1 enzyme and 0.33 nM Tb-anti-His.

As illustrated in Figure 4e, the signal displayed an increase with escalating T2-BDP-FL concentrations, reaching a plateau phase after 1 μM. The binding dissociation constant (Kd) values were determined by fitting the data to a one-site total binding equation by using GraphPad PRISM software. T2-BDP-FL consistently exhibited high affinity to hRIPK1, with corresponding Kd values of 60, 38, 36, 33, 34, 33, and 32 nM at incubation time points of 40, 80, 120, 160, 200, 240, and 280 min, respectively. The affinity signals were stable from incubation times of 160 to 280 min.

In addition, T3-BDP-FL consistently exhibited higher affinity to hRIPK1, with corresponding Kd values of 51, 27, 18, 14, 11, 10, and 9 nM at incubation time points of 40, 80, 120, 160, 200, 240, and 280 min, respectively. The affinity signals were stable from incubation times of 160 to 280 min (Figure 4f).

To strike a balance between the time required for dispensing and equilibration and the desired large dynamic range, a 4 h incubation period was chosen as the optimal incubation time for our TR-FRET assay.

Evaluation of DMSO Tolerance in the TR-FRET Assay

DMSO is a commonly used solvent for preparing stock solutions of compounds in early drug discovery, including those used in this study. Therefore, we conducted a DMSO tolerance study to assess the impact of DMSO on our TR-FRET assay, which is crucial for determining an optimal DMSO concentration. The final assay mixture comprised assay components along with 1 μM T2-BDP-FL, encompassing varying concentrations (0.011%, 0.033%, 0.11%, 0.33%, 1.11%, 3.33%, and 10%) of DMSO. TR-FRET signals were recorded after a 240 min incubation period. Figure 4g illustrates that the TR-FRET signal remained stable when the DMSO concentrations were below 1.11%. However, an increase in DMSO concentration from 1.11% to 3.33% resulted in a modest signal reduction of 16.5%, which became more substantial at higher concentrations of 10% (39.2% signal reduction). Consequently, the selected DMSO concentration for our TR-FRET assay was below 1%.

Measurement of Binding Affinity of RIPK1 Inhibitors by Established TR-FRET Displacement Assay

In accordance with the Cheng–Prusoff equation,48 the concentrations of probes were deliberately chosen at their Kd values to facilitate the measurement of inhibition constant (Ki) values for RIPK1 inhibitors (Ki = IC50/2). Specifically, the Kd values for T2-BDP-FL and T3-BDP-FL were determined to be 33 and 9.7 nM for hRIPK1, respectively (Figure S1). Employing the optimized conditions for the RIPK1 TR-FRET displacement assay, comprising the Kd concentrations of probes, 1 nM RIPK1 enzymes, 0.33 nM Tb-anti-His, and a 4-hour incubation time, we assessed the Ki values of various reported RIPK1 inhibitors. These inhibitors were classified into Type I (Tozasertib), Type II (compound 22b, PK68), and Type III (Nec-1s, Dihydropyrazole 77, GSK772) based on the binding model.27,28

The binding affinities of the tested RIPK1 inhibitors in our established TR-FRET displacement assay, alongside previously reported activities from FP and ADP-Glo kinase assays, are listed in Figure 5 and Table 1. In our TR-FRET displacement assay using T2-BDP-FL, the Ki values for Tozasertib, compound 22b, PK68, Nec-1s, Dihydropyrazole 77, and GSK772 for hRIPK1 were 23, 0.82, 1.1, 200, 0.86, and 2.4 nM, respectively (Figure 5a, Table 1). Similar results were obtained using T3-BDP-FL, which were 35, 0.93, 0.83, 220, 0.72, and 1.9 nM, respectively (Figure 5b, Table 1). Additionally, the binding affinity of ATP for human RIPK1 was evaluated to be 1.7 and 1.6 mM, respectively, in our TR-FRET displacement assays using T2-BDP-FL and T3-BDP-FL (Figure S2).

Figure 5.

Figure 5

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Dose–response curves of six RIPK1 inhibitors in the TR-FRET displacement assay. Dose–response curves of Type I (Tozasertib), Type II (compound 22b, PK68), and Type III (Nec-1s, Dihydropyrazole 77, GSK772) in the T2-BDP-FL (A) or T3-BDP-FL (B) mediated TR-FRET displacement assay for hRIPK1. (C) Correlation of Ki values obtained from the TR-FRET displacement assay using T2-BDP-FL and T3-BDP-FL.

Table 1. Summary of Binding Affinities of RIPK1 Inhibitors in TR-FRET, FP, and ADP-Glo Assay.

        TR-FRET
FP IC50 (nM) ADP-Glo IC50 (nM) T2-BDP-FL Ki (nM) T3-BDP-FL Ki (nM)
Type I Tozasertib NAa 20849 23 ± 3 35 ± 8
Type II Compound 22b NA 1126 0.82 ± 0.08 0.93 ± 0.04
PK68 NA 9019 1.1 ± 0.2 0.83 ± 0.1
Type III Nec-1s 200050 75449 200 ± 30 220 ± 30
Dihydropyrazole 77 2020 NA 0.86 ± 0.06 0.72 ± 0.09
GSK772 1622 122 2.4 ± 0.2 1.9 ± 0.2
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a

NA: not available.

In the previously reported FP assay, dihydropyrazole 77 and GSK772, both Type III inhibitors, exhibited IC50 values of 20 and 16 nM for hRIPK1, respectively.20,22 In FP assays, the lower boundary of IC50 values is 0.67 Kd,37 corresponding to 10 nM in their assays. The measured IC50 values for dihydropyrazole 77 and GSK772 approach the assay limit. In contrast, the Ki values of these two inhibitors in our TR-FRET displacement assay were 0.86 and 2.4 nM, respectively, representing an over 23- and 7-fold decrease compared with the FP assay. In the ADP-Glo kinase assay, five of these tested inhibitors were reported, with IC50 values of 208, 11, 90, 754, and 1 nM, respectively (Table 1).19,22,26,49 In our TR-FRET displacement assay, the Ki values for these inhibitors were 1–80 times lower. This data demonstrated the improved sensitivity of our TR-FRET displacement assay, capable of discriminating inhibitors within the single-digit nanomolar inhibition range. It is important to note that the theoretical lower limit of the Kd value in TR-FRET assays is dictated by the enzyme concentration employed, which is 1 nM in our assay.

Furthermore, we conducted a comparison between the Ki values generated in the two TR-FRET probe-mediated displacement assays. Notably, we observed a significant correlation between the Ki values obtained in the T2-BDP-FL mediated TR-FRET displacement assay and those in the T3-BDP-FL mediated TR-FRET displacement assay for hRIPK1, showing a slope of 1.07 and an R2 value of 0.99 (Figure 5c).

Development of NanoBRET Displacement Assay for RIPK1 Using T2-BDP589

A NanoBRET probe, T2-BDP589, derived from a Type II RIPK1 inhibitor, was developed to establish the NanoBRET assay for RIPK1 (Figure 3c). In this assay, HEK293T cells were transiently transfected with hRIPK1 and mRIPK1 enzymes fused to a NanoLuc tag on the N terminus, respectively. Subsequently, a titration of the T2-BDP589 probe (ranging from 0.32 nM to 5000 nM) was conducted to determine the apparent Kd values. As depicted in Figure 6a, T2-BDP589 exhibited high affinities to both hRIPK1 and mRIPK1 in cells, with apparent Kd values of 150 and 360 nM, respectively.

Figure 6.

Figure 6

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Measurement of intracellular RIPK1 target engagement by the NanoBRET displacement assay. (A) Dose–response curves of T2-BDP589 for hRIPK1 and mRIPK1. Dose–response curves of six RIPK1 inhibitors in the T2-BDP589 mediated NanoBRET displacement assay for hRIPK1 (B) and mRIPK1 (C). (D) Correlation of NanoBRET displacement assay and antinecroptosis cellular assay.

As illustrated in Figure 2, the competitive displacement of the T2-BDP589 probe by an unlabeled inhibitor facilitated the determination of the relative binding efficiency of RIPK1 inhibitors for intracellular RIPK1 enzymes. The addition of unlabeled inhibitors to cells, in conjunction with T2-BDP589, resulted in concentration-dependent attenuation of the BRET signal. The relative binding efficiencies of RIPK1 inhibitors became evident through the concentration required to displace a defined amount of the tracer. Employing a fixed concentration equivalent to the apparent Kd value of T2-BDP589 for competitive displacement, the relative binding efficiencies Ki were estimated utilizing the Cheng–Prusoff equation.

Measurement of RIPK1 Target Engagement of Inhibitors in Cells Using NanoBRET Displacement Assay

To underscore the applicability of the NanoBRET displacement assay for profiling intracellular RIPK1 target engagement, we utilized a diverse set of inhibitors with distinct structural and binding models. This set included one Type I RIPK1 inhibitor, two Type II RIPK1 inhibitors, and three Type III RIPK1 inhibitors.

As depicted in Figure 6 and summarized in Table 2, all six tested inhibitors demonstrated substantial engagement with hRIPK1 in cells, yielding apparent Ki values of 250, 6.5, 3.0, 250, 1.5, and 0.56 nM, respectively.

Table 2. Summary of Binding Affinities of RIPK1 Inhibitors in NanoBRET and Anti-necroptosis Cellular Assay.

    Antinecroptosis IC50 (nM)
   NanoBRET Ki (nM)
  
Human cells HT29 Mouse cells L929 IC50 ratio (m/h) Human Mouse Ki ratio (m/h)
Type I Tozasertib 25751 256151,a 10 250 ± 50 3200 ± 600 13
Type II Compound 22b 1.826 126 0.5 6.5 ± 1.0 22 ± 5 3
PK68 2319 1319 0.6 3.0 ± 0.4 15 ± 2 5
Type III Nec-1s 7751 80351,a 11 250 ± 50 3700 ± 1300 15
Dihydropyrazole 77 6320 320020 51 1.5 ± 0.2 350 ± 90 233
GSK772 6.322,b 130022 200 0.56 ± 0.06 1200 ± 400 2142
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a

MEF cells.

b

U937 cells.

Moreover, our NanoBRET displacement assay results revealed diminished target engagement for mRIPK1 in cells across all RIPK1 inhibitors, with apparent Ki values of 3200, 22, 15, 3700, 350, and 1200 nM, respectively (Figure 6c and Table 2). Notably, Type II inhibitors, compounds 22b and PK68, exhibited relatively small Ki ratios between hRIPK1 and mRIPK1 of 3 and 5, respectively. In contrast, Type III inhibitors, Nec-1s, Dihydropyrazole 77, and GSK772, demonstrated significantly large Ki ratios between hRIPK1 and mRIPK1 with 15, 233, and 2142, respectively. This observation aligns with the previously reported trend that Type III RIPK1 inhibitors commonly exhibit substantial species selectivity. This selectivity arises from the diminished flexibility of mouse RIPK1 in adopting the activation loop conformation required for these inhibitors to bind in their preferred Type III conformation.22

We further compared the IC50 ratios derived from the antinecroptosis potencies in both human and mouse cells, to our NanoBRET displacement assay. Notably, the ratio of target engagement between hRIPK1 and mRIPK1 in the NanoBRET assay exhibited a good correlation with the ratio of antinecroptosis potencies between human and mouse cells, demonstrating a slope of 0.99 and an R2 value of 0.87 (Figure 6d).

In summary, we have successfully developed two TR-FRET probes, T2-BDP-FL and T3-BDP-FL, derived from two distinct RIPK1 inhibitors with different binding models (DLG-out binding site and allosteric site, respectively), both exhibiting a high affinity for RIPK1 binding. Consistent results were obtained with these two probes in the TR-FRET displacement assay. Our TR-FRET displacement assay demonstrated superior sensitivity compared with previously reported FP and ADP-Glo kinase assays. Through these TR-FRET displacement assays, we conducted an initial screening of the binding affinities of various RIPK1 inhibitors with distinct structural and binding models to hRIPK1.

Additionally, we introduced a NanoBRET probe, T2-BDP589, to measure the intracellular RIPK1 target engagement via the NanoBRET displacement assay. This assay allowed us to profile the engagement and potency of RIPK1 inhibitors for both hRIPK1 and mRIPK1 in the cells. Notably, the ratio of target engagement between hRIPK1 and mRIPK1 exhibited a good correlation with the ratio of potencies between human and mouse cells.

Our established fluorescent probe displacement assays are characterized by their sensitivity and stability, enabling the characterization of inhibitors with a picomolar potency range. This screening platform provides a high-throughput approach to identify RIPK1 inhibitors based on both biochemical and cellular activities, undoubtedly facilitating the discovery and development of RIPK1 inhibitors.

Acknowledgments

The research was supported in part by National Institute of Health (R01-268518, and R01-CA250503 to J.W.), Cancer Prevention & Research Institute of Texas (CPRIT, RP220480 to J.W.), and the Michael E. DeBakey, M.D., Professorship in Pharmacology (to J.W.).

Glossary

ABBREVIATIONS

BODIPY

dipyrrometheneboron difluoride

ADP

adenosine diphosphate

NanoLuc

NanoLuc luciferase

HATU

2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

TEA

triethylamine

DMF

N,N-dimethylformamide

K2CO3

potassium carbonate

DCM

dichloromethane

BDP

BODIPY

DIPEA

N,N-diisopropylethylamine

ACN

acetonitrile

THF

tetrahydrofuran

LiOH

lithium hydroxide

nM

nanomolar

μM

micromolar

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00104.

  • Synthetic procedures, characterization data, biological assay procedures (PDF)

Author Contributions

D.L. and J.W. designed the experiments. X.Y. and H.L. performed the experiments. X.Y., D.L., and J.W. wrote the manuscript.

The authors declare the following competing financial interest(s): J.W. is the co-founder of Chemical Biology Probes LLC. J. W. has stock ownership in CoRegen Inc and serves as a consultant for this company.

Supplementary Material

ml4c00104_si_001.pdf (4.4MB, pdf)

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