Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase

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Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors Joel R. Walker, Mary P. Hall, Chad A. Zimprich, Matt B. Robers, Sarah J. Duellman, Thomas Machleidt, Jacquelynn Rodriguez, and Wenhui Zhou ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01129 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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ACS Chemical Biology

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors Joel R. Walker, Mary P. Hall, Chad A. Zimprich, Matthew B. Robers, Sarah J. Duellman, Thomas Machleidt, Jacquelynn Rodriguez, Wenhui Zhou a

Promega Biosciences LLC, 277 Granada Drive, San Luis Obispo, CA 93401; bPromega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399

Corresponding author: [email protected]

ABSTRACT: Novel engineered NanoLuc® (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42k PubChem compound library with low µM IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nM, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit® 1 ACS Paragon Plus Environment


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system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned. INTRODUCTION Bioluminescence enzymes from native luciferases, such as Firefly and Renilla luciferases, and their corresponding substrates have been extensively utilized as biological reporters in numerous applications.1-3 The widely recognized utility of bioluminescence motivates investigators to search for alternative luciferases with enhanced luminescence efficiency, improved signal stability, superior sensitivity, and broad linear dynamic range. The deep sea shrimp, Oplophorus, secretes a luciferase and its substrate in brilliant luminous clouds as a defense mechanism.4 This enzyme has been engineered as the newest commercially available luciferase, termed NanoLuc (Nluc) and is substantially brighter and smaller at 19 kDa than the native protein.5-6 Nluc catalyzes the oxidative conversion of furiamzine to furimamide with the apparent KM of 10 µM (Figure 1). It produces glow-type luminescence with signal half-life > 2 hour with specific activity ~150 fold greater than that of either firefly (Photinus pyralis) or Renilla luciferase. Nluc was found to be more robust than firefly luciferase under a variety of environment conditions, and showed greater thermal stability and more tolerance to salt and pH. Nluc, as a single species devoid of post-translational modifications, can be uniformly expressed in the experimental hosts without compartmental bias.5 Because of its brightness and stability, Nluc has been applied in the preclinical and clinical fields in terms of screening drug targets, drug target validation, disease detection, and in vivo imaging.7-19 And with the increased sensitivity and small size, Nluc is very useful as a reporter gene for monitoring cellular biology20-24 and can more closely mimic endogenous biology. A

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recent advancement enabled by Nluc is a real-time cell viability assay using a prosubstrate that is only processed to the active Nluc substrate by live cells.7 Nluc and the prosubstrate could be directly applied in the cell culture media, allowing the continuous detection of viable cells over 96 hours without interrupting cells and provides a great option to the cell viability assay community. In addition, Nluc is as an excellent energy transfer donor because it exhibits a narrow bioluminescence spectrum, allowing the distinct spectral discrimination from acceptor fluorophore. NanoBRETTM, having HaloTag-NCT red fluorophore (λemission 635 nm) as an optimal energy donor for Nluc.

NanoBRETTM was reported in quantifying protein-protein

interactions (PPI) using rapamycin-induced interaction between FKBP12 and FRB as a model.25 It was found that the NanoBRETTM assay maintained good sensitivity and response dynamics, even at the concentration of FKBP-HaloTag that were well below endogenous FKBP. It was further reported that co-expression of Nluc-BRD4 with human histone H3.3-HaloTag in mammalian cells produced a BRET signal that can monitor the binding of bromodomain proteins to acetylated histones for epigenetic regulation of transcriptional activity when the cells were treated with increasing doses of known BRD inhibitors.26 Moreover, drugs can be labelled with a red fluorescent dye to yield a cell permeable or impermeable tracer that can be used to examine the binding characteristics of selected targets in intact cells or on the cell surface, referring to the NanoBRET-based Target Engagement assay.27-28 Selective measurement of events only within intact and viable cells can be a challenge due to the presence of cell debris arising from cell culturing and handling, and due to the stability and brightness of Nluc. We envisioned that a highly potent Nluc inhibitor can provide several benefits: to selectively inhibit intracellular or extracellular Nluc to improve live cell assay



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performance; multiplex with the other luminescent or fluorescent assays to enhance screening throughput; or to create new assay concepts based on a ‘turn off-and-on’ Nluc activity. In this work, we report the design, development, and characterization of highly potent cell permeable and impermeable Nluc inhibitors based on a thieno-pyrrole high throughput screening hit. RESULTS and DISCUSSION Hit-to-Lead Development and Beyond. Nluc has been well characterized as a luciferase reporter system regarding to enzyme activity, glow kinetics, thermal stability, and suitability. However, even though an apo-Nluc crystal structure has been published,29 the lack of a Nlucsmall molecule co-crystal structure resulted in limited information about polar and non-polar residues between the binding site and the surface of protein. In addition, it was not possible to use molecular docking tools to assist fragment design for a Nluc inhibitor. Fortuitously, previous evaluations of hit rates from a collection of 42,460 PubChem compounds for orthogonal reporter gene assays revealed a few weak inhibitors of Nluc (Figure 2).30 We believed that this valuable information could be used to develop highly potent Nluc inhibitors. Particularly, the methyl thieno-pyrrole carboxylate 1 (PC 16011099), with a low µM IC50, was of interest for further exploration due to its feasibility of chemical derivatizations and non-apparent color quenching. Therefore, compound 1 was targeted for chemical modifications, aiming for improvement of the inhibition potency. We hypothesized that structure-activity relationship (SAR) studies at the four different substructure sites (Figure 2) would result in an understanding of the Nluc binding pocket and yield potent inhibitors. Further, we hoped the SAR would reveal a molecular site that could reach the surface of the protein, enabling additional functionalities for a broad range of applications while maintaining the desired inhibition potency.


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Modifications of Methyl Ester at 2-thienoyrrole (I). The first site that we investigated was the substituents around the methyl ester at the 2-position of the thienopyrrole. The syntheses of these analogues followed a 4-step procedure with generally excellent yields for each step (Schemes in Supporting Information). The targeted analogues (2–12) are listed in Table 1a. For IC50 measurements, a series of dilutions of the compound in 50 µL CO2-independent media containing 0.4 ng/ml of Nluc and 10% FBS were mixed with 50 µL NanoGlo buffer containing 20 µM furmiazine, and luminescence measured. Each sample was normalized to the no inhibitor control. The IC50 values were then determined using GraphPad Prism. Noticeably, the IC50 values indicated that substituents at the 2-thienopyrrole position prefers hydrophobic groups (Table 1a). Among this family of analogues, the free acid 2 and methyl amide 3 were the most polar compounds, having the lowest Log P (calculated) values, 3.0 and 2.6, respectively. Whereas their IC50 values were ~15 times and ~5 times less potent as compared to the more lipophilic compound 1 with the Log P value of 3.2. The same trend was observed for the secondary amides 3, 4, and 5 with the IC50 improving from 5.5, 0.54, and 0.26 µM while their Log P values increased from 2.6, 3.7 and 4.1, respectively. Tertiary cyclic fiveand six-member ring amides 6 and 7 having LogP values 3.1 and 3.5, respectively, were slightly less lipohilic than the corresponding secondary cyclic amides 4 and 5, but displayed fairly poor inhibition with an IC50 of 1.2 and 2.6 µM, respectively. The ~10-fold difference may indicate that either the amide N-H bond forms a hydrogen bond interaction or the tertiary amide is disfavored due to sterics in that sub pocket. Although the secondary aromatic phenyl amides 10, 11, and 12 were considered fairly hydrophobic molecules with LogPs above 4, all showed lowered IC50 values of 2.6, 11, and 4.7 µM, respectively, implying the flexibility of the cyclohexane ring might be preferred over a π−π stacking interaction. We then further introduced 5 ACS Paragon Plus Environment


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a methyl carboxylate to the secondary cyclohexane amide (5) to give trans- and cis-esters 8 and 9, respectively. Surprisingly, both compounds showed similar or even greater inhibition potency with an IC50 of 0.15 and 0.23 µM when compared to the IC50 of 0.26 µM for non-substituted cyclohexane amide 5. Trans-8 exhibited a slight increase in potency over cis-9 and infers the trans-confirmation is favored. Aniline N-Alkyl Chain Modifications (II).

Based on the above potent secondary

cyclohexane amides 5 and 8, we modified the N-ethyl of aniline with hydrogen, C6-alkyl, or (PEG)2. Clearly, replacing the ethyl group with a hydrogen exhibited a significant loss of potency with an IC50 of 45 µM (13, Table 1b). This result shows the hydrophobic interaction of a short ethyl chain is needed to maintain the necessary binding affinity. However, neither C6alkyl nor (PEG)2 alkyoxyl modifications showed improved potency, indicating the space in the binding pocket for this molecular site might be very restrictive, and the further modification of this chemical site was abandoned. Aromatic Aniline Substituents (III). So far compound 8 represented the most potent inhibitor with the combination of a trans methyl 4-aminocyclohexane 1-carboxylate and short Nethyl chain. Keeping these motifs constant, we further probed the 3-position methyl group on the aniline substituent. As showed in Table 1c, removal of meta-methyl on the aromatic ring, such as compound 18, exhibited no change of IC50 as compared to compound 8. However, placement of methyl onto the ortho or para position, such as compound 19 or 20, resulted in 5 to 10-fold loss of potency, illustrating the preference of meta substituent over other positions. A dramatic loss of the potency was observed when N-alkyl was locked onto the aromatic ring (21), indicating the need for rotational freedom around the aniline ring and ethyl chain. We further focused on the meta substitution to yield target compounds 22 – 27. In general, analogues with


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small groups (25 – 27) had little or no effect on the IC50 although they seemed more hydrophilic with lower LogP’s as compared to compound 8. This indicated the binding pocket at this site was tolerant to small meta substitutions. Surprisingly, compound 22 with a simple ethyl group on meta position resulted in a great enhancement of binding affinity with the IC50 of 3.9 nM. However when a bit more steric bulk is added with an isopropyl or isobutyl group (23 and 24), the binding affinity dropped 1-2 orders of magnitude as compared to 22, confirming the space around this sub pocket was sterically limited. Thieno-pyrrole Analogues (IV). Next we examined the core heterocycle around which the various substituents are attached (Table 1d). Compared to 8, the pyrole 28, where the thiophene ring was eliminated, resulted in a decrease of potency more than 50 fold. In contrast, furo[3,2b]pyrrole 31 showed only a 4-fold decrease in potency, similar to compound 32, where switching the fused thieno-ring from a 2, 3-pyrrole to a 3, 2-configuration. Interestingly, indole 29 showed a 4-fold enhanced potency with an IC50 of 0.036 µM. However, a 5-methoxy indole (30) exhibited a large decrease in potency with an IC50 of 3.4 µM. Undoubtedly, the aromatic ring fused to the pyrrole is one of the critical pieces for maintaining the desired binding affinity, possibly via π−π stacking interaction. Further, the subpocket around this molecular site could be very restrictive due to its intolerance of a small methoxy substitution (30).

Analogue 33

combines the meta ethyl on the aniline aromatic ring with the indole substitution to achieve a highly potent inhibitor with a single digit nM IC50. Comparison to 22 indicates the binding affinity of the ethyl group in this subpocket contributed more to the affinity than the pyrrolefused aromatic ring. Trans 1,4-Aminocyclohexane Carboxamides (V). Although Nluc fusion-proteins provides a variety of useful tools, capable of monitoring protein-protein interactions in live cells, or



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quantifying intracellular target engagement and the drug residence time in the intact cells, the luminescence generated by extracellular Nluc fusions from cell debris or secreted from the intact cells or both might interfere in the assay performance.31

In order to selectively suppress

extracellular Nluc activity, a potent cell-impermeable inhibitor is desired.

All the above-

mentioned small, neutral, and hydrophobic thienopyrrole or indole compounds are likely freely cell permeable, which could inhibit both intracellular and extracellular Nluc activities. Negatively charged compounds, such as carboxylic acids, phosphates or sulfonates are wellknown for preventing cell permeability. However, Nluc binding thus far have been driven by hydrophobic interactions and perhaps π−π stacking interactions. One could imagine that a highly polar and/or negative charge(s) to the central core structure would adversely impact the inhibition potency. We, therefore, envisioned if we could probe a specific molecular site that would allow attachment of a linker and further extend this linker toward the external Nluc protein surface. This linker could then be appended with a cell impermeable moiety and ideally without significant effect on the binding affinity because it would remain outside the binding pocket. Probing all four different molecular sites implied 3 out of 4 molecular sites resides in sterically confined, hydrophobic sub-pockets. However, the methyl ester at 2-pyrrole position exhibited flexibility for modifications and enhanced potencies were observed going from methyl (1), aminocyclopentane (4), aminocyclohexane (5) to methyl trans 1, 4-amino-cyclohexane carboxylate (8). It revealed this particular site might be capable of accommodating the linker throughout the binding pocket and onto the external protein surface. Therefore, we explored different linkers extending from the trans 1,4-aminocyclohexane carboxamide suitable for


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adapting a cell impermeable moiety and perhaps other functional groups without interfering the inhibition potency. We first prepared compounds 35–37 with various hydrophilic groups, such as hydroxyl, dimethyl amino, or carboxyl acid group attached to the other end of a short C2- or C3-alkyl linker and then compared to the hydrophobic C4-alkyl molecule 34 (Table 1e). These short linker compounds (35–37) exhibited a significant loss of binding affinity, especially the dimethyl amino compound 36 with an IC50 of 18 µM, indicating the hydrophilic moiety may still be located inside the binding pocket. We then increased the linker length to a C5- or C7-alkyl chain, such as compounds 39–43. Interestingly, all of the compounds with C5- or C7-alkyl linker linked to different hydrophilic groups, such as -OH, -NH2, -COOH, or -SO3H displayed ~2- to 5-fold enhanced binding affinity, implying these hydrophilic groups might approach toward the external protein surface. However, these polar groups may also facilitate the orientation of the linker to reach an optimal binding pose when compared to the hydrophobic C5-alkyl compound 38 with a lower binding affinity. Additionally, combining optimal substitutions, the N-ethyl and the indole scaffold, with polar capped linkers led to fully elaborated inhibitors with single digit nM potencies (44–45). To further prove our hypothesis, large cell impermeable moieties, such as poly aspartic acid or fluorescein dye, were attached to the other end of C6-aklyl linker (46–47). To our delight, those two compounds exhibited the similar double digit nM IC50‘s, strongly suggesting those moieties reside outside the binding pocket and the binding affinity is independent of size and polarity of the attached moiety. Characteristics of Thienopyrrole Inhibitors. The inhibition mode of the compounds was determined using compounds 1, 8, 34, and 37 as examples. Plotting turnover of luminescence versus furimazine concentrated at four different inhibitor concentrations gave four Michaelis-



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Menton plots (8, Figure 3a).

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The KM-apparent values were then plotted against inhibitor

concentration and the x-intercept gave the inhibition constant (KI) of 0.16 µM (8, Figure 3b). The graphs also indicate compound 8 is a competitive inhibitor. The calculated KI values, 1.9, 0.24, and 1.6 µM for compounds 1, 34, and 37, respectively, followed the same trend in potency as the IC50 determinations and showed competitive inhibition. The specificity of Nluc inhibition was further investigated with compounds 1, 5, 8 34, 38, and 41 and their ability to inhibit firefly luciferase (Fluc) activity, e.g., ULTRAGLO® luciferase. In a Corning 3570 assay plate, a solution containing 1µM luciferin in Luciferase Detection Reagent (Promega Corporation V865/859) was added to the assay wells. An equal volume of the Nluc inhibitor titrations was then added to the wells. The reactions were incubated at RT for 2 hours and luminescence was measured. Supplementary figure 1 (see supplemental matieral) demonstrates that these analogues do not inhibit the firefly luciferase activity. Also, these Nluc inhibitors do not have any activity against the binary NanoBit® enzyme system (supplementary figure 2) further showing that these compounds are ideal for multiplexing with other luciferases in future applications. Moreover, cytotoxicity of compounds 8 and 41 was investigated using a luciferase-coupled ATP method (CellTiter-GloTM, Promega). Typically, cells (HEK293 or Hela) were exposed to the doses of the inhibitor over a certain duration time, at the end of the incubation, CellTiterGloTM reagent was added, plates were incubated at room temperature for 30 minutes, and the luminescence intensity was measured on GloMax® luminometer (Promega). In general, there was no relationship between exposure time and concentration, indicating no toxicity. As shown in supplementary figure 3, when cells were exposed to compound 41 at 100 µM for over 18 hours, no obvious luminescence loss was observed, indicating 41 is non-toxic to cells. For


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compound 8, the luminescence began to drop slightly when the doses were greater than 50 µM, possibly indicating slight toxicity. However, the enhanced luminescence over 18-hour exposure for both compounds is possibly due to cell proliferation over long incubation times as compared to DMSO control. Nonetheless, both compounds showed little and no toxicity to cells, and can be used broadly in live cell-based assays. Furthermore, the chemical stability of thienopyrrole compound 43 was analyzed by HPLC. Differences of the peak area for compound 43 at 280 nm was used to evaluate its stability over time. Supplementary table 1 indicated both a 30mM DMSO stock solution and 90 µM solution in media (OptiMEM) were very stable at 20, 35, and 60 oC. No change of the peak area was observed in DMSO at 60 oC over 28 days and only a slight decrease was found in OptiMEM, indicating such a family of thienopyrrole inhibitors is positioned for use in a wide variety of applications. Evaluations of Cell Impermeability. Because of our interest in gating out Nluc found in media from dead or compromised cells, we assessed the permeability of these inhibitors using HEK293 or HeLa cells transiently transfected with C-terminal Beta-2 Adrenergic Receptor-Nluc (B2AR-Nluc) fusion protein to anchor and orient Nluc cytosolically.

After 24-hour post

transfection, cells were either left untreated or treated with 50µg/mL digitonin in order to compare inhibitor activity in living cells or cell lysates. The samples were then exposed to the inhibitor compound for up to 2 hours. After 10µM furimazine was added, the luminescence was measured. Ideally, if the inhibitor is permeable, the dose-response curves for both live and lysed cells be largely identical; however, if the inhibitor is less permeable or impermeable, the EC50 should be right-shifted in the live cells relative to the lysed cells.

Thus we assumed the

magnitude of EC50 shift should represent permeability. As showed in Figure 4, compounds 8,



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35, and 38 exhibited no change or slightly 2-fold shifted of EC50 for live cells as compared to lytic cells, suggesting these compounds are cell permeable. Especially, compound 38 with no EC50 shift indicated it might have passive permeability. In contrast, compounds 41, 43, 46, and 47 displayed significant EC50 shifts of 23, 34, 297 and 194-fold relative to EC50 values obtained in cell lysates, indicating impaired permeability for these compounds. As expected, the poly negative charged compound 46 and impermeable fluorescein-labeled 47 showed much less permeability than 41 and 43. It is worth mentioning that the previous toxicity study indicated compound 41 was less toxic than 8, also consistent with a cell impermeable compound being less toxic in general to cells than a permeable compound. We also monitored permeability as a function of exposure time in HEK293 cells transiently transfected with cytosolic Nluc using compounds 8 and 43 as representative examples. After 24 hours post transfection, cells were exposed to different doses of inhibitor for different time intervals, 10, 30, or 120 minutes, and the luminescence was measured after adding 10µM furimazine. As shown in supplementary figure 4, the dose response curves for either permeable inhibitor 8 or impermeable inhibitor 43 almost overlap for different exposure time intervals, suggesting the inhibition efficiency might not change over time. Specifically, impermeable 43 did not actively or passively enter the cell either in a dose-dependent or a time dependent manner.

Fortunately, this should ensure selective inhibition for extracellular luminescence

signals accumulated from cell debris and/or secreted Nluc, but should not affect the intracellular luminescence signal relative to an assay duration time. In addition, HeLa transiently transfected with N-terminal Nluc-Beta-2 Adrenergic Receptor (Nluc-B2AR) fusion protein which positions Nluc on the extracellular side of the plasma membrane. Cells were treated with various inhibitors (37, 38, 41) followed by bioluminescence


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imaging (Figure 5). As expected, the cell permeable compound 38 can effectively inhibit both intracellular and extracellular Nluc, whereas compounds 37 and 41 selectively inhibited extracellular Nluc on the cell surface. However Nluc-receptor fusion protein that is still retained inside the cell (undergoing trafficking to the cell membrane) and therefore inaccessible to compounds 37 and 41 is still clearly visible in vesicular compartments. SUMMARY A series of analogues were designed and synthesized to systematically probe four different molecular sites as NanoLuc luciferase inhibitors. We discovered a family of compounds by replacing the methyl ester of HTS hit 1 with various alkyl amide replacements. The combinations of optimal chemical modifications at the four different molecular sites allowed for potencies to reach single digit nM.

We discovered that the trans aminocyclohexane

carboxamide site allowed access to the external Nluc protein surface with an appropriate linker. We further synthesized a series of potential cell impermeable Nluc inhibitors by attaching negatively charged groups to the other end of the optimal hydrophobic C6-alkyl linker. Cell permeability of different Nluc inhibitors was further evaluated by comparing the EC50 shifts of live and lysed cells transiently transfected with C-terminal B2AR-Nluc fusion protein to anchor and orient Nluc cytosolically. Clearly, the negatively charged inhibitors exhibited a dramatic EC50 shift up to ~300 fold, indicating they were less cell-permeable or impermeable as compared to the non-charged inhibitors. Luminescence imaging further confirmed the cell permeable compound can inhibit both intracellular and extracellular Nluc, whereas extracellular inhibitors preferentially quenched extracellular Nluc. The inhibition of Nluc activity was demonstrated to be competitive against furimazine and also selective, not affecting Fluc or NanoBit® activity. Water soluble thienopyrrole compound 43 further showed the great chemical stability, and no apparent degradation was observed in DMSO and OptiMEM medium at 60oC over 1 month.



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Both permeable and less permeable Nluc inhibitors showed little to no toxicity to cells. These inhibitors will allow researchers the ability to control the bioluminescence from Nluc and Nluc fusion proteins in solution or with cell cultures. Additionally, the strategies used to modify the Nluc inhibitors to effect cell permeability can also be applied to append a variety of functional groups or biomolecules using an appropriate linker. This will allow opportunities toward the development of new relief-of-inhibition assays in the future. Supporting Information Available: This material is available free of charge via the Internet.

METHODS Synthesis of Thienopyrrole analogues. Details on the synthesis and characterization of the analogues 1–47 can be found in the supporting information. Inhibitor IC50 Determination. The following example provides the IC50 values for the compounds shown in Tables 1a–e. NanoLuc® enzyme was diluted to 0.4ng/ml in CO2 independent media+10% FBS to make the detection reagent. A 3x dilution series of each inhibitor was then made in the detection reagent. A “no inhibitor” control was also made for each sample. 50µL of each inhibitor dilution was mixed with 50 µL of NanoGlo buffer containing 20µM furimazine. (Final furimazine concentration is 10µM which is at KM.), and luminescence measured. Each sample was normalized to the “no inhibitor” control. The IC50 values were then determined using GraphPad Prism (log[inhibitor] vs. normalized response). Competitive Inhibition. A dilution 2X dilution series of furimazaine was prepared in NanoGlo buffer starting at 100µM. NanoLuc® enzyme was diluted to 0.4ng ml-1 in CO2 independent media+10% FBS to make a detection reagent. Inhibitor was diluted into detection reagent to prepare four solutions at the following concentrations: 3µM, 1 µM, 0.3 µM, no 14 ACS Paragon Plus Environment

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inhibitor.

50ul of each inhibitor dilution was combined in triplicate with each furimazine

dilution. Samples were incubated for three minutes at room temperature and then luminescence was measured using a GloMax®-Multi+ luminometer. KM-apparent values were calculated using GraphPad Prism Michaelis-Menten non-linear regression (Figure 3a). Calculated KMapparent values were re-plotted against inhibitor concentration as a means to calculate KI according to the following equation: KM-apparent=KM/KI[I]+KM where the Y intercept equals KM and X intercept equals –KI (Figure 3b). No inhibition of firefly or NanoBit luciferase. The following example describes the specificity of disclosed thienopyrrole compounds (1, 8, 5, 34, 38, and 41) for inhibiting Nluc luciferase activity and not firefly luciferase activity, e.g., ULTRAGLO® luciferase (supplementary figure 1). In a Corning 3570 assay plate, a solution containing 1µM luciferin in Luciferase Detection Reagent (Promega Corporation V865/859) was added to the assay wells. An equal volume of thienopyrrole compound titrations was then added to the wells.

The

reactions were incubated at RT for 2 hrs, and luminescence was measured on the Tecan M1000 Pro plate reader. Supplementary figure 1 demonstrates that the thienopyrrole compounds do not inhibit the firefly luciferase activity. NanoBit® enzyme was prepared with LgBit (10 nM final) and either SmBit (low affinity peptide at 10 µM) or HiBit (high affinity peptide at 0.1 nM) in CO2 independent media+10% FBS to make the detection reagent. A 3x dilution series of inhibitor 22 was then made in the detection reagent. A “no inhibitor” control was also made for each sample. 50µL of each inhibitor dilution was mixed with 10 µL of either SmBit or HiBit and 40 µL of NanoGlo buffer containing LgBit and furimazine. (Final furimazine concentration is 10µM which is at KM.), and luminescence measured. Each sample was normalized to the “no inhibitor” control. The IC50



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values were then determined using GraphPad Prism (log[inhibitor] vs. normalized response). This data is shown in supplementary figure 2. Thienopyrole cytotoxicity on HEK 293 cells. HEK293 WT cells were plated at 20K cells/well in DMEM + 10% FBS and incubated at 37°C in 5% CO2 independent media. Cells were then exposed to a dose-response curve of thienopyrole compounds for 18 hours, 120 minutes, 60 minutes, 30 minutes, or 10 minutes.

Viability was then measured by adding

CellTiter-Glo® and luminescence quantitated (8 and 41; supplementary figure 3). Cell permeability. Inhibition of C-terminal B2AR-NanoLuc in Live and Lytic cell format with HEK293 or HeLa cells were transiently transfected with B2AR-NanoLuc:pGEM3z (1:100), plated at 20K cells/well in DMEM + 10% FBS and incubated at 37° C in 5% CO2 independent media. 24 hours post-transfection cells were treated with vehicle DMSO (live) or 50µg/mL digitonin (lytic) cell format. Cells were then exposed to a thienopyrole compound dose-response for 30 minutes at 37°C. NanoGlo® Luciferase Assay Substrate was added and RLUs collected on luminometer (Figure 4; 8, 35, 38, 41, 43, 46, 47).


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cell Permeability with Bioluminescent Imaging. HeLa cells were transiently transfected with Nluc-B2AR (N-terminus Nluc) fusion protein to anchor and orient Nluc extracellularly. 24 hrs post-transfection, cells were treated with -/+ 30µM of a thienopyrrole compound, 41, 37, and 38. 10µM furimazine was added, and luminescence was detected by imaging on the Olympus LV200 equipped with a EMCDD camera (Hamamatsu ImagEM 9100-13), a temperature controlled stage and a 60x, 1.35 NA UPLAN S APO objective. All images were acquired at 37°C using an exposure time of 1.1 seconds and an EM gain setting of 500. Thienopyrrole compounds 41 and 37 were less permeable and inhibited extracellular Nluc and enhanced intracellular Nluc.

Compound 38 was cell permeable and inhibited both intracellular and

extracellular Nluc (Figure 5). Cell Permeability Time Course.

HEK293 cells were transiently transfected with

NanoLuc:pGEM3z (1:100), plated at 20K cells/well in DMEM + 10% FBS and incubated at 37°C in 5% CO2 independent media. 24 hours post-transfection cells were treated a doseresponse curve of thienopyrole compounds (8 and 41) for 10, 30, or 120 minutes. NanoGlo Luciferase Assay Substrate was added and RLUs collected on a luminometer (supplementary figure 4). Chemical stability. For the thermal stability, a stock solution at 30mM was prepared (from product as is and liquid DMSO) and 10µL of the solution was dispensed into 24 labeled, amber Eppendorf vials. The vials were stored at -20, 20, 35 or 60 °C (n=6 at each temperature). For analysis, vials were removed at designated test points. The content of the vial was diluted with 150µL of EtOH and 5µL of the solution was injected on the HPLC. For the thermal stability at 90µM in OptiMEM, a solution was made by diluting 5µL of the 30mM DMSO solution with 1.66 ml of OptiMEM media and 60µL of the solution was dispensed into 18 labeled, amber



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Eppendorf vials. The vials were stored at 20, 35 or 60 °C (n=6 at each temperature). For analysis, vials were removed at designated time points and a neat 20µL solution was injected on the HPLC (supplementary table 1).

ACKNOWLEDGEMENTS We thank the Analytical Services Group at Promega Biosciences, LLC., for the characterization of the synthesized compounds.

ABBREVIATIONS AcCN, acetonitrile; B2AR, beta-2 adrenergic receptor; BRET, bioluminescent resonance energy transfer; BTK, Bruton's tyrosine kinase; DIPEA, diisopropylethyalamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EA, ethyl acetate; EDC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; FBS, fetal bovine serum; Fluc, firefly luciferase; HATU, 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HOBT, hydroxybenzotriazole; LCMS, liquid chromatography mass spectrometer; MeOH, methanol, Nluc, NanoLuc luciferase; PPI, protein-protein interactions; RLU, relative light unit; SAR, structure activity relationships; TFA, trifluroacetic acid; TLC, thin layer chromatography; TSTU, N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 1. Reaction of Furimazine with Nluc

Figure 2. Chemical structures for Nluc hits from PubChem Library and chemical modification strategy for compound 1 (PC16011099).


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Table 1a. The IC50 values of 2-thienopyrrole analogues

Compound

R1 =

LogP

1 2 3

3.2 3.0 2.6

IC50 (µ µM) 1.10 16.4 5.50

4

3.7

0.54

5

4.1

0.26

6

3.1

1.20

7

3.5

2.60

8

3.5

0.15

9

3.9

0.23

10

4.2

2.60

11

4.1

10.7

12

4.3

4.7



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Table 1b. The IC50 values of aniline N-substituent analogues

Log P

IC50 (µ µM)

3.5

45.2

14

3.5

16.5

15

2.9

3.0

16

5.9

5.6

17

5.3

2.5

Compound

R2 =

13

H

R1 =


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Table 1c. The IC50 values of aromatic aniline substituent analogues

Log P

IC50 (µ µM)

18

3.1

0.11

19

3.5

0.69

20

3.5

1.3

21

3.6

20.2

22

4.0

0.0039

23

4.3

0.13

24

4.7

0.027

25

3.1

0.16

26

2.9

0.11

Compound

R3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.0

27

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0.24

Table 1d. The IC50 values of thieno-replaced pyrrole analogues

2.5

IC50 (µ µM) 7.7

29

3.6

0.036

30

3.4

3.40

31

2.2

0.59

32

NC

0.71

33

4.0

0.0054

Compound

A

28

H

R3

Log P


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Table 1e. The IC50 values of trans aminocyclohexane carbamide analogues

Compound

A

R3

R5

IC50 (µ µM)

34

0.30

35

0.83

36

17.8

37

0.89

38

0.16

39

0.030

40

0.063

41

0.094

42

0.031



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43

0.071

44

0.0023

45

0.0019

46

0.066

47

0.049


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RLU

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0.3(uM) 0.1(uM) 0.03(uM) 0(uM) Vmax 1476437 1742995 1830519 1995130 19.01 14.37 11.39 Km 33.39

inhibitor 8 Y-intercept 12 ± 0.28 X-intercept -0.16

Figure 3. Competitive study of inhibitor 8 with furimazine (Fz) against Nluc. (a) MichaelisMenton plot used to calculate KMapparent values; (b) Calculated KMapparent values were re-plotted against inhibitor concentration as a means to calculate KI according to the following equation: KMapparent=KM/KI[I]+KM where the Y intercept equals KM and X intercept equals –KI. In this example the calculated of KI for inhibitor 8 is 0.16 µM. Similar experiments with compounds 1, 34, and 37 gave calculated KI’s of 1.9, 0.24, and 1.6 µM (data not shown).



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(a) 1000000

Live Lytic

100000 10000 1000 100

10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4

Compound 8 [uM]

(b) 1000000

Live Lytic

100000 10000 1000 100

10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4

Compound 35 [uM]

(c) 1000000

Live Lytic

100000 10000 1000 100

10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4

Compound 38 [uM]


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(e) Live 1000000

Lytic

100000 10000 1000 100

10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4

Compound 43 [uM]

(f) Live Lytic

1000000 100000 10000 1000 100

10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4

Compound 46 [uM]



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(g) 1000000

Live Lytic

100000 10000 1000 100

10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3

Compound 47 [uM]

Figure 4. Inhibition of transiently transfected B2AR-Nluc fusion protein in HeLa (a-d) or HEK293 (e-g) cells with thienopyrrole compounds in live and lytic cell modes. (a) 8; (b) 35; (c) 38; (d) 41; (e) 43; (f) 46; (g) 47


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(a)

(b)

(c)

(d)

Figure 5. Bioluminescence images of HeLa cells transiently transfected with Nluc-B2AR treated with 30µM of a thienopyrrole compound, upon adding 10 µM furimazine. (a) no inhibitor; (b) 38; (c) 37; (d) 41



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Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase

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