Coelenterazine Analogue with Human Serum Albumin-Specifi c Bioluminescence
Ryo Nishihara,* Kazuki Niwa, Tatsunosuke Tomita, and Ryoji Kurita*
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ABSTRACT: A synthetic luciferin comprising an imidazopyrazinone core, named HuLumino1, was designed to generate specifi c bioluminescence with human serum albumin (HSA) in real serum samples. HuLumino1 was developed by attaching a methoxy- terminated alkyl chain to C-6 of coelenterazine and by eliminating a benzyl group at C-8. HSA levels were quantified within 5% error margins of an enzyme-linked immunosorbent assay without the need for any sample pretreatments because of the high specificity of HuLumino1.
ioluminescence (BL) is produced through an enzymatic reaction comprising a bioluminescent substrate (luciferin)
and an enzyme (luciferase), derived from luminous organisms such as firefl ies, Gaussia princeps, Oplophorus gracilirostris, and
1,2
sea pansy Renillareniformis. Luciferase-dependent BL assays, coupled with genetically engineered proteins, cells, and animals, have allowed monitoring of diverse molecular events
2-4
in living subjects.
enzymatic reactions such as Kemp elimination and hydrolysis of esters as a result of their unique properties for binding small hydrophobic molecules; however, the potential enzymatic
11,13
activities have not yet been elucidated.
Coelenterazine (CTZ), a luciferin of luminous marine organisms, is oxidized by bovine serum albumin (BSA) as well as various luciferases and forms coelenteramide in an excited state, with emission maxima in the range of 400-480
In some cases, a small exogenous molecule elicits the latent
14,15
nm (Figure 1a).
The nonspecific reaction of CTZ occurs
luciferase activity of a nonluminous protein. For instance, CycLuc2, a synthetic analogue of firefl y luciferin, emits light via the long-chain fatty acyl-CoA synthetase found in non-
5-7
luminous organisms rather than by fi refl y luciferase. Additionally, a study reported that in the heme-containing enzyme myeloperoxidase, which is expressed abundantly in neutrophils and monocytes, the luciferase activity (which does not occur naturally) is expressed by luminol, which is one of
8,9
the most widely used chemiluminescent reagents. These results imply that the introduction of an exogenous luminescent substrate can reveal potential luciferase activities that could diff er significantly from the normal functions of the enzymes and enable noninvasive detection of human proteins even without the luciferase derived from luminous organisms.
To investigate whether human proteins could show luciferase activity, at fi rst we focused on human serum albumin (HSA), which accounts for approximately 65% of serum proteins in the human body.10 This abundant protein is involved in a variety of important physiological functions, such as maintaining osmotic pressure, buff ering the blood pH levels, and transporting ligands, including hormones, amino acids, and
11,12
fatty acids, and delivering them to target organs or tissues. Especially, HSA-ligand complexes result in various efficient
mainly because it does not require any cofactor other than an oxygen molecule (Figure 1a and Table S1).1 The luminescent capacity of CTZ is due to its imidazopyrazinone ring. Cypridina Lase (CLuc), which cannot use CTZ as a luciferin, oxidizes only Cypridina luciferin, and the side chains at C-2, C- 6, and C-8 of the imidazopyrazinone core are dominant for enzymatic recognition (Table S1).16 A pair consisting of mutant Oplophorus Lase (NanoLuc) and furimazine, a CTZ analogue (CTZA), is known as a versatile BL reporter. Each of the BL probes has been individually developed with a synthetic imidazopyrazinone derivative that is suitable for the geometry of the active site in the pocket of the mutant luciferase. This enhances the optical intensity and enables it to perform the
17-22
multiplex BL assay (Table S2). We have also reported that RLuc8.6-535SG, a mutant Renilla reniformis Lase that
Received: September 25, 2020 Revised: November 12, 2020
© XXXX American Chemical Society
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Figure 1. (a) Reaction mechanism of coelenterazine-dependent bioluminescence. The imidazopyrazinone structure and modifiable substituent are highlighted in red and blue, respectively. (b-e) Chemical structures of coelenterazine analogues (CTZAs) upon (b) C-2, C-6, and C-8 substitutions, (c) HSA-specifi c substitution, (d) C-2 and C-6 substitution, and (e) C-2 or C-6 substitution. (f) Luminescence from serum albumins (0.1 or 1 mg/mL) treated with the indicated substrate (10 μM); error bars represent the standard deviations of three measurements. (g) Bioluminescence spectra of HuLumino1 in the presence or absence of HSA.
utilizes BottleBlue2.3 (BBlue2.3), a CTZA, which is permeable through the cell membrane, and displays bright emission suitable for deep-tissue imaging of cancer cells in vivo (Table S2).23
■ RESULTS AND DISCUSSION
First, in order to obtain a rational luciferin with HSA-specifi c bioluminescence, we assayed CTZ and 18 previously reported CTZAs where the p-hydroxyphenyl group at C-6 of the CTZ was modified by alkylation (Figure S1) with serum albumins (HSA and BSA). Furthermore, we found that BBlue2.3, a CTZA with a methoxy-terminated alkyl linker chain of three methylene units in length at C-6, displayed the brightest emission, producing 16.6-fold stronger luminescence when
combined with HSA (i.e., the BBlue2.3/HSA pair) compared with that of the CTZ/HSA pair (Figure S2). On the basis of these results, elimination of the benzyl group at C-8 of BBlue2.3 may have relieved its steric hindrance with the key amino acids in the substrate binding site of HSA and enhanced the enzymatic effi ciency of HSA. We then designed and synthesized a novel CTZA, named Human Luminophore 1 (HuLumino1), and an array of fi ve CTZAs, which included conventional analogues,24 in order to investigate the eff ect of substitution at C-2, C-6, and C-8 of CTZ on serum-albumin- dependent luminescence (Figure 1c-e). Moreover, we compared the CTZ luminescence of commercially available CTZAs (DeepBlueC and MCLA) and the synthesized CTZAs upon addition of fatty-acid-free HSA and BSA (Figure 1f).
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Figure 2. (a) Luminescence response in the presence (I) vs the absence (I0) of the binding drug concentration (0-100 μM). (b) Lineweaver- Burk plots indicating competitive inhibition by ibuprofen. V0 is defi ned as the luminescence intensity over the initial 30 s, and [S] is the substrate concentration. The concentrations of ibuprofen were 1 nM (squares) and 5 nM (triangles). Open circles are data for negative controls with no ibuprofen. (c) Ligand binding site 2 of HSA with HuLumino1 as posed by the Molecular Operating Environment (MOE) software. (d) Magnifi ed view of binding site 2. The ligand binding site in the blue region indicates the presence of a hydrophobic environment. (e) Predicted interaction between HuLumino1 and HSA.
Encouragingly, HuLumino1 showed signifi cantly enhanced luminescence with increasing HSA (but not BSA) concen- tration, as indicated by the 14.1-fold stronger emission compared with the BBlue2.3/HSA pair. The HuLumino1/
HSA pair displayed a flash-type luminescence with a peak
subtle conformational difference in the substrate binding site, although the overall sequence homology between HSA and BSA is 75.6%. To clarify the basis for light emission by HSA, the detailed kinetic properties (Km, kcat, and ØBL of CTZAs and HSA pairs were further investigated using a calibrated
wavelength of 432 nm (Figures 1g and S4). Unexpectedly,
25,26
luminometer (Table S3).
The Km of the HuLumino1/
HuLumino1 selectively activated HSA by recognizing the HSA pair is 4.3 μM, which is on the same order of magnitude
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Figure 3. (a) Variation in luminescence of HuLumino1 (10 μM) in the presence of proteins (0.1 mg/mL). Error bars represent the standard deviations of three measurements. (b, c) Linear relationships between the luminescence intensity of HuLumino1 (20 μM) and the concentration of HSA (0-0.1 mg/mL) in (b) PBS (squares) and (c) PBS-diluted plasma (100-fold, pH 7.4) (circles).
as for the NanoLuc system.27 In contrast, the kcat value of the NanoLuc system was 294-fold higher than that of the HuLumino1/HSA pair.18 However, in the HSA-catalyzed luminescence system, HuLumino1 showed a higher Km value than CTZA-1, but its kcat value was about 3-fold higher (Table S3), resulting in 4.2-fold stronger light emission compared with CTZA-1 (Figure 1f). In the case of a luciferin with high enzyme affinity (e.g., CTZA-1), there is a possibility of competitive inhibition by oxyluciferin, a product of the BL reaction.28 In addition, HuLumino1 has a much higher enzyme affi nity than CTZ, and its bioluminescence quantum yield (ØBL) is more than 100-fold higher than that of CTZ, suggesting that the structural characteristics derived from the alkyl linker chain at C-6 of the imidazopyrazinone core are dominant in an effi cient HSA-catalyzed luminescence reaction. Thus, although the kcat value of HuLumino1/HSA pair is much lower than that of the NanoLuc system, HuLumino1 is a relatively suitable luciferin for HSA than other existing luminescent substrates.
The steric structure of HSA, with or without binding to a
29,30
wide variety of drugs, was determined by crystallography.
To determine the specifi c site of the reaction between HSA and HuLumino1, a competitive assay was carried out with two site-specific HSA drugs (warfarin for site1 and ibuprofen for site2).30 Fatty-acid-free HSA PB solution was pretreated with the drugs (0-100 μM) to block the binding sites before addition of HuLumino1. The luminescence of the HuLumi-
no1/HSA pair dramatically decreased in the presence of ibuprofen (Figure 2a). In contrast, the eff ect of blocking site 1 with warfarin on luminescence was negligibly low, indicating that HuLumino1 specifically binds to the site 2 pocket of HSA. On detailed examination of the inhibitory kinetics, Line- weaver-Burk analysis demonstrated that ibuprofen compet- itively inhibits HuLumino1 (the Ki of ibuprofen is 6.3 nM) (Figure 2b). Therefore, the enzymatic luminous oxidation reaction of HuLumino1 occurs at binding site 2. Furthermore, the docking simulation results with the Molecular Operating Environment (MOE) software package shown in Figure 2c-e predicted the specific binding of HuLumino1 to the hydro- phobic microenvironment of site 2 through interactions with key amino acids such as R410, K414, and L453 (Figure 2d,e). In particular, R410, an important amino acid residue in esterase activity,11 which includes one of the enzymatic reactions of HSA, is strongly involved in the luminescence reaction via hydrogen bonding with the nitrogen atom of the imidazopyrazinone ring of HuLumino1 (Figure 2e). When 10 M guanidine hydrochloride, the common reagent used to denature the α-helix structure of proteins,31 was used to pretreat HSA, the luminescence of HuLumino1 resulted in a significant decrease following the addition of the unfolded HSA structure (Figure S5). Therefore, the enzymatic reaction of HuLumino1 completely depends on the microenvironment and steric constitution of HSA, which exhibits pockets at site 2 of the folding structure. These results suggest that nonspecific
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chemiluminescence does not cause the emission of HuLumi- no1, which is generally the case in other imidazopyrazinone compounds.
The normal level of HSA in blood serum is 35-55 mg/mL, and low levels of HSA in serum result in malnutrition, cirrhosis, and chronic hepatitis.32 Hence, the accurate detection of HSA in serum with high selectivity has great significance in clinical diagnostics.
Next, we demonstrated the sensitive and specific detection of HSA based on luciferase activity. Analysis of the reaction behavior of HuLumino1 with various biological proteins of interest revealed that only HSA led to a distinct luminescence enhancement. In contrast, other proteins (BSA, β-galactosi- dase, β-lactoglobulin, catalase, α-chymotrypsinogen, hemoglo- bin, human immunoglobulin G, porcine lipase, papain, pepsin, trypsin, γ-globulin, carbonic anhydrase, concanavalin A, glucosidase, histone, myoglobin, and RNase, 0.1 mg/mL) resulted in no emission (Figure 3a). The coexistence of most human proteins does not aff ect the bioluminescence reaction between HSA and HuLumino1. However, in the presence of some proteins, a slight decrease in luminescence intensity was observed (Figure S6). This suggests that HuLumino1 may bind to other human proteins; however, it does not exhibit bioluminescence. Hence, HuLumino1 can be used to detect HSA without interference from other proteins, as it exhibited an excellent selectivity for HSA in a complicated biological system. In the presence of 10 μM HuLumino1, a linear increase in luminescence intensity was observed in HSA, within the concentration range of 0-0.1 mg/mL (Figure 3b,c). The intensity gradually saturated at high concentration and showed a constant intensity at more than 10 mg/mL (Figure S7). The detection limit of HSA in the diluted serum was as
-1
low as 8.6 μg mL , which was equivalent to the standardized detection limit of HSA in a physiological system.33 Finally, two analytical methods, viz., our HuLumino1 BL-based assay and a conventional enzyme-linked immunosorbent assay (ELISA) were used to assay the human serum sampled from male AB plasma. As shown in Table 1, the HSA levels estimated with
6 and C-8 modifi cations, demonstrated high sensitivity, specificity, and rapid detection of HSA even in real human serum media containing various interfering biomolecular analytes. A detailed study of the enzymatic reaction revealed that the selectivity was derived from enzyme recognition of HSA drug binding site 2 to HuLumino1 and found that it reacted not only with the human-serum-derived albumin but also with the COS-1 cell-derived recombinant human albumin. Therefore, HuLumino1, either used alone or coupled with an ELISA protocol, could be useful as a probe for the early diagnosis of HSA-related diseases, enabling accurate detection and quantification of HSA in serum samples. In addition, the knowledge gained through the detailed investigation of the HuLumino1/HSA pair may greatly influence the extension of human protein analyses using enzymatic luminescent reactions without the need for genetic engineering approaches.
■ ASSOCIATED CONTENT
sı* Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.0c00536.
Detailed synthetic procedures and optical characteristics of novel compounds and additional information on luminescent properties (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Ryo Nishihara – National Institute of Advanced Industrial Science and Technology (AIST), DAILAB, DBT-AIST International Centre for Translational and Environmental Research (DAICENTER), Tsukuba, Ibaraki 305-8566, Japan; orcid.org/0000-0002-9539-596X;
Email: [email protected]
Ryoji Kurita – National Institute of Advanced Industrial Science and Technology (AIST), DAILAB, DBT-AIST International Centre for Translational and Environmental Research (DAICENTER), Tsukuba, Ibaraki 305-8566, Japan; Faculty of Pure and Applied Sciences, University of
Table 1. Assay of HSA in Human Serum Tsukuba, Tsukuba, Ibaraki 305-8573, Japan; orcid.org/
amount of HSA
added (mg/mL)
0
1
2.5
HSA (mg/mL) determined by the developed methoda
39.0 ± 3.1 44.5 ± 0.5 45.2 ± 0.5
HSA (mg/mL) determined by
ELISA 41.0 ± 3.6 NDb
ND
recovery (%)
95.2
106.1
104.1
0000-0001-5666-9561; Email: [email protected]
Authors
Kazuki Niwa – National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
Tatsunosuke Tomita – National Institute of Advanced
aConditions: HuLumino1 (20 μM) in PBS-diluted serum (1000-fold, 10 mM, pH 7.4). bND: not determined.
HuLumino1 agreed well with the levels measured by ELISA within 5% error. The spike and recovery test was also confined within the margin of 7% error (Table 1). Furthermore, we were able to observe an expression of recombinant HSA from living COS-1 cells (Figure S9), indicating that the HuLumino1/HSA pair has the potential to be used for molecular biology experiments in addition to its biomedical applications.
■ CONCLUSIONS
We developed the first synthetic luciferin for HSA based on an oxidative enzymatic reaction of an imidazopyrazinone molecule. HuLumino1, a CTZ-derived luminophore with C-
Industrial Science and Technology (AIST), DAILAB, DBT- AIST International Centre for Translational and Environmental Research (DAICENTER), Tsukuba, Ibaraki 305-8566, Japan
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.bioconjchem.0c00536
Author Contributions
All of the authors contributed to the writing of the manuscript and approved the final version of the manuscript.
Funding
This work was supported by JSPS KAKENHI Grant 20K15421, AMED under Grant JP191m0203012, the Shimazu Science Foundation, the Research Foundation for Opto- Science and Technology, the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical
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Engineering, a DAICENTER project grant from DBT (Government of India) to Renu Wadhwa, and a special strategic grant from AIST (Japan).
Notes
The authors declare no competing fi nancial interest.
■ ACKNOWLEDGMENTS
We thank Dr. Tsukasa Ishihara (Biomedical Research Institute, National Institute of Advanced Industrial International Science and Technology) for his help with the docking simulation experiment.
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