Potential applications of BPFP1 in Bcl-2 protein quantification, carcinoma cell visualization, cell sorting and early cancer diagnosis

Article abstract of DOI:10.1016/j.ejmech.2021.113725

Overexpression of the Bcl-2 protein has emerged as a hallmark of carcinoma cells and can be employed as a biochemical biomarker of these cells. Therefore, some Bcl-2 protein fluorescence probes (BPFPs) were designed for Bcl-2 protein quantification and carcinoma cells labeling. The high Bcl-2 protein binding affinity (K_i < 1 nM) and selectivity (over 50,000-fold Bcl-2 protein selectivity against Mcl-1 protein) of BPFP1 endow it with the ability to detect trace amounts of Bcl-2 protein. After being incubated with a range of concentrations of Bcl-2 protein, BPFP1 exhibited the desired fluorescence properties and its fluorescence intensity is proportional to Bcl-2 protein concentration. Therefore, BPFP1 provides a convenient approach for Bcl-2 protein quantification and we could determine the concentration of Bcl-2 protein based on the BPFP1's fluorescence intensity. Subsequent studies revealed that BPFP1 can fluorescently label carcinoma cells by binding to overexpressed Bcl-2 protein in living cells, and can distinguish carcinoma cells (HL-60 cells and ACHN cells) from normal-tissue cells (HUVECs) according to the different Bcl-2 protein expression levels between carcinoma cells and normal tissue cells. In the present study, BPFP1 represents a new tool for Bcl-2 protein quantification, carcinoma cell visualization and cell sorting. Moreover, BPFP1 can be used in the future for early cancer diagnosis by detecting carcinoma cells in patient tissues.

Full text of DOI:10.1016/j.ejmech.2021.113725

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European Journal of Medicinal Chemistry 224 (2021) 113725
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Potential applications of BPFP1 in Bcl-2 protein quantification,
carcinoma cell visualization, cell sorting and early cancer diagnosis
Tao Liang, Jia Li, Yi Zhou, Xuben Hou, Xinying Yang**, Hao Fang*
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44, West
Culture Road, 250012, Jinan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Overexpression of the Bcl-2 protein has emerged as a hallmark of carcinoma cells and can be employed
as a biochemical biomarker of these cells. Therefore, some Bcl-2 protein fluorescence probes (BPFPs)
were designed for Bcl-2 protein quantification and carcinoma cells labeling. The high Bcl-2 protein
binding affinity (Ki < 1 nM) and selectivity (over 50,000-fold Bcl-2 protein selectivity against Mcl-1
protein) of BPFP1 endow it with the ability to detect trace amounts of Bcl-2 protein. After being incu-
bated with a range of concentrations of Bcl-2 protein, BPFP1 exhibited the desired fluorescence prop-
erties and its fluorescence intensity is proportional to Bcl-2 protein concentration. Therefore, BPFP1
provides a convenient approach for Bcl-2 protein quantification and we could determine the concen-
tration of Bcl-2 protein based on the BPFP1's fluorescence intensity. Subsequent studies revealed that
BPFP1 can fluorescently label carcinoma cells by binding to overexpressed Bcl-2 protein in living cells,
and can distinguish carcinoma cells (HL-60 cells and ACHN cells) from normal-tissue cells (HUVECs)
according to the different Bcl-2 protein expression levels between carcinoma cells and normal tissue
cells. In the present study, BPFP1 represents a new tool for Bcl-2 protein quantification, carcinoma cell
visualization and cell sorting. Moreover, BPFP1 can be used in the future for early cancer diagnosis by
detecting carcinoma cells in patient tissues.
Received 29 December 2020
Received in revised form
3 July 2021
Accepted 26 July 2021
Keywords:
Bcl-2 protein fluorescence probe
Bcl-2 protein quantification
Carcinoma cell visualization
Cell sorting
Early cancer diagnosis
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Bcl-2 family proteins are the cardinal regulators of the mito-
chondrial pathway and they are subdivided into three groups ac-
The growing incidence of cancer, a major health problem, has
attracted people's attention around the world [1e6]. Decades of
efforts have led to the development of various significant thera-
peutic regimens for cancer, whereas the overall five-year survival
rate of patients remains low because of the delay in cancer diag-
nosis and the difficulty of cancer treatment [1,7e9]. Accumulating
evidence revealed that early cancer diagnosis is central to the
achievement of better outcomes and that effective clinical man-
agement of cancer is enabled by early cancer detection [10e13].
Although some methods have been developed for early cancer
diagnosis [14e19], the lack of effective tools and bureaucratic
procedures have greatly hindered the development of early cancer
diagnosis [20]. In the present study, we sought to develop an
effective and convenient tool for early cancer diagnosis.
cording to the functions and Bcl-2 homology (BH) domains: anti-
apoptotic proteins (Bcl-2, Mcl-1, Bcl-XL, and Bcl-w, etc.), pro-
apoptotic proteins (Bax, Bak, and Bok) and BH3-only proteins
(Bax, Bid, Bim, Noxa, and Puma, etc.) [21,22]. The complex in-
teractions between Bcl-2 family proteins regulate the apoptosis
process, whereas overexpression of anti-apoptotic proteins pre-
vents the activation of pro-apoptotic proteins, thereby abolishing
the apoptosis event [23e26]. Accumulating evidence indicates that
carcinoma cells frequently overexpress anti-apoptotic proteins,
especially anti-apoptotic protein Bcl-2, to escape apoptosis
[27e29]. Overexpression of Bcl-2 protein has emerged as a hall-
mark of carcinoma cells [30e34] and can be employed as a
biochemical biomarker to identify carcinoma cells. Moreover, car-
cinoma cells can be distinguished from normal-tissue cells
* Corresponding author.
** Corresponding author.
E-mail addresses: xinyingyang@sdu.edu.cn (X. Yang), haofangcn@sdu.edu.cn (H. Fang).
https://doi.org/10.1016/j.ejmech.2021.113725
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

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T. Liang, J. Li, Y. Zhou et al.
European Journal of Medicinal Chemistry 224 (2021) 113725
according to the expression level of Bcl-2 protein with normal-
tissue cells expressing less Bcl-2 protein than carcinoma cells.
Therefore, the tool for Bcl-2 protein quantification in living cells can
be also applied in carcinoma cell visualization, cell sorting and early
cancer diagnosis.
to obtain intermediate 6, which was converted into BPFPs1-4 by
coupling with different fluorescence group. The structures of in-
termediates and BPFPs1-4 were characterized by 1H NMR, 13C
NMR, and High-Resolution Mass Spectrometry (HRMS).
Fluorescence probes can be powerful tools and are widely used
in diverse fields [35e38]. Some Bcl-2 protein fluorescence probes
have been developed but their poor Bcl-2 protein affinities and Bcl-
2 protein selectivity make it difficult to achieve Bcl-2 protein
quantification [39]. Therefore, we need a Bcl-2 protein fluorescence
probe with very high Bcl-2 protein binding affinity, Bcl-2 protein
selectivity as well as the desired fluorescence properties to achieve
Bcl-2 protein quantification. Decades of efforts have developed
some compounds targeting Bcl-2 protein [40e43] and ABT-199
(Venetoclax) was identified as the molecule with the highest Bcl-
2 protein binding affinity (Ki < 1 nM) and selectivity [44]. To ach-
ieve above aims and improve the rationality of the Bcl-2 protein
fluorescence probe, we sought to modify the structure of ABT-199,
and develop an ABT-199-based Bcl-2 protein fluorescence probe.
Before that, the binding mode of ABT-199 to Bcl-2 protein (PDB ID:
2O2F) was studied, and the results suggested that the tetrahydro-
1,10-biphenyl fragment accommodates the P2 pocket and the 1H-
pyrrolo[[2,3-b]]pyridine fragment occupies the P4 pocket, whereas
the 4-chloro-3-nitrobenzenesulfonamide group is exposed to the
solvent (Fig. 1A). Given the importance of these functional frag-
ments, we set out to retain these fragments and replace the
solvent-exposed fragment with a fluorescence group (Fig. 1B) to
develop Bcl-2 protein fluorescence probes (BPFPs).
2.2. Bcl-2 protein binding affinities and selectivities
The Bcl-2 protein binding affinities and selectivities of BPFPs1-4
were evaluated using the competitive fluorescence polarization
binding assay with ABT-199 as the positive control. As shown in
Table 1, the Bcl-2 protein binding affinities of BPFP1 (Ki < 1 nM)
and BPFP2 (Ki < 1 nM) were comparable to the affinity of the
positive control, ABT-199 (Ki < 1 nM). However, BPFP3 and BPFP4
showed low Bcl-2 protein binding affinities due to the lack of the
acyl-sulfonamide group. None of the tested compounds showed
significant Mcl-1 protein binding affinity (Ki > 50000 nM). It's to be
noted that BPFP1 and BPFP2 exhibit very high Bcl-2 protein af-
finities and over 50000-fold Bcl-2 protein selectivity against Mcl-1
protein. Their high Bcl-2 protein binding affinities and selectivities
endowed them with the ability to specifically detect trace amounts
of Bcl-2 protein.
2.3. Proposed binding mode of BPFP1 and BPFP2 within Bcl-2
protein
BPFP1 and BPFP2 were docked into the Bcl-2 protein binding
site (PDB ID: 2O2F) by means of GLIDE (version 11.5, Schrodinger).
As shown in Fig. 2A, BPFP1 accommodates the P2 pocket as well as
P4 pocket well, and the binding mode of BPFP1 (docking
score, ꢀ7.042) is similar to that of ABT-199 (docking score, ꢀ7.179).
Moreover, a strong interaction was observed between the acyl-
sulfonamide group of BPFP1 and the W141 residues of Bcl-2 pro-
tein (Fig. 2B). In addition, BPFP2 (docking score, ꢀ4.398) can be
tolerated by P2 pocket and P4 pocket (Fig. 2D), whereas no
hydrogen bond interaction was monitored (Fig. 2D).
2. Results and discussion
2.1. Synthesis and characterization of BPFPs
The synthetic routes for target compounds are depicted in
Scheme 1. As shown in Scheme 1, methyl 4-bromo-2-fluorobenzoate
was reacted with 1-boc-piperazine to give intermediate 2. The
nucleophilic aromatic substitution reaction between intermediate
2 and 5-hydroxy-7-azaindole was performed to synthesize com-
pound 3. After removing the t-butyloxy carbonyl group, Borch
reduction was conducted to prepare key intermediate 5 using
compounds 4 and 40-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,10-
biphenyl]-2-carbaldehyde. The hydrolysis reaction was simply used
2.4. Spectroscopic properties of BPFPs
As summarized in Table 2, BPFP1 possesses an excitation
wavelength at 340 nm, an emission wavelength at 520 nm and a
good fluorescence quantum yield (28.3%). Moreover, BPFP2 ex-
hibits similar spectroscopic properties but its fluorescence
Fig. 1. (A) Binding mode of ABT-199 to Bcl-2 protein; (B) Design of the BPFPs.
2

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Scheme 1. Synthetic Route of BPFPs1-4a.
aRegents and Condition: (a) N-Boc-piperazine, Pd(AcO)2, toluene, 60 ꢁC, 6h, 81%; (b) 1H-pyrrolo[2,3-b]pyridin-5-ol, NaH, DMF, 120 ꢁC, 8h, 76%; (c) (i) HCl/ethyl acetate, ethyl acetate,
(ii) Saturated NaHCO3, ethyl acetate, 1h, 96%; (d) 40-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,10-biphenyl]-2-carbaldehyde, NaBH(AcO)3, rt, overnight, 79%; (e) KOH, THF/MeOH/
H2O ¼ 1:3:1, refluxed, 6h, 89%; (f) Isobutyl chloroformate, 4-methylmorpholine, NaH, DMF, 80 ꢁC, 8h, 60e62%; (g) HATU, DIEA, DMF, 60 ꢁC, overnight, 47e61%.
quantum yield is low (0.7%). Therefore, the relatively reasonable
fluorescence quantum yield of BPFP1 suggested that BPFP1 can be
selected for further studies.
2.5. Excitation and emission spectra of BPFP1 in different solvents
Subsequently, we explored the fluorescence excitation and
emission spectra of BPFP1 (1 mM) in different solvents. As we ex-
pected, the excitation spectra, emission spectra, and fluorescence
intensity of BPFP1 varied among the different solvents (Fig. 3). The
experimental results suggested that the fluorescent properties of
BPFP1 change with the change of its environment. We think that
BPFP1 in a free state and BPFP1 binding to Bcl-2 protein are in
different environments. BPFP1 in a free state not only has intra-
molecular motion, but also interacts with solvent molecules, which
leads to a small amount of energy of the excited BPFP1 molecule
released in the form of fluorescence. However, BPFP1 binding to
Bcl-2 protein abolishes its intramolecular motion as well as inter-
molecular interaction and more energy of the excited BPFP1
molecule is released in the form of fluorescence. Therefore, we
Table 1
Bcl-2 protein affinities and selectivities of BPFPs.
Compound
Bcl-2
Bcl-2 selectivity against Mcl-1
BPFP1
BPFP2
BPFP3
BPFP4
ABT-199
aKi < 1 nM
Ki < 1 nM
bNA
NA
Ki < 1 nM
>50000-fold
>50000-fold
̶
̶
>50000-fold
a
All compounds were assayed three times.
NA, no activity.
b
3

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Fig. 2. (A) Proposed binding mode of BPFP1 within the Bcl-2 binding site; (B) Interactions between BPFP1 and Bcl-2 (PDB ID: 2O2F); (C) Proposed binding mode of BPFP2 in the Bcl-
2 binding site; (D) Interactions between BPFP2 and Bcl-2 (PDB ID: 2O2F).
Table 2
To exclude the effect of Mcl-1 protein on the fluorescence in-
tensity of BPFP1, coincubation of BPFP1 with 0.125 mg/mL Mcl-1
protein was performed. Following incubation with Mcl-1 protein,
the fluorescence properties of BPFP1 were determined but no
change was observed (Fig. 4D). Thus, the presence of Mcl-1 protein
does not affect the fluorescence properties of BPFP1.
Spectroscopic properties of the tested BPFPs.
lmax (nm)
l
ex (nm)
l
em (nm)
F (%)
BPFP1
BPFP2
310
332
340
350
520
480
28.3
0.7
Combined with the above experimental results, we hypothe-
sized the mechanism of the fluorescence enhancement of BPFP1.
We think that BPFP1 in a free state possesses intramolecular mo-
tion which consumes the energy of excited BPFP1, and little energy
released as fluorescence. When BPFP1 binding to Bcl-2 protein,
restriction of intramolecular motion (RIM) of BPFP1 occurs and
more energy of excited BPFP1 released as fluorescence, followed by
the fluorescence enhancement of BPFP1. As the protein concen-
tration increases, more BPFP1 binds to the Bcl-2 protein and more
restriction of intramolecular motion (RIM) of BPFP1 occurs, that
results in the fluorescence of BPFP1 being proportional to the Bcl-2
protein concentration.
speculated that incubating BPFP1 with Bcl-2 protein might
enhance its fluorescence.
2.6. Bcl-2 protein quantification and fluorescent properties of
BPFP1 Co-incubated with Bcl-2 protein or Mcl-1 protein
To confirm our hypothesis, we measured the fluorescence in-
tensity of BPFP1, which was incubated with a range of concentra-
tions of Bcl-2 protein. As we expected, upon incubation with Bcl-2
protein, the fluorescence intensity of BPFP1 was enhanced
(Fig. 4A). Further studies revealed that BPFP1's fluorescence in-
tensity is proportional to Bcl-2 protein concentration (Fig. 4B).
Therefore, BPFP1 provides a convenient approach for Bcl-2 protein
quantification and we could calculate the concentration of Bcl-2
protein based on the BPFP1's fluorescence intensity.
2.7. Fluorescence imaging
Encouraged by the good properties of BPFP1, fluorescence im-
As a potent and selective Bcl-2 inhibitor, ABT-199 exhibits strong
Bcl-2 protein binding affinity (Ki < 1 nM) and it could compete with
BPFP1 for Bcl-2 binding sites. To determine whether the fluores-
cence enhancement was caused by the binding of BPFP1 to Bcl-2
aging of carcinoma cells (ACHN cells) and normal-tissue cells
(HUVECs) was performed. Upon incubation with 1
mM BPFP1,
ACHN cells were fluorescently labeled, and strong green fluores-
cence was observed (Fig. 5A), whereas the addition of ABT-199
protein, 10
m
M ABT-199 was co-incubated with 2
m
M BPFP1 and
(5
bating BPFP1 (1
ABT-199 (5 M), none of the HUVECs emitted fluorescence (Fig. 5C
and D). The distinct results of fluorescence imaging on carcinoma
cells (ACHN cells) and normal-tissue cells (HUVECs) indicated that
BPFP1 can selectively label carcinoma cells with green fluorescence
and spare normal-tissue cells.
m
M) quenched the fluorescence (Fig. 5B). Moreover, after incu-
0.125 mg/mL Bcl-2 protein. With the titration of ABT-199, BPFP1
was competed from the Bcl-2 protein binding site. Then, we
monitored a diminished fluorescence intensity, which is equivalent
to BPFP1's fluorescence intensity (Fig. 4C). The above results indi-
cated that the binding of BPFP1 to Bcl-2 protein enhances its
fluorescence intensity.
mM) with HUVECs in the presence or absence of
m
Fig. 3. Excitation spectra and emission spectra of BPFP1 (1 mM) in different solvents.
4