Antidiabetic activity in vitro and in vivo of BDB, a selective inhibitor of protein tyrosine phosphatase 1B, from Rhodomela confervoides

Luo Jiao (Orcid ID: 0000-0003-4810-2009)

Yu Rilei (Orcid ID: 0000-0001-6625-2014)

Title: Antidiabetic activity in vitro and in vivo of BDB, protein tyrosine phosphatase 1B

selective inhibitor from Rhodonela conferviodes

Running title: Antidiabetic effects of BDB as PTP1B inhibitor

Authorship: Jiao Luo^b,c*, Meiling Zheng^f, Bo Jiang^c,e, Chao Li^c,e, Shuju Guo^c,e, Lijun Wang^c,e,

Xiangqian Li^a,d, Rilei Yu^fand Dayong Shi^a,d*

^aState Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China;

^bSchool of Public Health, Qingdao University, Qingdao 266071, China;

^cCAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese

Academy of Sciences, Qingdao 266071, China;

^dLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine

Science and Technology, Qingdao 266071, China;

^eCenter for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao

266071, China;

^fKey Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and

Pharmacy, Ocean University of China, Qingdao 266003, China;

*Corresponding authors:

Jiao Luo, School of Public Health, Qingdao University, Qingdao 266071, China; E-mail:

luojiao2012@163.com.

Dayong Shi, State Key Laboratory of Microbial Technology, Shandong University, Jinan

250100, China; E-mail: shidayong@sdu.edu.cn.

Bullet point summary

- What is already known

Our previous study reported for the first time that BDB is a naturally occurring PTP1B

inhibitor.

-What this study adds

BDB is demonstrated to be a selective and cell-permeable inhibitor of PTP1B, and oral

administration of BDB is shown to prevent hyperglycemia and dyslipidemia in diabetic mice

and concurrently improve pancreatic islet architecture.

- Clinical significance

BDB could emerge as a clinical drug candidate for T2DM treatment in the near future.

Abstract

Background and Purpose

Protein tyrosine phosphatase (PTP) 1B (PTP1B) plays a critical role in the regulation of

obesity, type 2 diabetes mellitus (T2DM), and other metabolic diseases. However, drug

candidates exhibiting PTP1B selectivity and oral bioavailability are currently lacking. Here,

the enzyme inhibitory characteristics and pharmacological benefits of BDB were investigated

in vitro and in vivo.

Experimental Approach

Surface plasmon resonance (SPR) assay was performed to validate the direct binding of BDB

to PTP1B, and Lineweaver-Burk plotting of the enzymatic reaction was used to identify the

inhibition type of BDB. Both in vitro enzyme-inhibition assays and SPR experiments were

also conducted to study the selectivity exhibited by BDB toward four other PTP-family

proteins: TC-PTP, SHP-1, SHP-2, and LAR. C2C12 myotubes were used to evaluate BDB

cell permeability and effect on insulin signaling, and hypoglycemic and hypolipidemic effects

of BDB were investigated in diabetic BKS db mice by using oral gavage. The beneficial

effects of BDB on pancreatic islets were examined based on insulin and/or glucagon staining.

Key Results

BDB acted as a competitive inhibitor of PTP1B and demonstrated high selectivity for PTP1B

among the tested PTP-family proteins. Moreover, BDB was cell permeable and enhanced

insulin signaling in C2C12 myotubes. Lastly, oral administration of BDB produced effective

antidiabetic effects in spontaneously diabetic mice and markedly improved islet architecture,

which was coupled with an increase in the ratio of β-cells to α-cells.

Conclusion and Implications

BDB application offers a potentially practical pharmacological approach for treating T2DM

by selectively inhibiting PTP1B.

Key words:

Bromophenol; PTP1B inhibitor; Selectivity; Cell permeability; oral bioavailability

Abbreviations

AUC, area under the curve; ANOVA, one-way analysis of variance; BDB,

3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol; BKS, C57BLKS/J;

BKS db, BKS.Cg-Dock7^m+/+Lepr^db/J; CETSA, cellular thermal-shift assay; CMC-Na, sodium

carboxymethyl cellulose; DARTS, drug affinity responsive target stability; EDC,

N-ethyl-N’-(dimethylaminopropyl)-carbodiimide; FBG, fasting blood glucose; FFA, free

fatty acid; GSA, glycosylated serum albumin; HDL-C, high-density lipoprotein cholesterol;

IDT, International Diabetes Federation; INS, insulin; IR, insulin receptor; IRS, insulin

receptor substrate; ITT, insulin tolerance test; LDL-C, low-density lipoprotein cholesterol;

NHS, N-hydroxysuccinimide; pNPP, p-nitrophenyl phosphate; OGTT, oral glucose tolerance

test; PTP1B, Protein tyrosine phosphatase 1B; PTPs, protein tyrosine phosphatases; RBG,

random blood glucose; SPR, surface plasmon resonance; T2DM, type 2 diabetes mellitus; TC,

total cholesterol; TG, triglyceride; pTyr, phosphotyrosine.

Introduction

Diabetes is a worldwide epidemic that poses a major threat to global public health (Zheng,

Ley & Hu, 2018). The 9^thDiabetes Atlas released by the International Diabetes Federation

(IDF) identifies Asia as the epicenter of the diabetes crisis, with China being the country most

affected by diabetes (IDF, 2019). Type 2 diabetes mellitus (T2DM) accounts for 90%-95% of

diabetes mellitus, which is characterized by insulin resistance and/or inadequate insulin

secretion (American Diabetes, 2018; Chatterjee, Khunti & Davies, 2017). Although

considerable progress has been made in elucidating the molecular mechanisms of T2DM,

satisfactory treatment modalities for diabetes remain limited (Leroith & Accili, 2008;

Mittermayer et al., 2015).

Protein tyrosine phosphatase (PTP) 1B (PTP1B) downregulates insulin and leptin signaling

by catalyzing tyrosine dephosphorylation of insulin receptor (IR), insulin receptor substrate

(IRS), and leptin receptor (Byon, Kusari & Kusari, 1998; Kenner, Anyanwu, Olefsky &

Kusari, 1996; Zhang, Dodd & Tiganis, 2015), and PTP1B is thus a favorable drug target in

therapeutic interventions for obesity and T2DM (Johnson, Ermolieff & Jirousek, 2002;

Krishnan et al., 2018; Zhang & Zhang, 2007). Genetic evidence indicates a silent

heterologous single nucleotide polymorphism in exon8 and an insert variant in the 3ʹ-UTR of

PTP1B are associated with increased risk of developing T2DM as compared with the risk in

people who carry the wild-type PTP1B gene (Di Paola et al., 2002; Mok et al., 2002). Mice in

which the PTP1B gene is knocked out are healthy and fertile, and the mice exhibit increased

insulin sensitivity and resistance to high-fat diet-induced obesity (Delibegovic et al., 2009;

Elchebly et al., 1999; Haj, Zabolotny, Kim, Kahn & Neel, 2005; Klaman et al., 2000).

Because PTP1B is a highly validated therapeutic target, numerous academic institutions and

pharmaceutical companies have invested substantially in developing small-molecule

inhibitors of PTP1B, and although several natural and synthetic PTP1B inhibitors have been

reported, the USFDA has not yet approved any drug for marketing (Erbe et al., 2005; Ito,

Fukuda, Sakata, Morinaga & Ohta, 2014; Lantz et al., 2010; Zasloff et al., 2001). The main

challenge has been to identify PTP1B inhibitors that exhibit high selectivity, cell permeability

and oral bioavailability (Combs, 2010).

Discovery of lead compounds and new drugs from the ocean holds considerable potential for

yielding favorable therapeutic options for diverse diseases (Malve, 2016). Marine red algae of

the family Rhodomelaceae contain numerous bromophenol compounds, and many of these

compounds can produce various biological effects, including cytotoxic effect (Wu et al.,

2015), antioxidative effect (Li, Li, Ji & Wang, 2007), and glucose 6-phosphate

dehydrogenase inhibitory effect (Mikami, Kurihara, Kim & Takahashi, 2013). Notably,

several of these compounds exhibit PTP1B inhibitory activity, and ethanol extracts from the

red alga Rhodomela confervoides lower fasting glucose levels in the rat model of

streptozotocin-induced diabetes (Shi, Xu, He, Li, Fan & Han, 2008). Accordingly,

3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol (BDB, chemical

structure in Figure 1A), a natural bromophenol isolated from R. confervoides, was

demonstrated to act as a potent PTP1B inhibitor (Cui, Shi & Hu, 2011; Fan, Xu & Shi, 2003;

Shi, Xu, He, Li, Fan & Han, 2008). Considering these previous studies, we hypothesized that

BDB could represent a promising therapeutic agent against T2DM. Therefore, in this study

we aimed to elucidate the PTP1B inhibitory characteristics of BDB and evaluate the

hypoglycemic and hypolipidemic effects of BDB in spontaneously diabetic mice by using

oral gavage. Moreover, we sought to examine the protective effects of BDB on pancreatic

β-cells.

Methods

In vitro enzyme-inhibition assay

The inhibitory effects of BDB on PTP1B and TC-PTP were assayed using p-nitrophenyl

phosphate (p-NPP) as the substrate; the buffer solution contained 10 mM Tris-HCl (pH 7.5),

25 mM NaCl, 1 mM EDTA and 1 mM DTT, and the assay was performed as follows: 1μL of

enzyme (1 mg/mL) and 1μL of compounds (10 mM stock solution in DMSO) were added to

10μL of 50 mM p-NPP in 88μL of buffer solution in 96-well plates, and after the reaction

mixture was incubated for 30 min at 37 °C, the amount of p-nitrophenol, the catalytic product,

was determined by measuring the 405-nm absorbance in a microplate reader. The inhibitory

effects of the tested compounds are shown as the concentration that inhibited 50% of PTP1B

enzyme activity (IC₅₀). Assays for SHP-1, SHP-2, and LAR were performed using OMFP as

the substrate.

Lineweaver-Burk analysis

BDB and human PTP1B_1–321recombinant protein were preincubated at room temperature for

5 min, and then reaction mixtures (in 96-well plates) containing 10 mM Tris-HCl (pH 7.5), 25

mM NaCl, 1 mM EDTA, 1 mM DTT, and a series of p-NPP concentrations were incubated at

37 °C for 10 min; subsequently, the p-nitrophenol content was determined by measuring the

405-nm absorbance. In the presence of 1, 2, 4 and 8 μM BDB, the 1/[S] (mM^-1) and 1/[V]

(1/OD·s^-1) values were determined from the substrate concentration and initial reaction rate,

respectively, which were then plotted on the x-axis and y-axis. The enzyme inhibition type

was identified from the intersection characteristics of the linear-regression lines.

Surface plasmon resonance (SPR) assay

SPR experiments were conducted using a Biacore T200 SPR spectrometer. For surface

treatment of the CM5 sensor chip, PTP1B protein was immobilized on the chip by Amine

Coupling Kit (BR-1000-50, GE Healthcare). First, the chip was activated by injecting a 1:1

mixture of 0.2 M EDC (N-ethyl-Nʹ-(dimethylaminopropyl)-carbodiimide) and 0.05 M NHS

(N-hydroxysuccinimide) for 420 s at a flow rate of 10 μL·min^-1; the PTP1B protein was

diluted in 10 mM sodium acetate buffer (pH 5.0) to 10 μg·mL^-1, and ultimately ~10,000

response units (RU) were immobilized. Next, 1 M ethanolamine (pH 8.5) was applied for 7

min at a flow rate of 10 μg·mL^-1to block excess reactive esters on the matrix. For use as the

reference, the flow cell was subject to the aforementioned activation, immobilization and

blocking steps, but no protein was injected.

For kinetic analysis of small molecules and their corresponding protein interactions, the small

molecules were prepared as 10 mM stock solutions in DMSO and then diluted in PBS-P20

buffer containing 5% DMSO. All compounds were injected into the protein-immobilized and

reference flow cells for 60 s, which was followed by the allotted dissociation time of 60 s. To

correct for the excluded volume effect, we used a series of DMSO calibration solutions. For

kinetic evaluations, the reference data and blank data were subtracted by using the Biacore

T200 evaluation software, with the solvent correction being performed concomitantly, after

which curve-fitting (global fitting, 1:1 model) was performed for the entire dataset. For the

layout of all the graphical data, we used GraphPad Prism 6.01 software (GraphPad Software,

RRID: SCR_002798).

Cell culture and western blotting

C2C12 myoblasts (ATCC, Cat# CRL-1772, RRID: CVCL_0188) were purchased from ATCC

(Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum

(FBS). The cells were generally seeded into 6-well plate at 4 × 10⁵cells/well, and at 90%

confluence, the growth medium was changed to DMEM containing 10% horse serum (HS) to

induce differentiation. After 4 days of differentiation, C2C12 skeletal muscle cells were

starved overnight in serum-free DMEM, and then treated for 8 h with BDB at different

concentrations and either not stimulated or stimulated with insulin (100 nM) for 5 min.

Cells were lysed with RIPA buffer containing fresh PMSF, and after determining the

total-protein concentrations of the lysates by using a BCA kit, proteins were separated using

SDS-PAGE and transferred to PVDF membranes. To assess the phosphorylation levels of

insulin receptor substrate 1 (IRS1) and Akt, membranes were blocked with 5% skim milk and

incubated with primary antibodies overnight at 4 ℃ and then with secondary antibodies for 1

h at room temperature. Immunoreactive bands were detected using Western ECL Substrate,

and images were captured using a ChemiDoc™ XRS⁺System (Bio-Rad). To detect the total

protein levels of IRS1 and Akt, membranes were washed with stripping buffer (Thermo) and

immunoblotted as described above. Five independent experiments were performed. All

western blotting and experimental procedures conformed with British Journal of

Pharmacology (BJP) guidelines (Alexander et al., 2018).

HPLC analysis for measuring cell permeability

This HPLC-based experiment was performed as previously reported (das Neves, Sarmento,

Amiji & Bahia, 2012; Luo et al., 2018). Briefly, C2C12 skeletal muscle cells were pretreated

with BDB (10μ) for 8 h, fixed with methanol, lysed using sonication, and then centrifuged

to obtain the supernatant. The supernatant was evaporated under reduced pressure, and then

the precipitate was dissolved in methanol and subject to HPLC analysis (π-Nap, 75%

methanol/H2O + 2% acetic acid). DMSO-treated cells were used as the negative control.

Cellular thermal-shift assay (CETSA)

CETSA studies were performed based on the method described previously (Martinez Molina

et al., 2013). For cell-lysate CETSA, C2C12 skeletal muscle cells were collected in 1× PBS

containing fresh protease-inhibitor cocktail and then frozen and thawed thrice (with liquid

nitrogen being used for freezing cells). The lysates were divided into two aliquots, and one

aliquot was treated with 100 μM BDB and one with DMSO (control). After 20-min

incubation at room temperature, each lysate was divided into 14 fractions, which were heated

at different temperatures (42-68 °C) for 3 min and then cooled at room temperature for 3 min;

lastly, the lysates were boiled, separated using SDS-PAGE, and immunoblotted for PTP1B.

For intact-cell CETSA, C2C12 myotubes were pretreated with BDB (10 μM) for 8 h and then

heated and cooled as described above. After the cells were frozen and thawed twice, lysates

were collected by means of centrifugation and analyzed through western blotting.

Drug affinity responsive target stability (DARTS) assay

To determine PTP1B stability under protease treatment, the DARTS assay was performed as

described previously (Pai et al., 2015). Differentiated C2C12 myotubes were lysed using

M-PER buffer containing a protease-inhibitor cocktail and cell lysates were diluted in TNC

buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 10 mM CaCl₂) and then treated with

BDB at different concentrations (10, 30, and 100 μM) or DMSO (control). After incubating

the mixtures at room temperature for 30 min, pronase (12.5 μg·mL^-1) was added, and the

incubation was continued for another 15 min at room temperature. The reaction was

terminated by adding the protease-inhibitor cocktail, and the lysates were boiled and analyzed

through western blotting with anti-PTP1B antibody (Cat# sc-133259, RRID: AB_2174955).

GAPDH was used as a negative control.

Animal studies

Animals. We purchased 4-6-week-old male BKS.Cg-Dock7^m+/+Lepr^db/J diabetic mice (BKS

db mice, also called db/db mice; The Jackson Laboratory, stock number 000642; RRID:

IMSR_JAX:000642) and their lean C57BLKS/J wild-type controls (BKS mice; The Jackson

Laboratory, stock number 000662; RRID: IMSR_JAX:000662) from the Model Animal

Research Centre of Nanjing University (Nanjing, China).

Validity. BKS db mice replicate various aspects of human T2DM Phases I-III. The blood

glucose level of the BKS-background diabetic mice increased uncontrollably with age, and

their islet β-cells decreased substantially.

Housing and husbandary. Mice were housed in specific-pathogen-free conditions (2

mice/cage), under controlled temperature (24± 2 °C) and humidity (50-60%) and a regular

12/12-h light/dark cycle, and allowed free access to a normal chow diet and water.

Ethical statement. All animal care and experiments were performed according to the

guidelines approved by the Institute of Oceanology committees for the care and use of

laboratory animals (HAIFAJIZI-2013-3; approval date: December 09, 2013). Furthermore,

animal studies are reported in compliance with ARRIVE guidelines (Kilkenny, Browne,

Cuthill, Emerson, Altman & Group, 2010; McGrath, Drummond, McLachlan, Kilkenny &

Wainwright, 2010). Efforts were made to minimize animal suffering.

Blinding. Drugs (vehicle, metformin, and different concentrations of BDB) were prepared

and numbered by the designer. The operator was blinded to the drugs.

Studies conducted using BKS db mice. After 1 week of acclimatization, plasma glucose levels

of all mice were assessed to verify the diabetic status, and the qualified diabetic mice were

randomly divided into four groups (n =8): diabetic model group (BKS db, treated with

sodium carboxymethyl cellulose (CMC-Na) solution), metformin-treatment group

(metformin, 100 mg·kg^-1body wt·day^-1), low-dose BDB-treatment group (BDB-L, 50

mg·kg^-1body wt·day^-1), and high-dose BDB-treatment group (BDB-H, 100 mg·kg^-1body

wt·day^-1). Age-matched male BKS mice (treated with CMC-Na solution) were used as the

normal control (n = 8). All mice were administered drugs orally for 9 weeks. However, one

mouse from BKS db group died at the 5^thweek due to improper gastric administration during

the experimental period (n = 7 after the 5^thweek).

To measure the long-time and short-time fasting glucose (FBG) levels, mice were fasted for

12 and 6 h, respectively. Glucose levels were measured using tail-vein blood and an

ONETOUCH UtraEsay glucometer (Johnson & Johnson, USA).

At the end of the experiment, mice were anesthetized using 10% chloral hydrate and then

blood samples of all mice were collected through the venous plexus behind the eyeball.

Serum was separated using centrifugation and stored at -80 ˚C. Serum total cholesterol (TC),

triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), high-density

lipoprotein-cholesterol (HDL-C), and free fatty acid (FFA) levels were determined according

to the instructions of the assay-kit manufacturer. Because of insufficient serum samples, the

group size varied from 5 to 8 and detailed group size was shown in the figure legends. The

mice were then scarified, and tissues were separated immediately and fixed in 10% formalin

for subsequent immunohistochemical staining.

Oral glucose-tolerance test (OGTT) and insulin-tolerance test (ITT). Given the number of

participants and duration of the OGTT and ITT experiments, 6 mice were randomly chosen

from each group for the following tests. After 6 weeks of treatment, glucose (1.5 g·kg^-1) was

administered using gavage for the OGTT experiment after starvation for 12 h. After 7 weeks

of treatment, insulin (1 U·kg^-1) was injected subcutaneously for the ITT experiment after

fasting for 6 h. Blood glucose was measured using tail-vein blood at the specific times. The

area under the curves (AUC) was calculated from the data collected during the OGTT and

ITT experiments.

Immunohistochemistry. Pancreas from all treated mice were collected and fixed in 4%

paraformaldehyde solution overnight at 4 ℃. Subsequently, paraffin-embedded pancreas

sections were rehydrated and heated with sodium citrate buffer for antigen retrieval and then

blocked with 10% goat serum; to block endogenous peroxidases, the sections were treated

with 3% hydrogen peroxide solution. Next, the sections were incubated (overnight at 4 °C)

with following primary antibodies (from Abcam): guinea pig anti-insulin antibody (1:100,

Cat# ab7842, RRID: AB_306130), rabbit anti-glucagon antibody (1:8000, Cat# ab92517,

RRID: AB_10561971), and rabbiti anti-Ki67 antibody (1 µg/mL, Cat# ab15580, RRID:

AB_443209). For color development, we used the HRP-DAB or AP-Permanent Red system

or both, and then captured digital images and analyzed the results by using a Tissue FAXS

System (TissueGnostics, AT). Samples that showed dark staining and sections that appeared

damaged were excluded from the analysis, and the group size varied from 5 to 6 and detailed

group size was shown in the Figure 7 legend. The histological staining results were

statistically analyzed, in a blinded manner, by a technician from TissueGnostics. All

immunohistochemistry experimental procedures conformed with BJP guidelines (Alexander

et al., 2018).

Synthetic scheme for BDB and methylated BDB

BDB and its methylated derivative (Compound 6) were synthesized as follows. First, vanillin

was reacted with bromine in CH₃OH at 0 °C to obtain Compound 1 at 95% yield (Fürstner,

Stelzer, Rumbo & Krause, 2002). Compound 1 was methylated with CH₃I in DMF to

generate Compound 2(Fürstner, Stelzer, Rumbo & Krause, 2002) (97% yield), and then

Compound 2 was brominated a second time by using two equivalents of bromine in acetic

acid at 70 °C, which yielded Compound 3 (Ford & Davidson, 1993) (63% yield). Compound

3 was reduced with NaBH₄in methanol at 0 °C to obtain an alcohol, Compound 4 (93%

yield), and in the presence of AlCl₃, Friedel-Crafts reaction of Compound 4 with

1,2-dimethoxybenzene generated an intermediate compound, Compound 5 (90% yield).

Compound 5 was reacted with bromine in CH₂Cl₂at room temperature to generate

Compound 6 (81% yield). BDB was obtained at 92% yield (purity > 99%) by removing the

methyl groups from Compound 6 with BBr₃at 0 °C.

The following synthetic scheme was used:

The structural-elucidation data were as follows:

1

Methylated BDB (Compound 6): m.p. 178~180 ℃; H NMR (500 MHz, CDCl₃) δ: 6.60 (s,

1H, ArH), 6.45 (s, 1H, ArH), 6.14 (s, 1H, ArH), 4.20 (s, 2H, ArCH₂Ar), 3.96 (s, 2H,

ArCH₂Ar), 3.88 (s, 3H, OCH₃), 3.80 (s, 6H, OCH₃× 2), 3.79 (s, 3H, OCH₃), 3.71 (s, 3H,

OCH₃), 3.58 (s, 3H, OCH₃); ¹³C NMR (125 MHz, CDCl₃) δ: 152.4, 152.3, 152.2, 146.5,

146.0, 145.6, 136.0, 135.4, 135.2, 129.9, 122.8, 122.1, 121.7, 118.1, 117.5, 114.0, 113.8,

111.9, 60.5, 60.4, 56.2, 56.1, 42.1, 40.5. HR-EIMS m/z 831.7531 [M]⁺, Calc. 831.7527.

BDB: ¹H NMR (500 MHz, acetone-d₆) δ: 6.56 (s, 1H, ArH), 6.50 (s, 1H, ArH), 6.21 (s, 1H,

ArH), 4.05 (s, 2H, ArCH₂Ar), 3.78 (s, 2H); ¹³C NMR (125 MHz, acetone-d₆) δ: 145.7, 145.6,

145.2, 144.0, 143.6, 133.0, 131.9, 129.3, 117.0, 116.5, 129.3,115.1, 115.0, 114.0, 113.9, 41.0,

40.2. HR-EIMS m/z 742.6543 [M]⁺, Calc. 742.6551.

Data and statistical analyses

The data and statistical analyses complied with the recommendations on experimental design

and analysis in pharmacology (Curtis et al., 2018), and the data were analyzed in a blinded

manner. Sample size (animal experiments) was planned based on our previous study on

bromophenol compounds (Li et al., 2019). Group size was equal at design. However, in

certain cases, adequate blood samples or pancreas sections were not obtained during testing,

which explains the small sample size for recordings conducted with BDB. In the analysis of

band density in western blotting, data were normalized to the internal control to exclude

unwanted sources of variation. No data transformation was performed, and no outliers were

removed in data analysis and presentation.

Results are presented as the means ± SEM. Statistical analysis was performed only when

each group contained at least n = 5 independent samples, and the analysis was performed

using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test between

control and various drug-treated groups. Tukey tests were conducted only when F achieved a

p value of < 0.05 and there was no significant variance inhomogeneity. Data from small

group sizes of n < 5 was not subjected to statistical analysis. The declared group size is the

number of independent values, and statistical analysis was performed using these independent

values. Values were considered significant at p <0.05, and this holds for the data shown later

in Results. Analyses were performed using GraphPad Prism 6.01 software.

Materials

The following reagents were from commercial sources: DMEM containing

high-concentration glucose, penicillin-streptomycin, FBS, and HS, Hyclone (South Logan,

UT, USA); EDTA-free protease-inhibitor cocktail and pronase, Roche (Basel, Switzerland);

M-PER buffer, Thermo Fisher Scientific (Waltham, MA, USA); metformin, insulin, and

CMC-Na, Sigma-Aldrich (St. Louis, MO, USA); Series S CM5 sensor chips, EDC, NHS,

PBS-P20 buffer, 1.0 M ethanolamine-HCl (pH 8.5), and maintenance kit, GE (Mississauga,

ON, Canada); DMSO and sodium acetate, Solarbo (Beijing, China); IRS1 antibody (Cat#

2382, RRID: AB_330333), Akt antibody (Cat# 9272, RRID: AB_329827), and phospho-Akt

(Ser473) rabbit mAb (Cat# 4060, RRID: AB_2315049), Cell Signalling Technology (Danvers,

MA, USA); anti-phospho-IRS1 (Tyr608) antibody (Cat# 09-432, RRID: AB_1163457) ,

Millipore (Bedford, MA, USA); insulin antibody (Cat# ab7842, RRID: AB_306130),

glucagon antibody (Cat# ab92517, RRID: AB_10561971 ), and Ki67 antibody (Cat# ab15580,

RRID: AB_443209), Abcam (Cambridge, MA, USA); GAPDH antibody (Cat# 60004-1-Ig,

RRID: AB_2107436), Proteintech (Wuhan, China); BCA Protein Assay Kit, Thermo

(Waltham, MA, USA); Western ECL Substrate, Bio-Rad (Hercules, CA, USA); TG, TC,

LDL-C, HDL-C, and FFA assay kits and glycosylated serum protein assay kit, Nanjing

Jiancheng Bioengineering Institute (Nanjing, China). Recombinant hPTP1B_1-321protein was

purified in our laboratory. Recombinant TC-PTP, SHP-1, SHP-2 and LAR proteins were from

Sino Biological (Beijing, China).

Nomenclature of Targets and Ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in

http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS

Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the

Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

Results

BDB is a competitive inhibitor of PTP1B

Recombinant hPTP1B_1-321was used for measuring the in vitro inhibitory activity of BDB.

BDB potently inhibited PTP1B, with an IC₅₀of 1.864 μM (Figure 1B). To exclude the

possibility that false-positive data were generated from the in vitro enzyme assay, direct

interaction between BDB and PTP1B was detected using SPR analysis, which revealed that

BDB bound to PTP1B with an affinity of 1.812 μM and in a slow-on and slow-off manner

(Figure 1C).

Small-molecule inhibitors can form ligand-protein complexes with their targets and thereby

disrupt the function and increase the stability of target proteins. We used two

target-engagement assays to investigate whether BDB binds to and enhances the stability of

PTP1B. The results of DARTS assay showed that BDB treatment could protect PTP1B from

pronase-induced proteolysis (Figure 1D), and the results of CETSA performed using C2C12

myotube lysates indicated that BDB protected PTP1B against temperature-dependent

degradation (Figure 1E).

Next, the inhibitory type of BDB was studied through Lineweaver-Burk analysis of the

enzyme reaction (Figure 1F); in the generated plot, the regression lines intersected on the

y-axis, and whereas the K_Mvalues increased in a dose-dependent manner, the V_maxvalues did

not change, indicating that BDB is a competitive inhibitor. The measured K_iof BDB was 1.1

μM (Figure 1G).

To ascertain the manners in which BDB binds to PTP1B, we constructed 3D binding models

in an open conformation of PTP1B (PDB code: 2HNP) through docking simulation (Figure

S1 and 1H), which revealed that BDB is embedded in the active site and a dense network of

hydrogen bonds is established between the phenolic hydroxyl groups of BDB and Pro180,

Cys215 and Asp265 of PTP1B; moreover, Lys116 and Arg221 participate in the formation of

cation-π interactions with the phenyl rings. Because of these multiple interactions, BDB can

effectively self-adapt to the active site of the enzyme and function as a potent inhibitor.

Lastly, we again used SPR analysis to examine PTP1B interaction with a methylated

derivative of BDB, which was synthesized in our laboratory. The methylated derivative did

not bind to PTP1B (Figure 1I), which suggests that hydrogen bonding constitutes the main

interaction force that maintains the binding of BDB and PTP1B.

BDB is a specific inhibitor of PTP1B

Considering that PTP-family members harbor a highly conserved catalytic domain, we next

investigated the selectivity of BDB for PTP1B relative to four other PTPs: TC-PTP, SHP-1,

SHP-2, and LAR. At 20 μg·mL^-1, BDB caused 93.97% inhibition of PTP1B but was

markedly less effective against the other tested PTPs (all were inhibited by <50%) (Figure

2A). The catalytic domain of TC-PTP shares 74% sequence identity with that of PTP1B

(Figure S1), but the BDB IC₅₀for TC-PTP was 13.73 μM, which is ~7.5-fold higher than that

for PTP1B (Figure 2B). Moreover, the IC₅₀values for SHP-1, SHP-2, and LAR were

respectively 41.85, 33.43, and 27.61 μM (Figure 2C-E). These results indicate that BDB is a

specific inhibitor of PTP1B.

To further verify this selectivity of BDB, we immobilized the aforementioned four PTPs on

CM5 chips and performed SPR analysis. The affinity of BDB binding to TC-PTP was 16.13

μM (Figure 2F), which is ~8.9-folds lower than the affinity of BDB binding to PTP1B (1.812

μM, Figure 1C). Similarly, BDB exhibited considerably lower binding affinity for SHP-1 and

SHP-2 than PTP1B (Figure 2G-H), and, furthermore, showed no detectable binding to LAR

(Figure 2I).

BDB is cell permeable in C2C12 skeletal muscle cells

To function at cellular and whole-animal levels, BDB must penetrate the cell membrane;

therefore, detection of the cell-membrane permeability of BDB is essential. First, we

calculated the theoretical octanol-water partition coefficient to predict the lipophilicity of

BDB. The theoretical cLogP was 6.94, which indicated preferential distribution of BDB into

a hydrophobic environment rather than a hydrophilic environment; thus BDB is likely to pass

through the cell membrane. Next, we treated C2C12 skeletal muscle cells with 10 μM BDB

for 8 h and then used HPLC to measure the content of BDB in cell lysates (Figure S3 and 3A).

The average ratio of BDB in the cell lysates was 12.76% in the five independent repetitions

of the experiment (Figure 3B), which indicates that BDB successfully passed through the cell

membrane and accumulated intracellularly.

Given that BDB demonstrated the ability to cross the plasma membrane, we next examined

the cellular activity of BDB as a PTP1B inhibitor. We used immunoblotting and analyzed the

phosphorylation level of Tyr608 of IRS1, one of the direct substrates of PTP1B, and Ser473

of Akt, a downstream effector of IRS1 (Figure 3C). Notably, after BDB treatment,

insulin-stimulated phosphorylation of IRS1 Y608 was increased (Figure 3D), as was that of

Akt S473 (Figure 3E); by contrast, PTP1B levels were unaffected by BDB exposure (Figure

3F). These results suggest that BDB enhances insulin signaling directly without altering the

protein level of PTP1B.

Here, we also tested whether BDB can increase the thermal stability of PTP1B in intact cells;

CETSA results indicated that BDB treatment effectively protects PTP1B protein from

temperature-dependent degradation at the intact-cell level (Figure 3G).

Oral administration of BDB prevents hyperglycemia in diabetic BKS db mice

To evaluate the potential antidiabetic effect of BDB in vivo, we used genetically diabetic mice:

BKS db mice (db/db mice); the mice were orally administered BDB (50 and 100

mg·kg^-1body wt·day^-1) for 9 weeks, with metformin (100 mg·kg^-1body wt·day^-1) serving as

the positive control.

Diabetic mice are characterized based on obesity, polyphagia, and polyuria. During the 9

weeks of treatment, BDB did not affect either body weight or food intake of the diabetic mice

(Figure 4A-B). However, water intake of the mice in the BDB-treatment group showed a

decreasing tendency starting from the 5^thweek (Figure 4C) and was significantly decreased at

the 5^thand 7^thweek.

Long-time and short-time fasting glucose levels were significantly decreased starting from

the 2^ndweek of high-dose BDB treatment (Figure 4D-E). Metformin effectively lowered the

blood glucose level from the 3^rdweek onward, which was one week later than when BDB

treatment produced the effect. At the end of the 9^thweek, random blood glucose (RBS) levels

and 12-h fasting blood glucose (FBG) levels were measured, which revealed that BDB

treatment led to significant glycemic control in the diabetic mice (Figure 4F-G). Moreover,

serum glycosylated serum albumin (GSA) levels were ameliorated at the 9^thweek of BDB

treatment (Figure 4H).

To examine the effects of BDB on glucose tolerance and insulin sensitivity, OGTT and ITT

were performed: BDB improved glucose clearance in diabetic mice, with the quantified data

showing that the AUC was decreased by ~25.6% (Figure 5A-B). BDB treatment also

significantly decreased insulin resistance in BKS db mice, as indicated by the ~36.7%

decrease in the AUC (Figure 5C-D).

Collectively, our results suggest that oral administration of BDB prevented hyperglycemia

and improved insulin sensitivity in diabetic BKS db mice.

BDB treatment ameliorates hepatic steatosis and dyslipidemia

As compared with non-diabetic control mice, vehicle-treated diabetic mice exhibited overt

hepatic steatosis, with vacuolated and swollen hepatocytes being detected (Figure 6A). BDB

treatment ameliorated these hepatic disorders and caused significant reductions in circulating

TC, TG, and LDL-C levels relative to the levels after vehicle treatment (Figure 6B-D).

Moreover, HDL-C levels were effectively increased after 9 weeks of BDB treatment (Figure

6E). However, BDB did not affect circulating FFA levels (Figure 6F). These results suggest

that BDB treatment also produces mitigative effects on metabolic abnormalities associated

with T2DM, such as hepatic steatosis and hyperlipidemia.

BDB treatment improves pancreatic islet architecture in BKS db mice

PTP1B inhibitors can effectively reduce blood glucose levels in diabetic animal models, but

the long-term effect of PTP1B inhibition on pancreatic islets remains incompletely studied.

Therefore, we performed multiple-antibody staining of pancreas and acquired digital images

of whole-pancreas sections to characterize islet morphology and measure cell composition

(Figures 7 and S4).

Pancreatic islet architecture and cell composition clearly differed between the nondiabetic

and diabetic groups (Figure 7). In nondiabetic control mice, the islets harbored a large tight

insulin-positive β-cell core surrounded by a layer of slightly glucagon-positive α-cells,

whereas the islets in vehicle-treated diabetic mice contained relatively more

glucagon-positive α-cells, which penetrated the entire islet, including the central core (Figure

7B-D). Notably, 9 weeks of BDB treatment significantly increased the number of β-cells

(Figure 7 B), reduced the number of α-cells in the islets (Figure 7C), and increased the ratio

of β-cells to α-cells (Figure 7D). More importantly, BDB-treatment restored the distribution

pattern of β-/α-cells in the islets: theα-cells in the islet core were markedly decreased and

theα-cells at the islet edges were effectively increased (Figure 7D). Although islet PTP1B

expression did not differ between nondiabetic and diabetic individuals (Figure S4D), our

results indicate that the efficacy of the small-molecule PTP1B inhibitor potentially extends

beyond blood glucose control and helps improve islet architecture.

Lastly, we analyzed β-cell proliferation in islets by double staining for Ki67, a marker of

cellular proliferation, and insulin (Figure 7E). Relative to the level in healthy BKS mice,

fewer Ki67-positive β-cells present in the islets of vehicle-treated diabetic mice, but

Ki67-positive β-cells were readily detected in the islets of BDB-treated BKS db mice (Figure

7F). These results indicate that BDB can enhance the islet architecture and β-cell mass by

promoting the proliferation of β-cells.

Discussion and Conclusions

Over the past decade, PTP1B has been regarded as a favorable drug target for therapeutic

interventions designed for obesity and T2DM (He, Y u , Zhang & Zhang, 2014). Thus, intense

pharmaceutical research has been conducted to identify small-molecule inhibitors of PTP1B

for the treatment of T2DM and obesity-related metabolic diseases. However, most of the

identified inhibitors are negatively charged phosphotyrosine (pTyr) mimics, such as

phosphonates, carboxylic acids, and sulfamic acids, which exhibit poor PTP1B selectivity

and insufficient in vivo efficacy because of low cell permeability and bioavailability (Thareja,

Aggarwal, Bhardwaj & Kumar, 2012). To date, only five small-molecule PTP1B inhibitors

have reached the clinical-trial stage, ertiprotafib, TTP814, JTT-551, MSI-1436, and KQ-791,

and no drug has as yet been approved for treatment.

To overcome the aforementioned challenge, we have focused on investigating small-molecule

PTP1B inhibitors obtained from marine natural products and derivatives (Fan, Xu & Shi,

2003; Jiang, Guo, Shi, Guo & Wang, 2013; Jiang, Shi, Cui & Guo, 2012; Li et al., 2019; Luo,

Jiang, Li, Jia & Shi, 2019; Luo et al., 2018). BDB is a natural bromophenol isolated from the

marine red alga R. confervoides (Fan, Xu & Shi, 2003). Here, we identified BDB as a

competitive inhibitor of PTP1B by using in vitro enzyme-inhibition assays. Moreover, to

eliminate any false-positive data that might be obtained in enzymatic reaction analyses, we

used the SPR assay to examine the direct binding of BDB with PTP1B. The Biacore SPR

method is a rapid, label-free, and real-time technique for detecting biomolecular interactions

(Schasfoort, 2017), and continuous enhancement of the sensitivity of commercial SPR

instruments has resulted in SPR becoming a well-established and necessary research tool for

studying the binding of small-molecule compounds (Liao et al., 2017). The binding affinity

between proteins and small molecules generally ranged from 10^-3to 10^-6M, and the affinity

of BDB for PTP1B was measured to be 1.8×10^-6M. This suggests that BDB is a potent

PTP1B inhibitor.

The results of molecular dynamics simulations revealed that BDB is deeply embedded in the

pocket of the PTP1B catalytic domain and that strong hydrogen-bonding interactions exist

between BDB and the residues of PTP1B. Importantly, by using SPR to examine the

interaction between a methylated derivative of BDB and PTP1B, we determined that the

hydrogen bonds provide the main interaction force underlying the binding of BDB and

PTP1B. Thus, we speculate that BDB inhibits PTP1B by competing with the substrate for

interaction at the enzyme’s catalytic domain, with the BDB binding being mediated by

hydrogen bonding.

The results of docking analyses indicated that BDB interacts with the PTP1B residues Pro180,

Cys215, and Asp265. Intriguingly, these residues are the same as the corresponding residues

in TC-PTP (Figure S2). PTP-family proteins harbor a highly conserved catalytic domain, and

this is particularly relevant here in the case of TC-PTP, which shares 74% sequence identity

with PTP1B in its catalytic domain. Moreover, the active sites of these two proteins are

identical (Andersen et al., 2001; Johnson, Ermolieff & Jirousek, 2002). Poor selectivity can

cause target-related side effects, and selectivity is one of the major challenges faced in the

development of PTP1B inhibitors. Nevertheless, we showed here that BDB acted as a

specific inhibitor of PTP1B, and further that BDB exhibited lower binding affinity for

TC-PTP, SHP-1, and SHP-2 and showed no binding to LAR. The mechanism underlying this

selectivity is unclear, and this could be addressed in future studies by performing 2D-HNMR

and co-crystallization of BDB with PTP1B; however, currently, the selectivity exhibited by

inhibitors toward closely related phosphatases remains challenging to explain.

PTP1B anchors on the cytoplasmic face of the endoplasmic reticulum through a C-terminal

hydrophobic sequence (Frangioni, Beahm, Shifrin, Jost & Neel, 1992) and catalyzes protein

dephosphorylation by means of its charged active site (Xiao et al., 2014). Several compounds

have been found to inhibit PTP1B highly effectively in vitro, but poor cell permeability limits

the ability of these compounds to reach their intracellular target. For example, many of the

inhibitory compounds are highly charged or strongly polar because they contain pTyr

mimetics, and thus these compounds cannot pass through the cell membrane and access

intracellular PTP1B. By contrast, BDB, unlike pTyr mimetics, is an uncharged compound and

its theoretical cLogP was calculated here to be 6.94; thus, BDB is expected to show

preferential distribution into a hydrophobic rather than a hydrophilic milieu and is likely to

pass through the cell membrane. Accordingly, our HPLC analysis of BDB accumulation in

C2C12 myotubes yielded an average BDB ratio in the myotubes of nearly 13%, which

indicates that BDB successfully passed through the cell membrane and accumulated

intracellularly. However, a limitation of the HPLC method is that the nonspecific adsorption

of BDB on the cell membrane cannot be excluded, and thus we performed immunoblotting

and intact-cell CETSA to further assess the ability of BDB to interact with intracellular

PTP1B and enhance PTP1B-mediated insulin signaling. We identified that BDB protected

intracellular PTP1B from temperature-dependent degradation and that BDB directly

increased the phosphorylation levels of IRS1 and Akt without altering the protein level of

PTP1B. These results indicate that BDB is cell permeable. However, high cLogP value

could affect the solubility of BDB, and sequentially may influence the absorption of BDB. It

is essential to perform pharmacokinetic study measure the absorption, distribution, and

elimination of BDB in mice model in our future research.

The in vivo efficacy of several PTP1B inhibitors does not agree with their observed in vitro

effects; thus, pharmacodynamic studies on these inhibitors in diabetic animal models are

indispensable. More importantly, oral bioavailability is one of the major challenges

encountered in the development of PTP1B inhibitors (Combs, 2010). In this study, we

investigated the effects of long-term oral administration of BDB. BDB markedly reduced

fasting glucose levels starting from the 2^ndweek, and this demonstrated that the BDB effect

was measurable one week earlier than the effect of metformin. OGTT results further showed

that BDB increased the ability of diabetic mice to clear glucose, and ITT results showed that

BDB notably decreased insulin resistance in BKS db mice. Taken together, our results

indicated high bioavailability of BDB, and oral administration of BDB prevented

hyperglycemia and concurrently enhanced glucose tolerance and insulin sensitivity.

Dyslipidemia, which affects ~50% of T2DM patients, is a risk factor for cardiovascular

complications. Diabetic dyslipidemia is characterized by a high plasma TG concentration,

low HDL-C concentration, and high LDL-C concentration (Mooradian, 2009). We found that

BDB markedly reduced the circulating levels of TC, TG, and LDL-C and effectively

increased the level of HDL-C, which indicates that BDB exerted ameliorative effects on

aberrant blood lipid levels.

Diabetes mellitus is a progressive disease in which β-cells counts start to decrease from as

early as the diagnosis of pre-diabetes, and these cell numbers in pancreatic islets decrease at a

rate of ~4% per year as the disease progresses (Halban et al., 2014; Wang, Fiaschi -Taesch,

V a savada, Scott, Garcia -Ocana & Stewart, 2015). In T2DM patients, β-cell failure occurs due

to β-cell dedifferentiation, which results in the decrease in β-cell numbers (Halban et al.,

2014). Given this new understanding of the mechanism of β-cell failure, identification of

effective methods to reverse the dedifferentiation of β-cells has emerged as the key to the

treatment of diabetes mellitus. Among the currently approved oral hypoglycemic drugs,

insulin secretagogues can induce β-cell apoptosis (Maedler, Carr, Bosco, Zuellig, Berney &

Donath, 2005) and glinides can damage the β-cell function (Takahashi et al., 2007). Moreover,

long-term use of insulin sensitizers and glycosidase inhibitors does not help improve the

function of β-cells (Halban et al., 2014). Therefore, urgent demand exists for effective

therapeutic drugs to restore β-cell amounts in T2DM patients.

The role of PTP1B in regulating the insulin signaling pathway is widely recognized, but few

investigations to date have focused on the function of PTP1B in β-cells. A previous study

showed that relative to control mice, mice with PTP1B deletion in the pancreas displayed

earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion

under high-fat-diet feeding (Liu et al., 2014). However, highly contrasting results were

obtained in a contemporary study: isolated islets from 8-week-old PTP1B^-/-mice showed

enhanced glucose-stimulated insulin secretion (Fernandez -Ruiz, Vieira, Garcia -Roves &

Gomis, 2014), and pancreatic islets from PTP1B^-/-mice also exhibited, relative to control,

increased β-cell area coupled with higher β-cell proliferation and lower β-cell apoptosis

(Fernandez -Ruiz, Vieira, Garcia -Roves & Gomis, 2014). Here, we used a small-molecule

inhibitor of PTP1B to further investigate the precise role of PTP1B in regulating the

pancreatic β-cell mass. Intriguingly, BDB treatment improved pancreatic islet architecture

and concurrently increased the percentage of β-cells and decreased percentage of α-cells.

Moreover, BDB administration increased the number of Ki67-positive β-cells, which implies

that the treatment promoted β-cell proliferation. Thus, our study indicates that selective

inhibition of PTP1B is not only beneficial for glycemic control, but also conducive to the

proliferation/viability of pancreatic β-cells. A recent study confirmed that the protective effect

exerted by PTP1B on islets represents another mechanism of action of the enzyme: targeting

of pancreatic islet PTP1B improved islet graft revascularization and transplant outcomes

(Figueiredo et al., 2019). Collectively, these findings reveal that targeting PTP1B can

improve the structure and function of diabetic islets.

As compared with kinase research, phosphatase research is still in its infancy. Several early

studies led many to believe that phosphatases were “undruggable” targets because of the high

conservation rate of their active site. Here we have provided proof-of-concept results

indicating that these challenges associated with active-site-directed inhibitors of PTP1B can

be overcome. We have identified BDB as a specific PTP1B inhibitor that exhibits high cell

permeability, and we have also presented evidence that BDB is orally bioavailable and exerts

antidiabetic effects in a spontaneously diabetic mouse model and concurrently enhances

glucose tolerance and insulin sensitivity in these mice. More importantly, we have reported

that BDB improves pancreatic islet architecture and concomitantly increases β-cell amounts.

The current study also has some limitations. Additional investigations into the mechanism

underlying BDB specificity for PTP1B and further testing of BDB selectivity by including

additional PTPs and PTP1B-knockout cells/animals need to be addressed in our future work.

Pharmacokinetic study and BDB toxicity should also be evaluated in mice model.

In conclusion, this study has shown that oral administration of BDB successfully produced

antidiabetic effects by selectively inhibiting PTP1B. Our study has also provided new

insights into the role of PTP1B in pancreatic β-cells. Our hope is that BDB will emerge as a

candidate drug for clinical treatment of T2DM in the near future.

Acknowledgments

This work was supported by the Natural Science Foundation of China (No. 81703354), Key

research and development project of Shandong province (2018GSF118200, 2018GSF118208),

Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC014) and National

Program for Support of Top-notch Young Professionals, Fund of Taishan scholar project,

Shandong Provincial Natural Science Foundation for Distinguished Young Scholars

(JQ201722), NSFC-Shandong Joint Fund (U1706213), Qingdao Marine Biomedical Science

and Technology Innovation Center project (2017-CXZX01-1-1, 2017-CXZX01-3-9).

Author contribution

Jiao Luo conceived, designed and conducted this study. Meiling Zheng and Rilei Yu

performed the molecular docking. Jiao Luo and Bo Jiang drafted and revised the manuscript.

Chao Li, Shuju Guo, Lijun Wang, Xiangqian Li provided great helps for the laboratory

technique, experiments, and data analysis. Dayong Shi provided the financial support and

supervised the study. All authors reviewed the results and approved the final version of the

manuscript.

Conflict of interest

The authors confirm that there are no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent

reporting and scientific rigour of preclinical research as stated in the BJP guidelines for

Natural Product Research, Design and Analysis, Immunoblotting and Immunochemistry, and

Animal Experimentation, and as recommended by funding agencies, publishers and other

organisations engaged with supporting research.

Reference

Alexander SPH, Kelly E, Mathie A, Peters JA, Veale EL, Armstrong JF, et al. (2019). THE CONCISE GUIDE TO

PHARMACOLOGY 2019/20: Introduction and Other Protein Targets. Br J Pharmacol 176 Suppl 1: S1-S20.

Alexander SPH, Roberts RE, Broughton BRS, Sobey CG, George CH, Stanford SC, et al. (2018). Goals and

practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of

Pharmacology. Br J Pharmacol 175: 407-411.

American Diabetes A (2018). 2. Classification and Diagnosis of Diabetes: Standards of Medical Care in

Diabetes-2018. Diabetes Care 41: S13-S27.

Andersen JN, Mortensen OH, Peters GH, Drake PG, Iversen LF, Olsen OH, et al. (2001). Structural and

evolutionary relationships among protein tyrosine phosphatase domains. Mol Cell Biol 21: 7117-7136.

Byon JC, Kusari AB, & Kusari J (1998). Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin

signal transduction. Mol Cell Biochem 182: 101-108.

Chatterjee S, Khunti K, & Davies MJ (2017). Type 2 diabetes. Lancet 389: 2239-2251.

Combs AP (2010). Recent advances in the discovery of competitive protein tyrosine phosphatase 1B inhibitors

for the treatment of diabetes, obesity, and cancer. J Med Chem 53: 2333-2344.

Cui Y, Shi D, & Hu Z (2011). Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new

synthetic bromophenols and their methoxy derivatives. Chinese Journal of Oceanology and Limnology 29:

1237.

Curtis MJ, Alexander S, Cirino G, Docherty JR, George CH, Giembycz MA, et al. (2018). Experimental design and

analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. Br J Pharmacol

175: 987-993.

das Neves J, Sarmento B, Amiji M, & Bahia MF (2012). Development and validation of a HPLC method for the

assay of dapivirine in cell-based and tissue permeability experiments. J Chromatogr B Analyt Technol Biomed

Life Sci 911: 76-83.

Delibegovic M, Zimmer D, Kauffman C, Rak K, Hong EG, Cho YR, et al. (2009). Liver-specific deletion of

protein-tyrosine phosphatase 1B (PTP1B) improves metabolic syndrome and attenuates diet-induced

endoplasmic reticulum stress. Diabetes 58: 590-599.

Di Paola R, Frittitta L, Miscio G, Bozzali M, Baratta R, Centra M, et al. (2002). A variation in 3' UTR of hPTP1B

increases specific gene expression and associates with insulin resistance. Am J Hum Genet 70: 806-812.

Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, et al. (1999). Increased insulin sensitivity

and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283: 1544-1548.

Erbe DV, Wang S, Zhang YL, Harding K, Kung L, Tam M, et al. (2005). Ertiprotafib improves glycemic control and

lowers lipids via multiple mechanisms. Mol Pharmacol 67: 69-77.

Fürstner A, Stelzer F, Rumbo A, & Krause H (2002). Total Synthesis of the Turrianes and Evaluation of Their

DNA‐Cleaving Properties. Chemistry–A European Journal 8: 1856-1871.

Fan X, Xu NJ, & Shi JG (2003). Bromophenols from the red alga Rhodomela confervoides. J Nat Prod 66:

455-458.

Fernandez-Ruiz R, Vieira E, Garcia-Roves PM, & Gomis R (2014). Protein tyrosine phosphatase-1B modulates

pancreatic beta-cell mass. PloS one 9: e90344.

Figueiredo H, Figueroa ALC, Garcia A, Fernandez-Ruiz R, Broca C, Wojtusciszyn A, et al. (2019). Targeting

pancreatic islet PTP1B improves islet graft revascularization and transplant outcomes. Science translational

medicine 11.

Ford PW, & Davidson BS (1993). Synthesis of varacin, a cytotoxic naturally occurring benzopentathiepin isolated

from a marine ascidian. J Org Chem 58: 4522-4523.

Frangioni JV, Beahm PH, Shifrin V, Jost CA, & Neel BG (1992). The nontransmembrane tyrosine phosphatase

PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. Cell 68: 545-560.

Haj FG, Zabolotny JM, Kim YB, Kahn BB, & Neel BG (2005). Liver-specific protein-tyrosine phosphatase 1B

(PTP1B) re-expression alters glucose homeostasis of PTP1B-/-mice. J Biol Chem 280: 15038-15046.

Halban PA, Polonsky KS, Bowden DW, Hawkins MA, Ling C, Mather KJ, et al. (2014). beta-cell failure in type 2

diabetes: postulated mechanisms and prospects for prevention and treatment. Diabetes Care 37: 1751-1758.

Harding SD, Sharman JL, Faccenda E, Southan C, Pawson AJ, Ireland S, et al. (2018). The IUPHAR/BPS Guide to

PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY.

Nucleic acids research 46: D1091-D1106.

He RJ, Yu ZH, Zhang RY, & Zhang ZY (2014). Protein tyrosine phosphatases as potential therapeutic targets. Acta

Pharmacol Sin 35: 1227-1246.

International Diabetes Federation (2019). IDF Diabetes Atlas - Nineth Edition. Retrieved from

https://idf.org/e-library/epidemiology-research/diabetes-atlas.html.

Ito M, Fukuda S, Sakata S, Morinaga H, & Ohta T (2014). Pharmacological effects of JTT-551, a novel protein

tyrosine phosphatase 1B inhibitor, in diet-induced obesity mice. J Diabetes Res 2014: 680348.

Jiang B, Guo S, Shi D, Guo C, & Wang T (2013). Discovery of novel bromophenol

3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol as protein tyrosine

phosphatase 1B inhibitor and its anti-diabetic properties in C57BL/KsJ-db/db mice. Eur J Med Chem 64:

129-136.

Jiang B, Shi D, Cui Y, & Guo S (2012). Design, synthesis, and biological evaluation of bromophenol derivatives as

protein tyrosine phosphatase 1B inhibitors. Arch Pharm (Weinheim) 345: 444-453.

Johnson TO, Ermolieff J, & Jirousek MR (2002). Protein tyrosine phosphatase 1B inhibitors for diabetes. Nature

reviews Drug discovery 1: 696-709.

Kenner KA, Anyanwu E, Olefsky JM, & Kusari J (1996). Protein-tyrosine phosphatase 1B is a negative regulator

of insulin- and insulin-like growth factor-I-stimulated signaling. J Biol Chem 271: 19810-19816.

Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG, & Group NCRRGW (2010). Animal research: reporting

in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577-1579.

Klaman LD, Boss O, Peroni OD, Kim JK, Martino JL, Zabolotny JM, et al. (2000). Increased energy expenditure,

decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.

Mol Cell Biol 20: 5479-5489.

Krishnan N, Bonham CA, Rus IA, Shrestha OK, Gauss CM, Haque A, et al. (2018). Harnessing insulin- and

leptin-induced oxidation of PTP1B for therapeutic development. Nature communications 9: 283.

Lantz KA, Hart SG, Planey SL, Roitman MF, Ruiz-White IA, Wolfe HR, et al. (2010). Inhibition of PTP1B by

trodusquemine (MSI-1436) causes fat-specific weight loss in diet-induced obese mice. Obesity 18: 1516-1523.

Leroith D, & Accili D (2008). Mechanisms of disease: using genetically altered mice to study concepts of type 2

diabetes. Nature clinical practice Endocrinology & metabolism 4: 164-172.

Li C, Luo J, Guo S, Jia X, Guo C, Li X, et al. (2019). Highly Selective Protein Tyrosine Phosphatase Inhibitor,

2,2',3,3'-Tetrabromo-4,4',5,5'-tetrahydroxydiphenylmethane, Ameliorates Type 2 Diabetes Mellitus in BKS db

Mice. Molecular pharmaceutics 16: 1839-1850.

Li K, Li XM, Ji NY, & Wang BG (2007). Natural bromophenols from the marine red alga Polysiphonia urceolata

(Rhodomelaceae): structural elucidation and DPPH radical-scavenging activity. Bioorg Med Chem 15:

6627-6631.

Liao LX, Song XM, Wang LC, Lv HN, Chen JF, Liu D, et al. (2017). Highly selective inhibition of IMPDH2 provides

the basis of antineuroinflammation therapy. Proceedings of the National Academy of Sciences of the United

States of America 114: E5986-E5994.

Liu S, Xi Y, Bettaieb A, Matsuo K, Matsuo I, Kulkarni RN, et al. (2014). Disruption of protein-tyrosine

phosphatase 1B expression in the pancreas affects beta-cell function. Endocrinology 155: 3329-3338.

Luo J, Jiang B, Li C, Jia X, & Shi D (2019). CYC27 Synthetic Derivative of Bromophenol from Red Alga Rhodomela

confervoides: Anti-Diabetic Effects of Sensitizing Insulin Signaling Pathways and Modulating RNA

Splicing-Associated RBPs. Marine drugs 17.

Luo J, Xu Q, Jiang B, Zhang R, Jia X, Li X, et al. (2018). Selectivity, cell permeability and oral availability studies of

novel bromophenol derivative HPN as protein tyrosine phosphatase 1B inhibitor. Br J Pharmacol 175: 140-153.

Maedler K, Carr RD, Bosco D, Zuellig RA, Berney T, & Donath MY (2005). Sulfonylurea induced beta-cell

apoptosis in cultured human islets. The Journal of clinical endocrinology and metabolism 90: 501-506.

Malve H (2016). Exploring the ocean for new drug developments: Marine pharmacology. Journal of pharmacy

& bioallied sciences 8: 83-91.

Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, et al. (2013). Monitoring drug target

engagement in cells and tissues using the cellular thermal shift assay. Science 341: 84-87.

McGrath JC, Drummond GB, McLachlan EM, Kilkenny C, & Wainwright CL (2010). Guidelines for reporting

experiments involving animals: the ARRIVE guidelines. Br J Pharmacol 160: 1573-1576.

Mikami D, Kurihara H, Kim SM, & Takahashi K (2013). Red algal bromophenols as glucose 6-phosphate

dehydrogenase inhibitors. Marine drugs 11: 4050-4057.

Mittermayer F, Caveney E, De Oliveira C, Gourgiotis L, Puri M, Tai LJ, et al. (2015). Addressing unmet medical

needs in type 2 diabetes: a narrative review of drugs under development. Curr Diabetes Rev 11: 17-31.

Mok A, Cao H, Zinman B, Hanley AJ, Harris SB, Kennedy BP, et al. (2002). A single nucleotide polymorphism in

protein tyrosine phosphatase PTP-1B is associated with protection from diabetes or impaired glucose tolerance

in Oji-Cree. The Journal of clinical endocrinology and metabolism 87: 724-727.

Mooradian AD (2009). Dyslipidemia in type 2 diabetes mellitus. Nature clinical practice Endocrinology &

metabolism 5: 150-159.

Pai MY, Lomenick B, Hwang H, Schiestl R, McBride W, Loo JA, et al. (2015). Drug affinity responsive target

stability (DARTS) for small-molecule target identification. Methods Mol Biol 1263: 287-298.

Schasfoort RB (2017) Handbook of surface plasmon resonance. Royal Society of Chemistry.

Shi D, Xu F, He J, Li J, Fan X, & Han L (2008). Inhibition of bromophenols against PTP1B and anti-hyperglycemic

effect of Rhodomela confervoides extract in diabetic rats. Chin Sci Bull 53: 2476-2479.

Takahashi A, Nagashima K, Hamasaki A, Kuwamura N, Kawasaki Y, Ikeda H, et al. (2007). Sulfonylurea and

glinide reduce insulin content, functional expression of K(ATP) channels, and accelerate apoptotic beta-cell

death in the chronic phase. Diabetes Res Clin Pract 77: 343-350.

Thareja S, Aggarwal S, Bhardwaj TR, & Kumar M (2012). Protein tyrosine phosphatase 1B inhibitors: a

molecular level legitimate approach for the management of diabetes mellitus. Med Res Rev 32: 459-517.

Wang P, Fiaschi-Taesch NM, Vasavada RC, Scott DK, Garcia-Ocana A, & Stewart AF (2015). Diabetes

mellitus--advances and challenges in human beta-cell proliferation. Nat Rev Endocrinol 11: 201-212.

Wu N, Luo J, Jiang B, Wang L, Wang S, Wang C, et al. (2015). Marine bromophenol bis

(2,3-dibromo-4,5-dihydroxy-phenyl)-methane inhibits the proliferation, migration, and invasion of

hepatocellular carcinoma cells via modulating beta1-integrin/FAK signaling. Marine drugs 13: 1010-1025.

Xiao P, Wang X, Wang HM, Fu XL, Cui FA, Yu X, et al. (2014). The second-sphere residue T263 is important for

the function and catalytic activity of PTP1B via interaction with the WPD-loop. Int J Biochem Cell Biol 57: 84-95.

Zasloff M, Williams JI, Chen Q, Anderson M, Maeder T, Holroyd K, et al. (2001). A spermine-coupled cholesterol

metabolite from the shark with potent appetite suppressant and antidiabetic properties. Int J Obesity 25:

689-697.

Zhang S, & Zhang ZY (2007). PTP1B as a drug target: recent developments in PTP1B inhibitor discovery. Drug

Discov Today 12: 373-381.

Zhang ZY, Dodd GT, & Tiganis T (2015). Protein Tyrosine Phosphatases in Hypothalamic Insulin and Leptin

Signaling. Trends Pharmacol Sci 36: 661-674.

Zheng Y, Ley SH, & Hu FB (2018). Global aetiology and epidemiology of type 2 diabetes mellitus and its

complications. Nat Rev Endocrinol 14: 88-98.

Figure 1

Chemical structure and PTP1B inhibitory activity of BDB. (A) Chemical structure of BDB.

(B) IC₅₀of BDB inhibitory activity against PTP1B. Data are shown by plotting relative

inhibition rate of PTP1B against log-values of inhibitor concentration. (C) SPR

characterization of binding affinity between BDB and PTP1B, which was immobilized on a

CM5 chip. (D) Drug affinity responsive target stability (DARTS) assay performed using

whole-cell lysates of C2C12 skeletal muscle cells, which were pretreated with 10 μM BDB.

(E) Cellular thermal shift assay (CETSA) performed using lysates of C2C12 myotubes,

which were exposed to 100 μM BDB. (F) Lineweaver-Burk analysis of PTP1B inhibition by

BDB, which was applied at 1, 2, 4, and 8 μM BDB; the reciprocal of reaction velocity (1/V_i)

is plotted against the reciprocal of substrate concentration (1/[p-NPP]). (G) Ki of BDB

inhibitory activity against PTP1B. Data are shown by plotting K_mappvalues against the

inhibitor concentrations. (H) Mode of BDB binding to PTP1B active site (PDB code 2HNP).

The inhibitor BDB and key residues of PTP1B are shown in a capped stick representation.

Carbon is shown in green for ligands and gray for PTP1B, oxygen is in orange, and bromine

is in red. (I) SPR analysis of binding affinity between methylated BDB and PTP1B.

Article Doi

Antidiabetic activity in vitro and in vivo of BDB, a selective inhibitor of protein tyrosine phosphatase 1B, from Rhodomela confervoides

DOI: 10.1111/bph.15195

Source and publish data:

Authors:

Article abstract of DOI:10.1111/bph.15195

Full text of DOI:10.1111/bph.15195

Products guided by the article

R&D Labs maybe for 70625-31-9

Relevant to this article

Hot Product