Binding properties of marine bromophenols with human protein tyrosine phosphatase 1B: Molecular docking, surface plasmon resonance and cellular insulin resistance study

doi:10.1016/j.ijbiomac.2020.06.263

International Journal of Biological Macromolecules

Volume 163, 15 November 2020, Pages 200-208

https://doi.org/10.1016/j.ijbiomac.2020.06.263 Get rights and content

Highlights

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Molecular docking study of marine bromophenols with protein tyrosine phosphatase 1B (PTP1B) of open and closed conformations.
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Surface plasmon resonance (SPR) assay on the interaction of bromophenols and PTP1B protein.
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Effect of compound 3 on insulin resistance was studied in palmiate-treated HepG2 cells.

Abstract

Protein tyrosine phosphatase 1B (PTP1B) is a highly validated target for the treatment of type 2 diabetes and obesity. Previous studies have shown that bromophenols from marine red alga Rhodomela confervoides can inhibit PTP1B activity. However, traditional in vitro enzymatic assays may result in false positive activity. Here, we reported a successful application of molecular docking and surface plasmon resonance (SPR) assay for the characterization of small-molecule PTP1B inhibitors with high affinity. First, molecular docking study indicated that six bromophenol compounds preferred to bind PTP1B with open conformation rather than one with closed conformation. Next, SPR study indicated that compound 3 was the most potent and stable PTP1B inhibitor at the nanomolar level. Then Lineweaver–Burk plot data showed that compound 3 was a competitive PTP1B inhibitor. Moreover, compound 3 could improve palmitate-induced insulin resistance in HepG2 cells. Taken together, molecular docking and SPR-based methodology could apply in the development of PTP1B inhibitors with high affinity.

Introduction

Diabetes is a world epidemic which poses a great threat to people's health around the world [1]. The 9th IDF Diabetes Atlas shows that an estimated 463 million adults (aged 20–79 years) worldwide suffer from diabetes, and one quarter of them are from China [2]. Type 2 diabetes mellitus (T2DM) accounts for 90%–95% of diabetes mellitus, which is characterized by insulin resistance and insufficient insulin secretion [3]. However, the existing treatment methods cannot meet all the needs of type 2 diabetes treatment, and some of them are often ineffective [4]. Therefore, it is urgent to develop new therapeutic agents.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin signaling pathways. And now it is still an effective target for T2DM and obesity treatment interventions for the following reasons [5,6]. First, biochemical studies have shown that PTP1B down-regulated insulin signaling by dephosphorylating the tyrosine residues of the insulin receptor (IR) β subunit and insulin receptor substrate (IRS) [[7], [8], [9]]. Second, PTP1B gene deletion produced healthy and fertile mice with increased insulin sensitivity and resistance to high fat-induced weight gain [10,11]. Liver-specific or muscle-specific PTP1B deletion also proved a major role of PTP1B in regulating insulin sensitivity, lipid metabolism, and glucose homeostasis [[12], [13], [14], [15]]. Finally, small-molecule PTP1B inhibitors and antisense oligonucleotides showed strong anti-diabetic and anti-obesity effects both in vitro and in vivo [[16], [17], [18], [19]]. Unfortunately, although many natural and synthetic PTP1B inhibitors have been reported, no compound has been approved by US FDA. Currently, only MSI-1436, KQ-791, and ISIS-PTP1BRX are in clinical trials. Lack of affinity and selectivity, and low cell permeability are the major obstacles in the development of PTP1B inhibitors [20].

The lead compound, bis-(2,3-dibromo-4,5-dihydroxy-phenyl)-methane, is a natural PTP1B inhibitor isolated from the marine red alga Rhodomela confervoides [21]. Recently research has reported that this lead compound could improve hyperglycemia and dyslipidemia in diabetic model mice [22]. In order to discover more potent PTP1B inhibitor, our PTP1B inhibitor team is dedicated to find and optimize small molecule compounds. Six derivatives (structures shown in Fig. 1) were synthesized [23] and their PTP1B inhibitory activities were further determined. Data showed that these compounds displayed similar inhibitory activity against PTP1B and we could not distinguish which was the best one. More needed to be done to find the most potent PTP1B inhibitors.

Surface plasmon resonance (SPR) is a fast, label-free, and real-time technique to detect biomolecular interaction [24]. With the continuous improvement of the sensitivity of commercial SPR instruments, today SPR is a well-established research tool to study the binding between small molecule compounds and target protein [25]. However, there are few reports of the use of SPR for systematic screening and characterization of small molecule PTP1B inhibitors.

In the present study, molecular docking was performed to predict the binding of the marine bromophenols with PTP1B. SPR assay was used to characterize the interaction kinetics of small molecule inhibitors to PTP1B. Lineweaver–Burk plots were used to determine the inhibitor type of compound 3. Moreover, the cellular effect of compound 3 on insulin resistance was evaluated by immunoblotting.

Section snippets

Materials

N-ethyl-N′-(dimethylaminopropyl)-carbodiimide (EDC), Series S CM5 sensor chips, PBS-P buffer, N-hydroxysuccinimide (NHS), 1.0 M ethanolamine-HCl, pH 8.5, and maintenance kit were purchased from GE Healthcare (Mississauga, ON, Canada). DMSO and sodium acetate were obtained from Solaibo (Beijing, China). Palmitate (PA) and insulin (INS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). IRS1 antibody, Insulin Receptor β antibody, phospho-IRβ (Tyr1146) antibody, and phospho-Akt (Ser473)

Inhibitory effects of compounds against PTP1B

Inhibitory activities of compounds against PTP1B were first conducted. The enzyme activity was tested by using p-NPP as substrate and the amount of catalytic product was measured spectrophotometrically. As shown in Table 1, all these bromophenols displayed similar inhibition activity against PTP1B at the dose of 20 μg/mL.

Molecular docking study of compounds against PTP1B

A positive effort was made to explain the activity of these compounds against PTP1B by performing molecular docking (Fig. 2, Fig. 3). Two crystal structures of PTP1B were

Discussion

PTP1B is a highly validated therapeutic target for metabolic syndromes, such as type 2 diabetes and obesity. However, the discovery of pharmaceutically acceptable inhibitors has proven to be a big challenge. One of the primary obstacles is the discovery of stable PTP1B inhibitors with high affinity. In the present study, we combined molecular docking and SPR study to identify positive PTP1B inhibitor with potent affinity.

In our previous study, in vitro enzymatic assay was carried out to measure

Authors' contributions

Jiao Luo: Conceptualization, Writing-original draft. Renshuai Zhang: Software. Xuehe Wang: Data Curation. Zhanhui Hou: Methodology. Shuju Guo: Supervision, Funding acquisition and Data Curation. Bo Jiang: Conceptualization, Resources, Project administration, Writing-review & editing.

Acknowledgement

This research was funded by grants from NSFC-Shandong Joint Fund (U1706213). The authors are grateful to all members of the laboratory for their continuous technical advice and helpful discussion.

Declaration of competing interest

All the authors declared no competing interest.

References (39)

F. Ahmad et al.
Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway
J. Biol. Chem.
(1995)
F.G. Haj et al.
Liver-specific protein-tyrosine phosphatase 1B (PTP1B) re-expression alters glucose homeostasis of PTP1B−/− mice
J. Biol. Chem.
(2005)
D. Shi et al.
Bromophenols as inhibitors of protein tyrosine phosphatase 1B with antidiabetic properties
Bioorg. Med. Chem. Lett.
(2012)
F. Fathi et al.
Kinetic and thermodynamic studies of bovine serum albumin interaction with ascorbyl palmitate and ascorbyl stearate food additives using surface plasmon resonance
Food Chem.
(2018)
F. Fathi et al.
Kinetic and thermodynamic insights into interaction of albumin with piperacillin: spectroscopic and molecular modeling approaches
J. Mol. Liq.
(2019)
J.B. Baell
Redox-active nuisance screening compounds and their classification
Drug Discov. Today
(2011)
R.N. Shinde et al.
Geometrical criteria for characterizing open and closed states of WPD-loop in PTP1B
J. Mol. Struct.
(2012)
X. Li et al.
Toward a treatment of diabesity: in vitro and in vivo evaluation of uncharged bromophenol derivatives as a new series of PTP1B inhibitors
Eur. J. Med. Chem.
(2019)
Y. Zheng et al.
Global aetiology and epidemiology of type 2 diabetes mellitus and its complications
Nat. Rev. Endocrinol.
(2018)
International Diabetes Federation
IDF Diabetes Atlas - nineth edition

A. American Diabetes

2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2018

Diabetes Care

(2018)

F. Mittermayer et al.

Addressing unmet medical needs in type 2 diabetes: a narrative review of drugs under development

Curr. Diabetes Rev.

(2015)

T.O. Johnson et al.

Protein tyrosine phosphatase 1B inhibitors for diabetes

Nat. Rev. Drug Discov.

(2002)

M. Feldhammer et al.

PTP1B: a simple enzyme for a complex world

Crit. Rev. Biochem. Mol. Biol.

(2013)

J.C. Byon et al.

Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction

Mol. Cell. Biochem.

(1998)

B.L. Seely et al.

Protein tyrosine phosphatase 1B interacts with the activated insulin receptor

Diabetes

(1996)

M. Elchebly et al.

Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene

Science

(1999)

L.D. Klaman et al.

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

Mol. Cell. Biol.

(2000)

M. Delibegovic et al.

Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B

Mol. Cell. Biol.

(2007)

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Binding properties of marine bromophenols with human protein tyrosine phosphatase 1B: Molecular docking, surface plasmon resonance and cellular insulin resistance study

Highlights

Abstract

Introduction

Section snippets

Materials

Inhibitory effects of compounds against PTP1B

Molecular docking study of compounds against PTP1B

Discussion

Authors' contributions

Acknowledgement

Declaration of competing interest

J. Biol. Chem.

J. Biol. Chem.

Bioorg. Med. Chem. Lett.

Food Chem.

J. Mol. Liq.

Drug Discov. Today

J. Mol. Struct.

Eur. J. Med. Chem.

Global aetiology and epidemiology of type 2 diabetes mellitus and its complications

Nat. Rev. Endocrinol.

IDF Diabetes Atlas - nineth edition

2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2018

Diabetes Care

Addressing unmet medical needs in type 2 diabetes: a narrative review of drugs under development

Curr. Diabetes Rev.

Protein tyrosine phosphatase 1B inhibitors for diabetes

Nat. Rev. Drug Discov.

PTP1B: a simple enzyme for a complex world

Crit. Rev. Biochem. Mol. Biol.

Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction

Mol. Cell. Biochem.

Protein tyrosine phosphatase 1B interacts with the activated insulin receptor

Diabetes

Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene

Science

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

Mol. Cell. Biol.

Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B

Mol. Cell. Biol.