Introduction

Type-2 Diabetes Mellitus (T2DM), together with obesity, is a growing threat to public health and accounts for multitudes of chronic deaths worldwide each year. According to the World Health Organization (WHO), the toll of diabetes incidence have grown exponentially from 108 million in the 1980s to 422 million in 20141 and is expected to exceed 600 million by 20352. Even children were affected with diabetes as early as preschool years4, 3. More than 90 percent of these diabetes cases were reported to be T2DM, which is the ultimate result of insulin resistance causing abnormal glucose homeostasis. Metabolic insulin signal transduction is initiated by the tyrosine phosphorylation of insulin receptor, which then recruits a series of downstream signaling molecules5, starting from insulin receptor substrate (IRS) to glucose transporter type 4 (GLUT4), which helps transport circulating glucose into cells in need of it. Signal arrest at any point of these signaling cascade events may lead to insulin resistance followed by an aggravation in blood glucose level. Leptin resistance, metabolic inflammation, mitochondrial disfunction and endoplasmic reticulum stress, which are the characteristics of obesity too, contribute to insulin resistance7, 6. Thus, insulin resistance has been regarded to be the most common pathophysiological link between T2DM and obesity.

Besides lifestyle modification, pharmacological interventions can be of great help in finding a prominent solution to this life-threatening metabolic disorder. Throughout these recent decades, many therapeutic approaches have become available as the traditional insulin therapy, on its own, have not been sufficient to enhance insulin sensitivity. Injectable insulin and oral anti-diabetic agents (OAD), such as metformin, sulfonylureas (or the structurally similar meglitinides), gliflozins, dipeptidyl peptidase-IV inhibitors, new insulin analogues (lispro, aspart and glargine), inhalational insulin drug, bromocriptine, thiazolidinediones (pioglitazone is the only one currently available), incretin mimetics and α-glucosidase inhibitors, are among the few current treatments12, 11, 10, 9, 8. There are several limitations, however, when using these drugs either in mono- or poly-therapy. The side effects, for instance, have been well-described14, 13, 11. Besides this, different responses to OAD among patients have also been recorded due to an array of single-nucleotide polymorphisms (SNPs) at several important genes such as insulin, leptin and sodium-glucose co-transporter-2 (SGLT2)8. Due to the complexity and progressive nature of T2DM, it is essential to explore new and alternative therapeutic pathways and drugs.

Protein tyrosine phosphatases (PTPs) have received immense attention since 1988 for their role in biological systems- as either positive or negative regulators of many important signal transductions15. Among the hundreds of phosphatases that exist, protein tyrosine phosphatase 1B (PTP1B) was the first enzyme to be purified and studied16. The involvement of this enzyme in many cellular signaling events, especially in the downregulation of insulin and leptin signaling pathways, have led to novel approaches for diabetes and obesity treatment17. In line with that, many research studies have been carried out in search for novel phosphotyrosine mimetics, such as lead PTP1B inhibitors. PTP1B inhibitors might potentially reverse insulin and leptin resistance and normalize plasma leptin, glucose and insulin18. Therefore, PTP1B could be a convincing candidate at the genetic, molecular, biochemical and physiological levels for drug design in combating the global epidemic of diabetes and obesity.

Natural products and traditional medicines serve a promising platform for drug discovery with a wide range of secondary metabolites, enabling access to various lead compounds. Many well-accepted drugs available on the markets today have been developed from natural products19. Taxol from , Orlistat, a derivative of lipstatin from bacterium , vincristine from , morphine from berberine from , and papaverine from opium poppy25 are a few examples of potent drugs that have emerged from natural products. Following that, our current study involved screening of potent PTP1B inhibitors from the Natural Product Discovery System (NADI) database (http://www.nadi-discovery.com) using virtual screening. Plant species whose compounds give best hits and strong interactions were shortlisted for an assessment against PTP1B enzyme.

Methods

In vitro PTP1B inhibition assay

The PTP1B assay kit (Cat. No.: 539736-1KIT) was purchased from Merck (Kenilworth, NJ, USA). The test was carried out according to the manufacturer’s protocol. Briefly, 5 μL PTP1B enzyme at 0.5 ng/μL (final quantity was 2.5 ng per well) and 50 μL IR5 substrate solution at 150 μM (final concentration in each well was 75 μM) were prepared in assay buffer and added into the wells of a 96-well microtiter plate (provided in the kit). Instantly, 2.5 μL crude extracts dissolved in 2.5% DMSO (dimethylsulfoxide) at 4000 ppm were added to the wells with the reaction mixture to give a final concentration of 100 ppm. Assay buffer was added into each reaction mixture so that the total volume in the well was 100 μL. The mixture was incubated for 30 minutes at 30°C. Red Reagent was pipetted into each well to terminate the enzyme-substrate reaction after 30 min, and incubated for another 30 min at 30°C before the absorbance was measured. “Time zero” absorbance was measured by mixing 25 μL Red Reagent directly with 50 μL PTP1B enzyme at a concentration 1 ng/μL, with 50 μL IR5 substrate solution at 150 μM. Suramin and plain assay buffer were used as positive and negative controls, respectively, while plain 2.5% DMSO was used as blank or sample control. Absorbances were converted to nmol POby using the standard curve of phosphate. Lastly, the percentage of inhibition was calculated using the equation below:

Type of interaction	Distance (Å)	Interacting Residues(H-Donor to H-Acceptor)	OAI’s atom	Amino Acid Residue
Hydrogen Bond	2.70	A:LYS120:NZ –A:OAI301:O20	O20	NZ:LYS120
Hydrogen Bond	2.88	A:SER216:N –A:OAI301:O16	O16	N:SER216
Hydrogen Bond	3.33	A:ALA217:N –A:OAI301:O16	O16	N:ALA217
Hydrogen Bond	2.75	A:GLY220:N –A:OAI301:O17	O17	N:GLY220
Hydrogen Bond	3.07	A:ARG221:N –A:OAI301:O15	O15	N:ARG221
Hydrogen Bond	2.78	A:ARG221:NE - A:OAI301:O15	O15	NE:ARG221
Hydrogen Bond	3.14	A:ARG221:NH2 - A:OAI301:O16	O16	NH2:ARG221
Hydrogen Bond	3.24	A:SER216:CB –A:OAI301:O19	O19	CB:SER216
Hydrophobic	4.80	A:OAI301 –A:VAL49	Pi-Orbital	Alkyl:VAL49
Hydrophobic	4.74	A:OAI301 –A:ALA217	Pi-Orbital	Alkyl:ALA217

RESULTS

Control docking and virtual screening

From a total of 126 registered PTP1B crystal structures, the structures with PDBID: 1C83 and 1C85 were shortlisted after comparing the X-ray resolution, classification of enzyme, and the objectives of the original literature. These structures were studied with the presence of ligands that inhibited PTP1B. Despite the comparably low andvalue of 1C83 and 1C85 ranging from 0.183 to 0.197 and 0.231 to 0.267, respectively, the high-resolution structure 1C83 (1.80 Å) was favored, as compared to 1C85 (2.75 Å). The interactions between protein crystal 1C83 with OAI (Figure 1 and 2) are listed in Table 1. Control docking was performed to confirm the accuracy of parameters to be used in docking simulation (Figure 1). A dock conformation (Run 21) in the top ranked cluster with the lowest binding energy of -10.15 kcal/mol, mean binding energy of -9.45 kcal/mol, and reference RMSD of 0.50 Å was obtained. The low reference RMSD (<2.0 Å) indicated only a minute difference29 between the docked pose and originally published pose30. Therefore, the docking parameters can mimic the experimental condition to reproduce a similar docking environment and were used for further virtual screening. Figure 2 showed the stacking of OAIs in the original protein crystal pose (yellow) and re-docked pose (green).

NADI Code	Lowest Binding Energy	Scientific name	Compound name
MSC759	-10.03	Piper longum	Chabamide
MSC3271	-9.87	Dryobalanops aromatica	α-Viniferin
MSC2289	-9.68	Momordica charantia	Kuguacin B
MSC665	-9.67	Stemona tuberosa	9α-bisdehydrotuberostemonine A
MSC341	-9.56	Garcinia mangostana	Garcinone E
MSC3480	-9.53	Cymbopogon nardus	6C-pentosyl luteolin
MSC2437	-9.53	Calophyllum inophyllum	Caloxanthone B
MSC3270	-9.43	Shorea gibbosa	Ampelopsin A
MSC770	-9.32	Clausena excavata Burm.	Clausarin
MSC3221	-9.25	Jatropha Curcas	24 -Methylenecycloartenol
MSC254	-9.20	Garcinia mangostana	Mangostenone B
MSC1647	-9.14	Manilkara zapota	23,26-Dihydroxy-tirucalla-7,24-dien-3-one
MSC3019	-9.14	Dysoxylum hainanense	α-Spinasterol acetate
MSC1383	-9.13	Vitex trifolia	17-6-Acetoxy-9-hydroxy-13(14)-labden-16,15-olide
MSC606	-9.11	Oryza sativa	n-β-D-Glucopyranosyl-glutamate-indole-3-acetic acid
MSC348	-9.09	Garcinia mangostana	1,3,7-Trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone
MSC1946	-9.07	Brucea javanica	Dihydrobruceajavanin A
MSC248	-9.06	Morinda citrifolia	Caloxanthone A
MSC1670	-9.06	Calophyllum inophyllum	Americanin A
MSC347	-9.03	Garcinia mangostana	Tovophyllin B
MSC607	-8.91	Oryza sativa	n-β-D-Glucopyranosyl-aspartate-indole-3-acetic acid
MSC2036	-8.90	Tinospora tuberculata	Cycloeucalenone
MSC2297	-8.87	Momordica charantia	Kuguacin H
MSC2101	-8.87	Boesenbergia rotunda	Rotundol
MSC645	-8.86	Stemona tuberosa	Bisdehydrostemoninines A
MSC664	-8.85	Stemona tuberosa	9α-Bisdehydrotuberostemonine
MSC767	-8.85	Caesalpinia pulcherrima	6β-Cinnamoyl-7β-hydroxyvouacapen-5α-ol
MSC2308	-8.82	Momordica charantia	Kuguacin S
MSC2081	-8.81	Boesenbergia rotunda	Nicolaioidesin C
MSC611	-8.77	Oryza sativa	Ergosterol peroxide
MSC1238	-8.76	Blumea balsamifera	3-O-7''-biluteolin
MSC1047	-8.75	Jasminum sambac	Sambacin
MSC3497	-8.74	Calophyllum inophyllum	Caloxanthone O
MSC2067	-8.73	Boesenbergia rotunda	Panduratin B1
MSC2656	-8.72	Murraya paniculata	Genistin-6'-O-malonate
MSC1665	-8.71	Eugenia cumini	2-Hydroxy-3-(hydroxymethyl)anthraquinone
MSC1254	-8.71	Morinda citrifolia	Myricetin 3-O-(4''-O-acetyl)-α-L-rhamnopyranoside
MSC3241	-8.69	Tinospora tuberculata	Diptoindonesin G
MSC2748	-8.69	Etlingera elatior	16-Hydroxylabda-8(17),11,13-trien-16,15-olide
MSC2035	-8.69	Hopea mengarawan	Cycloeucalenol
MSC3461	-8.67	Baeckea frutescens	Inophyllin A
MSC3418	-8.67	Calophyllum inophyllum	6-Methylquercetin
MSC735	-8.65	Piper sarmentosum	Horsfieldin
MSC1639	-8.65	Dysoxylum hainanense	ent-18-Acetoxy-16-hydroxy-8(14)-pimaren-15-one
MSC2567	-8.62	Murraya paniculata	Marmesin-4'-O-α-L-arabinopyranoside
MSC876	-8.61	Pandanus amaryllifolius	Pandamarilactonine-B
MSC1369	-8.60	Boesenbergia rotunda	Cudraflavone C
MSC2068	-8.60	Artocarpus champeden	Panduratin B2
MSC659	-8.59	Stemona tuberosa	Tuberocrooline
MSC2296	-8.59	Momordica charantia	Kuguacin G

Sample	% Inhibition	Sample	% Inhibition
Suramin	87.54 ± 1.02	Manilkara zapota (L)	39.16 ± 18.88
P. amaryllifolius (L)	94.38 ± 2.43	Psidium guajava (L)	30.73 ± 8.22
Vitex negundo (L)	89.03 ± 3.65	Oryza sativa (WP)	30.24 ± 6.75
Piper nigrum (F)	81.39 ± 10.38	Garcinia mangostana (L)	25.54 ± 15.98
Cymbopogon nardus (L)	79.78 ± 6.12	Artocarpus heterophyllus (B)	9.83 ± 32.23
Cymbopogon nardus (P)	66.90 ± 6.43	Myristica fragrans (F)	2.81 ± 6.97
Cymbopogon nardus (L)	66.02 ± 13.20	Barringtonia asiatica (F)	2.28 ± 23.82
Manilkara zapota (F)	62.06 ± 24.87	Murraya koenigii (L)	CBD
Calophyllum inophyllum (L)	48.58 ± 21.05	Artocarpus heterophyllus (L)	CBD
Momordica charantia (F)	42.80 ± 14.62	Anacardium occidentale (L)	CBD
Morinda citrifolia (L)	42.18 ± 7.71	-	-

The validated docking parameters were used to screen 4000 natural compounds from NADI database through virtual screening. The highest ranked ligand was MSC759 with FEB of -10.03 kcal/mol, comparable to the binding of OAI (-10.15 kcal/mol). From a pool of 148 ligands (4.93% from sample pool) that exhibited FEB higher than -8.00 kcal/mol (±2 kcal/mol from control)31, 26, we grouped 50 ligands with top hits and documented them in Table 2, together with their respective plants of origin as listed in NADI. All these 50 ligands were docked inside the catalytic site of PTP1B in order to scrutinize the protein-ligand interactions and the data were cross-examined and compared to the control and literature. Figure 3 showed that the docking conformations of the 5 top ranked ligands inside the binding site were similar to the docking conformation of the control, even though their FEB varied. The compounds that were listed among the 50 top hits and which showed preferable protein-ligand interactions are illustrated in Figure 4. Their interactions with PTP1B are detailed in Figure 5 and Figure 6.

DISCUSSION

Screening for effective drugs can be staggeringly time-consuming and costly. In fact, the success rate that the tested molecule turns out to have activity at the biological target of interest lies below 0.5%32. The author described the process of screening for a drug likened to searching for a needle in a haystack. Computational aided methods of drug design has become a promising approach to research since late 1990’s33. Since then, advancements in cheminformatics and Computer-Aided Drug Designing (CADD) have revolutionized the process of drug discovery into a fast, cost-effective and reliable approach. Such approaches are reasonably more efficient such that they can be used as a scoring function to select the small molecules, among millions or even billions of secondary molecules thought to exist, with high affinity towards biological targets35, 34. Furthermore, through computer modeling, the small molecules or ligands can be optimized, tested for side effects, or even modified to yield properties more akin to the ideal drug. The success story of CADD in many research discoveries have been reviewed38, 37, 36. Herein, we applied virtual screening through molecular docking in search for promising PTP1B phosphatase inhibitors among 4000 natural compounds from the NADI database, which we believe could serve as a preliminary step for future drug development for T2DM and obesity.

From the 4000 natural compounds screened, 50 compounds with the highest hits were redocked back into PTP1B crystal structure, 1C83, to inspect the interaction between PTP1B and each compound. The catalytic domain of PTP1B enzyme is comprised of the amino acids 30-278. From these, residue Cys215 (active site nucleophile), WPD loop (amino acids 79-187), YRD loop (Tyr46 and charged residues: Arg47 and Asp48), and the secondary aryl-phosphate binding site were reported to be the most significant regions in the PTP1B enzyme structure39. Residues His214-Arg221 (His-Cys-Ser-Ala-Gly-Ile-Gly-Arg) that surrounded the catalytic residue Cys215 form a cradle-like structure that favors substrate binding40. Meanwhile, WPD loop acts like an arm that can be either found in closed or opened motif with regards to substrate binding to the enzyme. In addition to assisting in stronger binding of substrate to enzyme, the WPD loop can be involved in catalysis by protonating the tyrosyl leaving group of the substrate by Asp18141. On the contrary, the charged residues in the YRD loop is known to be important in inhibitor specificity (via a salt bridge formation between the residues) to potent inhibitors that have basic nitrogen, thus offering selective inhibition over other PTPs43, 42.

Most of the compounds from the NADI database bound to the PTP1B catalytic site (similarly to OAI) and interacted with amino acids at the catalytic site and surrounding active site loops, WPD and YRD (Figure 4). Chabamide (MSC759; ), the compound with the highest hit in the virtual screening interacted with three amino acids at the catalytic region of PTP1B through hydrogen bonding: Arg47, Arg221, and Gly221 (Figure 5). Conformation with the lowest FEB ( highest affinity towards the enzyme) of this compound might be due to the interaction with Arg47, the charged residue in the YRD loop that is specific for inhibitor molecules. Furthermore, 6-C-pentosyl luteolin (MSC2437) from displayed four promising hydrogen bonding with Arg 47, Asp 181, Ser 216 and Arg 221. The compound that formed the greatest hydrogen bonding with the enzyme compared to OAI (inhibitor in the crystal structure) was n-β-D-Glucopyranosyl-glutamate-indole-3-acetic acid (MSC606), which was isolated from . This ligand formed eight hydrogen interactions (with residues in the catalytic, cradle-like domain and in the charged region of YRD loop), thereby indicating the strong bonding with PTP1B (Figure 6).

Another compound that showed significant interaction with the enzyme was pandamarilactonine-B (MSC876), an alkaloid with two lactones and one pyrrolidine ring structure44, from . This ligand was the only one (among the 50 top hits) to form hydrogen bonding with Cys215, the catalytic residue (Figure 6). Upon binding, pandamarilactonine-B was clamped between Asp181 and Ala217 through pi-interaction, which showed good penetration into the catalytic site and inhibition characteristics through interference with protonation activity to form the cysteinyl phosphate intermediate41. In addition, a carbon-hydrogen bonding with Tyr46 and Arg47, the charged residues in YRD loop, was also observed between pandamarilactonine-B and PTP1B. Furthermore, kuguacin B (MSC2289) from , a-spinasterol acetate (MSC3019) from , horsfieldin (MSC735) from , and garcinone E (MSC341) from all interacted with PTP1B through non-covalent bonding, such as pi-pi stacking and pi-alkyl interactions. These interactions were believed to be insignificant as they were considerably further compared to hydrogen bonding and electrostatic interactions45.

Taking into consideration the strength of protein-ligand interactions, we selected a few plants to be studied for PTP1B inhibition activity. Based on the test, methanol leaf extracts of exerted the highest enzyme inhibition, which was never reported elsewhere, although is known to be rich with nitrogen-containing alkaloids. As discussed earlier in this section, the nitrogen molecule could serve as the basic entity to interact with the charged YRD loop through a salt bridge formation43. In support of our observation, Sasidharan .46 have reported robust anti-hyperglycemic activity of ethanol extract of in streptozotocin (STZ)-induced diabetic rats. Similar to , and showed greater than 80% inhibition. The anti-diabetic properties of and have been well-documented by many research studies51, 50, 49, 48, 47, but most of them involved studies and no inhibitory pathways were illustrated. Betulinic acid, one of the compounds present in leaves, was reported to have potent PTP1B inhibition activity52. Nonetheless, the methanol leaves and peel extracts of , and aqueous leaves and fruit extracts of . and , respectively, showed moderate activity towards PTP1B enzyme.

The computer-aided drug designing approach in the search of PTP1B inhibitors often targets the catalytic site of the protein. Though the catalytic region is important for the activity of the enzyme, looking outside of the catalytic pocket of the protein structure may expedite the discovery of drugs with more selectivity towards PTP1B. This is important since all PTPs share nearly 50-80% homology in the catalytic domain which could be a challenge in providing selectivity to PTP1B53. Moreover, the charged catalytic domain (YRD loop) poses a challenge in designing PTP1B inhibitors, such as oral drugs. It is for these reasons that many computationally designed and validated drugs have failed to yield the same expected outcomes . The non-catalytic phosphotyrosine-binding domain offers an interesting and alternative option to target PTP1B54 and should be considered for drug development.

CONCLUSION

Our virtual screening study and enzymatic assays indicated the promising PTP1B inhibitory activity of leaves, leaves and fruit, (aqueous and methanol extracts of leaves and peel), and leaves. The good correlation between our method and enzyme assay shows the validity of our test parameters. Moreover, cross-examination and comparison of our results to the published literature show complementary ethnopharmaceutical properties of the genus and. Further fractionation or isolation of active principles from these plants can provide a good platform to develop promising anti-diabetic or anti-obesity drugs through PTP1B-targeted approaches. However, taken into account the limitations of targeting only the catalytic region of PTP1B, future investigations should include all possible binding pockets.

Abbreviations

CADD: Computer-aided drug designing

DMSO: Dimethyl sulfoxide

FEB: Free energy of binding

GLUT4: Glucose transporter type 4

IPHARM: Institute of Pharmaceuticals & Nutraceuticals

IRS: Insulin receptor substrate

NADI: Natural product discovery system

OAD: Oral anti-diabetic agents

PTP1B: Protein tyrosine phosphatase 1B

PTPs: Protein tyrosine phosphatases

RAM: Random access memory

RMSD: Root-mean-square deviation

SGLT2: Sodium-glucose co-transporter-2

SNP: Single-nucleotide polymorphism

STZ: Streptozotocin

T2DM: Type-2 diabetes mellitus

WHO: World Health Organization

Competing Interests

Authors declare that there are no conflicts of interests.

Authors' Contributions

Ho Yueng Hsing carried out the experiment. Selestin Rathnasamy wrote the manuscript with the assistant of Roza Dianita. Habibah A. Wahab edited the manuscript and supervised the project.

Acknowledgment

We thank to USM and RIKEN for financial support from USM RU TOP-DOWN project entitled Catalogue of USM-RIKEN Natural Product (CURINaP) Library for the Discovery of Bioactive Molecules on Ageing and Ageing-Related Diseases, 1001/PFARMASI/870031.