INTRODUCTION:

Protein phosphorylation is a post-translational modification involved in regulating cellular signaling and homeostasis. Aberrant levels of cellular protein phosphorylation are often associated with a range of diseases, including metabolic disorders and cancers. Cellular protein phosphorylation is regulated by the orchestrated activities of two classes of enzymes, kinases and phosphatases, and the abnormal activity of either protein class can lead to or indicate a disease.

Consequently, kinases have been established as molecular targets for a plethora of diseases, primarily cancers, and have been effectively targeted by small molecules and biologics developed as therapeutics for a variety of diseases.^1,2 Meanwhile, recent studies have also shown that phosphatases have a nearly equal role in regulating cellular protein phosphorylation levels as kinases and are equally attractive molecular targets.³Despite this, phosphatases are severely underrepresented as therapeutic drug targets, and the development of potent, selective, and efficacious phosphatase inhibitors is critical for expanding treatment modalities and therapeutic options for many diseases.

One particular phosphatase of interest is PTP1B, the founding and most well-characterized member of the protein tyrosine phosphatase (PTP) superfamily. PTP1B is a non-receptor type phosphatase, most highly distributed in insulin-responsive tissues. PTP1B has been identified as a promising therapeutic target for type II diabetes mellitus and obesity due to its role in the attenuation of the insulin and leptin signaling pathways, with its overactivity being linked to downstream effects in these respective pathways, most notably insulin resistance.⁴ Recent studies have also uncovered its role in the regulation of anti-apoptotic pathways, pro-metastatic signaling, cell-to-cell adhesion, endothelial dysfunction, neuroinflammation, and neurodegeneration, presenting PTP1B as a potential therapeutic target for multiple forms of cancer as well as Rett syndrome, a rare neurodegenerative disease.^5,6 Undoubtedly, the development of molecules that modulate the activity of PTP1B can provide additional therapeutic modalities for these various diseases.⁷

Despite the availability of over 800 known experimental PTP1B inhibitors, drug discovery efforts have proven mainly ineffective in developing an efficacious and selective PTP1B-targeting drug, likely due to the highly conserved catalytic site of PTPs.⁸ However, recent studies have highlighted the inhibitory potential of numerous medicinal plants against PTP1B, suggesting that they could serve as a potential source of bioactive components that can be used as novel scaffolds for drug optimization efforts.^9-13 Medicinal plants have been utilized in traditional medicine for centuries and have exhibited potential for treating various diseases. They produce diverse secondary metabolites, which act in synergy to give the plant extract its therapeutic effect.^{14, 15} This synergistic effect of plant metabolites is crucial, as it enhances the overall potency and efficacy of the extract while also providing a distinct approach towards drug discovery. Over the past few decades, there has been a significant increase in the use of medicinal plants for health promotion and disease treatment, not only in developing countries but also in developed countries. This trend is particularly evident in countries such as the UK, Germany, China, and France, where many medicinal plant extracts have gained recognition and are now utilized as prescription drugs. This growing acceptance and integration of medicinal plants into mainstream healthcare systems reflects the increasing acknowledgment of their therapeutic potential and the desire to explore natural remedies for various health conditions.^16-20 Therefore, in this study, we aim to investigate the inhibitory effect of Eriodictyon californicum on PTP1B.

E. californicum (Hook. and Arn.) Torr., also known as yerba santa or “holy herb,” is an evergreen shrub that belongs to the Boraginaceae family. This plant is native to California, Oregon, and Northern Mexico. Yerba santa has a long history of traditional use by Native Americans and Spanish settlers who recognized its medicinal properties. It was used to treat various ailments, including headaches, rheumatism, inflammation, coughs, fevers, asthma, and lung infections.^21-23 The FDA considers yerba santa safe, and it is used in food and pharmaceutical applications as a flavoring agent and taste modifier.^24-26 A recent study has highlighted the potential benefits of SantEnergy™ Nu, a nutritional food supplement containing yerba santa plant extracts, which can help reduce body weight and body fat in women with obesity over three months, with a good safety profile and tolerance.²⁷ Moreover, yerba santa has been recognized for its diverse therapeutic properties. It has been shown to possess anticarcinogenic, neuroprotective, anti-inflammatory, iron-chelating, and antioxidant properties.^28-32 The plant has been extensively documented in literature as a rich source of bioactive flavonoids.^{24,25,28,29,32} E. californicum exhibits a promising therapeutic potential, necessitating further study to uncover the scope of its wide-ranging effects and potential biomedical applications. This study investigates the potential of phytochemicals in E. californicum ethanolic leaf extract to inhibit PTP1B phosphatase activity. We employed in vitro phosphatase inhibition assays, enzyme kinetics, and molecular docking to examine the extent and nature of PTP1B inhibition by the extract. These methodologies helped us better understand the combinatorial effects of the phytochemicals in ECE and their interactions with the enzyme. The findings of this study demonstrate that E. californicum has significant potential as a natural source of PTP1B inhibitors, contributing uniquely to the growing body of research on plant-based agents targeting PTP1B.

MATERIALS AND METHODS:

Plant material and chemicals:

Whole wild-crafted dry leaves of Eriodictyon californicum were obtained in an FDA-approved, non-gas-permeable 3 mm poly structure bag from Monterey Bay Spice Company, Watsonville, CA, USA, Lot No. 19G212-287. The plant sample was identified and deposited in the plant material repository at the Department of Chemistry, Bellarmine University, voucher number BU-2019-001. All chemicals utilized were of analytical grade and procured from Sigma-Aldrich., Inc. in St. Louis, MO, USA, except for 4-mercaptoethanol, which was purchased from Bio-Rad Laboratories, Inc., Hercules, CA, USA, and p-nitrophenyl phosphate (pNPP), which was acquired from Thermo Fisher Scientific, Inc., Waltham, MA, USA.

Preparation of extract:

The extraction of plant material was performed using the Soxhlet extraction method.³¹ Briefly, 50g of coarse powder of E. californicum dried leaves were subjected to exhaustive extraction with 500ml of 95% ethanol. The extraction was carried out at 60-80°C for 12hours. The resulting extract was subjected to distillation under reduced pressure using a Buchi-type rotary evaporator, and the solvent-free extract was placed in a desiccator at room temperature until a constant weight was obtained. The percentage yield of ECE was 35.07%. The extract was diluted to a known concentration (w/v) in dimethyl sulfoxide (DMSO) for biochemical assays.

Recombinant expression and purification of PTP1B:

The plasmid encoding for human PTP1B was a gift from Wolfgang Peti (Addgene Plasmid #102719).³³ This construct covers residues 1-301 and has an N-terminal 6X-His tag. PTP1B was recombinantly expressed in Rosetta (DE3) by growing transformed cells in LB medium (Miller formulation) at 37°C and 250 RPM until the optical density at 600nm reached 0.60, upon which protein expression was induced by the addition of 1mM IPTG. The culture was allowed to express for an additional 5hours at 37°C. Cells were harvested by centrifugation at 6,000 rcf for 10 minutes and stored at -20°C until purification. Cells were resuspended in lysis buffer (50mM Tris, pH 8.0, 500mM NaCl, 3mM NaN3), lysed by ultrasonic homogenization, and clarified by centrifugation at 35,000 x g for 45mins. Lysate was loaded onto a pre-equilibrated Ni-affinity column, followed by gradient elution using a high imidazole buffer (50mM Tris, pH 8.0, 500mM NaCl, 500mM Imidazole, 3mM NaN3) in an Akta Prime FPLC (flow rate of 2ml/min). Fractions containing His-tagged PTP1B were pooled, incubated with 5mg of TEV protease, and dialyzed overnight at 4°C into the TEV protease reaction buffer (30mM Tris, pH 7.2, 150mM NaCl, 2mM TCEP) to cleave the tag and reduce the imidazole concentration. The sample was then re-loaded into a Ni-affinity column to remove TEV protease and tagged PTP1B. Finally, the protein was purified by size exclusion chromatography (Superdex 16/60 200pg) and exchanged into the final buffer (30mM Tris, pH 7.2, 100mM NaCl, 2mM TCEP). Sample purity was assessed based on monodispersity from the gel filtration chromatogram and by SDS-PAGE. The typical protein yield was 20-30mg per liter of LB medium.

PTP1B inhibitory assay:

Phosphatase inhibition assay was conducted based on a previously reported method with minor modifications.³⁴Briefly, 1.0µM of recombinant PTP1B in reaction buffer (10mM Tris, pH 7.3, 25mM NaCl, 5mM BME) and varying concentrations of ECE were incubated at room temperature for 15minutes in a 96-well plate. After the incubation period, 1.5mM pNPP, a synthetic substrate for PTP1B, was added to the reaction mixture and further incubated for 25minutes. The reaction was terminated by the addition of 10M NaOH. The amount of product (p-nitrophenol) was measured at 405nm using a microplate plate reader (Tecan US, Inc., Raleigh, NC, USA). Sodium orthovanadate (Na₃VO₄) was used as a positive control, while DMSO was the negative control. Percent inhibition (%I) was calculated using the following equation:

%I = ((Ac-As)/Ac)*100

where Ac is the absorbance of the negative control (DMSO) and As is the absorbance of the corresponding sample (ECE-treated).

Study of inhibition kinetics:

To investigate the inhibition mechanism and the impact of ECE on the kinetic parameters of PTP1B, both K_m and V_max were determined using Michaelis-Menten (MM) and Lineweaver-Burk (LB) plots. The kinetic assays were conducted following the same method as the aforementioned PTP1B inhibitory assay. The MM plot for PTP1B inhibition was obtained by varying the concentrations of the substrate pNPP within the range of 0.25–2.5mM while keeping the concentrations of ECE constant at 2.5µg/ml, 5.0µg/ml, and 12.5µg/ml. The enzyme's initial velocity (V_o) was determined using the standard curve of p-nitrophenol. A double reciprocal LB plot (1/[S] versus 1/V_o) was generated to determine the inhibition mode.

Molecular docking of known bioactive compounds from E. californicum:

Molecular docking was performed using Autodock VINA (version 1.1.2) to predict and quantify the binding interactions between PTP1B and compounds known to be present in yerba santa.^35-38Recently, the most comprehensive chemical characterization of E. californicum to date was published.³⁹Due to the relatively understudied nature of this plant, we utilized the chemicals identified in the before-mentioned study for our molecular docking analysis. For the receptor file, both the open (PDB: 7KEY) and closed (PDB: 1L8G) conformations of PTP1B were used.^40,41These structures were converted from PDB to PDBQT format using Autodock Tools (version 1.5.6). Compound 3D structures were generated using OpenBabel (version 3.0) and converted into PDBQT using MGLTools (v. 1.5.6). An unbiased, blind docking approach was employed, where the docking grid was placed at the center of the receptor and made sufficiently large to cover the entire protein. The exhaustiveness parameter in VINA was set to 100. Molecular docking results were visualized using PyMol.

Statistical Analysis:

Statistical analysis was conducted using GraphPad Prism (version 9.5.1 for Windows, GraphPad Software, San Diego, CA, USA). The experiments were performed in triplicate, and the results are expressed as mean± standard deviation. The dose-dependence curve and IC₅₀ quantification were determined by non-linear regression analysis. The data were analyzed by a student’s t-test. Statistical significance was assigned if p<0.05.

RESULT:

Effect of ECE on PTP1B activity:

The results in Table 1 provide insights into the inhibitory effect of ECE on the phosphatase activity of PTP1B at various concentrations. The data show that ECE significantly inhibited PTP1B activity in a concentration-dependent manner, ranging from 21.3± 8.7% at 1.3µg/ml to 101.8±1.9% at 50µg/ml, the highest concentration tested. The IC₅₀ was determined to be 4.19 µg/ml, as shown in Figure 1A. Na₃VO₄, a general inhibitor of protein tyrosine phosphatases, was used as a control, and its IC₅₀ was found to be 0.022µg/ml, as shown in Figure 1B.

Table 1. Inhibitory effect of ECE on PTP1B

ECE (µg/ml)	% Inhibition
1.3	21.3 ± 8.7*
2.5	42.8 ± 4.7*
5.0	57.2 ± 9.8*
12.5	91.2 ± 4.9*
25.0	94.4 ± 6.5*
50.0	101.8 ± 1.9*

Each value represented is expressed as mean ± standard deviation (n=3), *p<0.05

Figure 1. Dose-response curve of test samples’ effect on PTP1B activity. (A) ECE (B) Na3VO4. Data are expressed as mean± standard deviation (n=3), p<0.05

Inhibition kinetics of ECE on PTP1B activity:

Kinetic assays were conducted to gain further insights into the potential inhibitory effects of ECE on PTP1B activity. Although the ECE contains diverse phytochemicals, the data obtained from MM and LB plots were significant and informative. These findings provided a comprehensive overview of the ECE inhibitory profile, consistent with our molecular docking results.

The MM curve (Figure 2A) shows a concentration-dependent inhibitory effect of ECE on the initial velocity V_o of PTP1B. In addition, the LB plot (Figure 2B) revealed an apparent mixed inhibition mechanism, suggesting that compounds in ECE may interact with both the free enzyme and enzyme-substrate complex. Table 2 presents the K_m and V_max values obtained from the LB plot, which indicate a dose-dependent decrease in V_max with increasing ECE concentration. At a concentration of 12.5µg/ml, there was a 40.96% decrease in V_max, suggesting that ECE has a potent inhibitory effect on PTP1B activity. At lower concentrations, ECE did not significantly affect K_m. At the highest concentration, K_m increased slightly, but the change was not statistically significant.

Figure 2. Kinetics analysis of ECE's effect on PTP1B. (A) MM curve (B) LB plot. Data are expressed as mean±standard deviation (n=3).

Table 2. Effect of ECE on K_m and V_maxof PTP1B

ECE (µg/ml)	Vmax	Km
0.0	7.9 ± 0.9	1.0 ± 0.3
2.5	7.1 ± 1.7*	1.1 ± 0.6*
5.0	5.4 ± 1.4*	1.0 ± 0.7*
12.5	4.6 ± 1.1**	1.9 ± 0.9*

Each value represented is expressed as mean ± standard deviation (n=3), **p<0.02, *NS

Although the use of crude extract may obscure precise conclusions regarding the mechanism of inhibition, it strongly supports the inhibitory activity of ECE on PTP1B. These preliminary findings suggest that phytochemicals in ECE interact with PTP1B, altering its kinetics. Further research with pure compounds is necessary to determine the exact mechanism of enzyme inhibition.

Analysis of molecular docking studies:

To further explore the apparent inhibition mechanism of ECE, molecular docking simulations were performed on known bioactive compounds present in yerba santa.^38,39 We performed simulations with all 41 known compounds, which are listed in Table 3. Remarkably, all compounds exhibited modest predicted binding energies, irrespective of whether they were docked onto the open or closed conformation of PTP1B, as shown in Table 3. Docking results revealed that the majority of the compounds bind to a similar pocket near the P loop of the active site of PTP1B in the open conformation (Figure 3A). Most molecules also dock to the same, but distinct, pocket in the closed conformation (Figure 3B). This pocket overlaps with the active site of PTP1B. Closer examination of the binding of eriodyctiol, which exhibited the most favorable predicted free energy of binding at -8.1 kcal/mol to the open conformation of PTP1B, revealed interactions, particularly hydrogen bonding with N44 and Y46 (Figure 4). Meanwhile, chryseriol is one of the best binders in the closed conformation, with a binding energy of -7.40kcal/mol. Within this pocket, it directly interacts with active site residues, such as through hydrogen bonding with G220 in the P loop and D181 in the WPD loop (Figure 4). While these predicted interactions are relatively modest, these docking results show how known bioactive compounds, particularly flavanones likely present in the ECE, may bind with PTP1B, whether in the open or closed conformation. This information complements the kinetic studies by providing a molecular-level understanding of how ECE phytochemicals interact with the enzyme. Combining kinetic and docking results offers a comprehensive view of ECE's potential as a source of PTP1B inhibitors

Table 3. Docking results of known yerba santa compounds

Compound	SMILES	Open (kcal/mol)	Closed (kcal/mol)
3,4-hydroxyphenyl-2-propenoic acid	C1=CC(=CC=C1/C=C/C(=O)O)O	-6.00	-6.30
4'-isobutyryl homoeriodictyol	CC(C)C(=O)OC1=C(C=C(C=C1)[C@@H]2CC(=O)C3=C(C=C(C=C3O2)O)O)OC	-7.20	-6.40
6-methoxy homoeriodictyol	COC1=C(C=CC(=C1)[C@@H]2CC(=O)C3=C(O2)C=C(C(=C3O)OC)O)O	-6.90	-7.30
6-methoxy naringenin	COC1=C(C2=C(C=C1O)OC(CC2=O)C3=CC=C(C=C3)O)O	-7.10	-6.80
6-methoxy sakuranetin	COC1=C(C(=C2C(=O)C[C@H](OC2=C1)C3=CC=C(C=C3)O)O)OC	-7.30	-6.70
acacetin	COC1=CC=C(C=C1)C2=CC(=O)C3=C(C=C(C=C3O2)O)O	-7.60	-7.20
apigenin	C1=CC(=CC=C1C2=CC(=O)C3=C(C=C(C=C3O2)O)O)O	-7.30	-7.20
chryseriol	COC1=C(C=CC(=C1)C2=CC(=O)C3=C(C=C(C=C3O2)O)O)O	-7.30	-7.40
chrysin	C1=CC=C(C=C1)C2=CC(=O)C3=C(C=C(C=C3O2)O)O	-7.40	-7.30
cirsimaritin	COC1=C(C(=C2C(=C1)OC(=CC2=O)C3=CC=C(C=C3)O)O)OC	-7.30	-7.00
eriodictyol dimethylether	COC1=CC(=C2C(=O)C[C@H](OC2=C1)C3=CC(=C(C=C3)O)OC)O	-7.40	-7.30
eriodictyol	C1[C@H](OC2=CC(=CC(=C2C1=O)O)O)C3=CC(=C(C=C3)O)O	-8.10	-7.20
eriolic acid A	CC(=CCC(C(=CCC1=CC(=CC(=C1OC)CC=C(C)CO)C(=O)O)C)O)C	-7.40	-6.60
eriolic acid B	CC(=CCC(C(=CCC1=CC(=CC(=C1O)CC=C(C)C)C(=O)O)C)O)C	-6.70	-6.40
eriolic acid C	CC(=CCC(/C(=C/CC1=CC(=CC(=C1O)C/C=C(\C)/CO)C(=O)O)/C)O)C	-7.30	-7.00
eriolic acid D	CC(=CCC(C(=CCC1=CC(=CC(=C1O)CC=C(C)C)C(=O)O)C)O)C	-6.70	-6.50
erionic acid A	CC(CCC1=C(C(=CC(=C1)C(=O)O)C/C=C(\C)/CO)O)C(=O)/C=C/C(C)(C)O	-7.40	-6.50
erionic acid B	CC(CCC1=CC(=CC2=C1OC(C(C2)O)(C)C)C(=O)O)C(=O)/C=C/C(C)(C)O	-7.80	-7.20
erionic acid C	CC(CCC1=CC(=CC(=C1O)CC=C(C)CO)C(O)=O)C(=O)CC=C(C)C	-6.70	-6.20
erionic acid D	CC(CCC1=CC(=CC2=C1OC(C(C2)O)(C)C)C(=O)O)C(=O)CC=C(C)C	-7.10	-7.00
erionic acid E	CC(CCC1=C(C(=CC(=C1)C(=O)O)CC=C(C)C)O)C(=O)/C=C/C(C)(C)O	-7.80	-6.10
erionic acid F	CC(=CCC(C(CCC1=CC(=CC(=C1O)CC=C(C)C)C(=O)O)C)=O)C	-6.80	-6.20
genkwanin	COC1=CC(=C2C(=C1)OC(=CC2=O)C3=CC=C(C=C3)O)O	-7.20	-7.30
hesperetin	COC1=C(C=C(C=C1)[C@@H]2CC(=O)C3=C(C=C(C=C3O2)O)O)O	-7.20	-7.00
hispidulin	COC1=C(C2=C(C=C1O)OC(=CC2=O)C3=CC=C(C=C3)O)O	-7.40	-6.90
homoeriodictyol	COC1=C(C=CC(=C1)C2CC(=O)C3=C(C=C(C=C3O2)O)O)O	-8.10	-6.90
isorhamnetin	COC1=C(C=CC(=C1)C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O)O	-7.50	-7.10
jaceosidin	COC1=C(C=CC(=C1)C2=CC(=O)C3=C(O2)C=C(C(=C3O)OC)O)O	-7.50	-7.20
kaempferol-3-glucoside	C1=CC(=CC=C1C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)O	-7.30	-6.70
luteolin	C1=CC(=C(C=C1C2=CC(=O)C3=C(C=C(C=C3O2)O)O)O)O	-7.70	-7.30
melitric acid	C1=CC(=C(C=C1C[C@H](C(=O)O)OC(=O)/C=C/C2=CC(=C(C=C2)O/C(=C/C3=CC(=C(C=C3)O)O)/C(=O)O)O)O)O	-7.80	-7.40
naringenin	C1[C@H](OC2=CC(=CC(=C2C1=O)O)O)C3=CC=C(C=C3)O	-7.70	-7.40
nepetin	COC1=C(C2=C(C=C1O)OC(=CC2=O)C3=CC(=C(C=C3)O)O)O	-7.60	-6.80
pectolinaringenin	COC1=CC=C(C=C1)C2=CC(=O)C3=C(O2)C=C(C(=C3O)OC)O	-7.50	-6.80
pinocembrin	C1[C@H](OC2=CC(=CC(=C2C1=O)O)O)C3=CC=CC=C3	-7.30	-7.20
quercetin 3-O-glucoside	C1=CC(=C(C=C1C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)O)O	-7.80	-7.00
rosmaniric acid	C1=CC(=C(C=C1C[C@H](C(=O)O)OC(=O)/C=C/C2=CC(=C(C=C2)O)O)O)O	-7.50	-6.70
sakuranetin	COC1=CC(=C2C(=O)C[C@H](OC2=C1)C3=CC=C(C=C3)O)O	-7.30	-7.30
salvionolic acid H	C1=CC(=C(C=C1CC(C(=O)O)OC(=O)/C=C/C2=CC(=C(C=C2)O)O/C(=C\C3=CC(=C(C=C3)O)O)/C(=O)O)O)O	-7.80	-6.50
sterubin	COC1=CC(=C2C(=O)C[C@H](OC2=C1)C3=CC(=C(C=C3)O)O)O	-7.50	-7.20
velutin	COC1=CC(=C2C(=C1)OC(=CC2=O)C3=CC(=C(C=C3)O)OC)O	-7.20	-7.30

Figure 3. Known bioactive compounds from yerba santa are docked to the open (A, PDB: 7KEY) and closed (B, PDB: 1L8G) conformations of PTP1B.

Figure 4. Representative interactions between ECE bioactive compounds and PTP1B. Eriodictyol binds to a pocket near the P loop (left), while chryseriol binds to the active site, directly interacting with the P and WPD loops (right).

DISCUSSION:

Research on natural products has revolutionized the field of medicine, making remarkable contributions to drug development. More than 50% of the drugs approved by the FDA in the past four decades have been derived from natural sources.⁴² Despite this, lack of treatment for significant health concerns, including obesity, diabetes, and cancers, necessitates continued exploration of natural resources for potential therapeutic strategies. Pharmacological investigations focusing on the combined effects of phytochemicals have provided valuable insights into multiple facets of drug development, including molecular targets, disease pathways, cellular processes, and patient responses.⁴³ While extensive research has been conducted in this area, it is crucial to emphasize adopting a comprehensive and non-reductionist approach to innovative drug discovery from natural products. This approach allows us to fully understand their complex mechanisms of action at the molecular level. Particularly, crude extracts from natural sources are often advantageous over isolated single chemicals. These extracts have potential synergistic effects, exert a broad spectrum of activity, demonstrate safety, and often have a rich history of traditional use.^16,44-46 By harnessing the diverse chemical components and complex biological activities present in natural products through using crude extracts, we can pave the way for developing novel therapeutic strategies. This approach holds enormous potential in improving health outcomes and addressing the challenges posed by various diseases. It recognizes the complexity and richness of natural products and maximizes their inherent advantages, leading to the discovery of effective and safe treatments.

In this study, we investigated the unexplored potential of E. californicum as a source of inhibitors for the phosphatase activity of PTP1B, a pivotal protein implicated in obesity, diabetes, and cancers. We explored the effects of ECE on the activity of PTP1B, considering the potential synergistic interactions among multiple compounds within plant extracts. Our study underscores the importance of the holistic and synergistic properties of natural products in drug discovery. The findings reveal that ECE possesses potent inhibitory effects on PTP1B activity, as evidenced by an IC₅₀ value of 4.19µg/ml. While the IC₅₀ value of ECE was higher than the positive control used in the study (Na₃VO₄ with an IC₅₀ of 0.022µg/ml), it was significantly lower than the IC₅₀ value of pure natural compounds such as ursolic acid (0.13mg/ml) and orientin (0.18mg/ml), reported in a previous study.⁴⁷This remarkable inhibitory activity could be attributed to the abundant presence of flavonoids in E. californicum leaves.^24,27,28 Flavonoids have been extensively studied and reported to exhibit inhibition of PTP1B.^48,49 Additionally, our previous study demonstrated a substantial presence of phenolic compounds in ECE, along with a notable flavonoid content.³¹

Results from this study reveal an apparent mixed inhibition pattern by ECE, suggesting that phytochemicals in ECE may bind to both the free enzyme and the enzyme-substrate complex, thereby modulating the enzyme's kinetics and reducing its activity. The inhibitory effect was more pronounced at higher concentrations of ECE, as evidenced by a decrease in V_max and a slight increase in apparent K_m. This suggests that ECE may bind to the enzyme and induce a conformational change, making it less favorable for catalyzing the dephosphorylation reaction. The observed inhibitory effects could be attributed to the abundance of flavonoids present in ECE, which is supported by existing literature demonstrating the ability of flavonoids to exhibit a mixed type of inhibition against PTP1B.^47,50,51

To further understand the underlying inhibitory mechanism and validate the promising inhibitory effect of ECE, we performed molecular docking simulations using known bioactive phytochemicals present in ECE. Molecular docking has been used extensively in drug discovery to study the interactions between ligands and proteins.^52,53 Our findings indicate that the E. californicum flavanones exhibited interactions in shallow pockets near or at the active site of PTP1B, suggesting a potential mechanism for the inhibitory effects of ECE on PTP1B. Molecules were docked against the open and closed conformation, revealing distinct binding sites. In the open conformation, the pocket is adjacent to the P loop, although there is no direct interaction. Meanwhile, the molecules directly interact with the active site loops when bound to the closed conformation, providing a possible mechanism for competitive inhibitors. Interestingly, luteolin, one of the molecules docked, has previously been characterized as a competitive PTP1B inhibitor.⁵⁴ The modest predicted free energies of binding and the apparent mixed inhibition mechanism revealed by the kinetics study suggest the presence of additional PTP1B inhibitors in the ethanolic crude extract. Alternatively, the similarities in flavanone structures and their binding poses within the active site of PTP1B highlight the potential for a range of phytochemicals in ECE to exert synergistic inhibitory effects on PTP1B. The potent inhibition observed in this study may result from a combinatorial effect of several weak inhibitors, including flavanones or other compounds. Overall, the findings of our study suggest that ECE holds significant potential as a source of PTP1B inhibitors. These results contribute to the growing body of evidence supporting the use of natural products and crude extracts as inhibitors of PTP1B.^{47,51,52,55-58}

CONCLUSION:

In the present study, we investigated the potential of Eriodictyon californicum as a source of inhibitors for PTP1B. Our findings indicate that the ethanolic crude extract of E. californicum possesses a potent inhibitory effect on the phosphatase activity of PTP1B, exhibiting an apparent mixed inhibition pattern as revealed by kinetics analysis. Additionally, molecular docking simulations provide additional support for the potential of yerba santa flavanones as active site inhibitors of PTP1B. These findings emphasize the importance of adopting a comprehensive and holistic approach to drug discovery from natural sources, particularly in harnessing the potential synergistic effects of multiple compounds within plant extracts. The remarkable inhibitory activity observed in this study highlights the promising therapeutic potential of E. californicum as a source of PTP1B inhibitors.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

This work was supported by the KAS Special Research Awards granted by the Kentucky Academy of Science, 40202, KY, USA. Additional funding was provided through start-up funds from the College of Science and Mathematics and the Department of Biochemistry, Chemistry, and Physics at Georgia Southern University.

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