In Silico Evaluation of Diketopiperazine (DPK) Derivatives as Potential Inhibitors for G-Protein-Coupled Receptors (GPCRs) ^†

Abstract

G-protein-coupled receptors (GPCRs) are a group of various membrane proteins that mediate essential physiological processes by translating extracellular signals into intracellular responses. The β2-Adrenergic Receptor (β2-AR), a key GPCR, plays a critical role in smooth muscle relaxation, bronchodilation, and cardiovascular function, making it an important therapeutic target for diseases such as asthma and hypertension. Diketopiperazines (DPKs), as cyclic peptides, have shown promise as scaffolds for inhibiting protein interactions and modulating receptor activity, offering a potential alternative to traditional small-molecule inhibitors with reduced side effects. In this study, five DPK derivatives were selected from the PubChem database and evaluated for their binding affinity to the 3D structure of β2-AR (PDB ID = 2RH1) through molecular docking studies using AutoDock 4.6 and MGLTools. The binding energy and hydrogen bond formation of each compound were evaluated to determine their interaction efficiency. Among the compounds, tryptophan-proline diketopiperazine (compound 3) exhibited the highest binding affinity with a binding energy of −5.89 kcal/mol. This enhanced interaction is attributed to the aromatic nature of tryptophan, which promotes strong π-π stacking interactions, and the rigidity of proline, which optimally fits within the receptor’s binding pocket. Hydrophobic interactions further stabilized the complex. These findings highlight compound 3 as a promising β2-AR modulator, providing valuable insights for the design of peptide-based inhibitors targeting β2-AR-related pathologies.

1. Introduction

G-protein-coupled receptors (GPCRs) are one of the most diverse and largest families of membrane proteins, playing essential roles in mediating a wide range of physiological processes. GPCRs are involved in various biological functions, including neurotransmission, immune response modulation, sensory perception, and regulation of metabolism, making them critical targets in the treatment of numerous diseases [1]. These receptors work by transducing extracellular signals, such as hormones, neurotransmitters, and sensory stimuli, into intracellular responses through the activation of G-proteins and downstream signaling pathways. Their ability to regulate cellular processes and function across diverse biological systems makes GPCRs critical to maintaining homeostasis in the human body [2,3].

One of the most studied GPCRs is the β2-Adrenergic Receptor (β2-AR). This receptor plays a pivotal role in mediating smooth muscle relaxation, bronchodilation, and regulating cardiovascular functions. As such, β2-AR is a crucial pharmacological target for treating a variety of diseases, including asthma, hypertension, and chronic obstructive pulmonary disease (COPD). Its widespread role in modulating critical biological processes, particularly in the respiratory and cardiovascular systems, has led to its prominence in drug discovery efforts. In particular, agonists and antagonists targeting β2-AR are commonly used to manage conditions such as asthma and other respiratory disorders, underscoring the therapeutic significance of β2-AR [4,5,6].

Despite the success of small-molecule drugs targeting GPCRs, traditional therapeutics face several challenges, such as receptor desensitization, limited specificity, and off-target effects. These issues often arise from the inherent flexibility of GPCRs, which can lead to unwanted interactions with other proteins and receptors. Moreover, the prolonged use of small molecules may result in receptor downregulation or reduced efficacy over time. In contrast, peptide-based inhibitors have emerged as an attractive alternative, offering several advantages in terms of structural stability, high specificity, and a reduced likelihood of off-target effects. These peptides can be designed to target specific receptor sites with high affinity, minimizing systemic side effects and enhancing therapeutic efficacy [7].

Among the peptide-based inhibitors, cyclic peptides such as diketopiperazines (DPKs) have garnered significant attention due to their ability to modulate protein–protein interactions effectively. DPKs, a class of cyclic peptides, have been recognized for their stability, rigidity, and ability to interact with protein targets in a highly specific manner. These features make DPKs excellent candidates for modulating GPCR function. DPK derivatives can adopt rigid, well-defined conformations that allow for optimal fitting within receptor binding pockets, providing a robust mechanism for binding and potentially inhibiting receptor activity. Their structural diversity also allows for the design of peptide-based ligands with a high degree of specificity for particular GPCR subtypes, enabling the development of targeted therapies with fewer side effects [8,9,10].

Molecular docking studies have become a powerful tool in the drug discovery process, particularly in evaluating the interaction and affinity between ligands and receptors at the atomic level. Regarding GPCR-targeted therapies, docking studies are useful for the prediction of binding affinities and the identification of key interactions between ligand molecules and the receptor’s binding pocket. These computational approaches highlight the identification of potential drug candidates by simulating how ligands interact with the receptor and by providing insights into their stability and binding efficacy. In this study, we used molecular docking to evaluate the binding potential of selected DPK derivatives to β2-AR. The goal is to identify peptide-based drug candidates that can modulate β2-AR activity and, ultimately, provide an overview for developing safer and more effective treatments for β2-AR-related diseases [11].

2. Methods

To evaluate the binding efficiency of diketopiperazines (DPKs) to β2-adrenergic receptor (β2-AR), molecular docking studies were performed using AutoDock 4.6 and MGLTools [11]. Five DPK derivatives, selected from the PubChem database based on their structural diversity and predicted interactions with GPCRs, were considered in this study. These compounds were pre-processed to ensure compatibility with the docking simulations. Specifically, the DPK derivatives were converted into appropriate file formats, and the necessary Gasteiger charges and Kollman charges were added, along with the addition of polar hydrogens, to ensure correct preparation for docking.

The crystal structure of β2-AR (PDB ID: 2RH1) was obtained from the Protein Data Bank and prepared for docking analysis [12]. Before docking, the receptor was prepared by removing unnecessary molecules, adding hydrogen atoms, and assigning the right charges. The final structure was saved in PDBQT format, which works with AutoDock 4.2 and makes sure the receptor’s atoms, charges, and flexible parts are handled correctly during the simulation [13].

Each DPK derivative was subjected to energy minimization to optimize its structure and ensure its stability prior to the docking procedure. Docking simulations were carried out using the Lamarckian Genetic Algorithm (LGA), which is a well-established method in AutoDock for predicting binding affinity. The LGA provided binding energy values for each compound, which were used to evaluate the interaction efficiency. Key parameters assessed during the docking process included binding energy (kcal/mol), the formation of hydrogen bonds, and the presence of hydrophobic contacts. These parameters are essential for understanding the stability and strength of the receptor–ligand interaction and were used to determine the most favorable binding interactions between each DPK and β2-AR.

Visualization of the molecular structures and docking results was performed using Chimera and PyMOL 1.8 for 3D structural analysis [14,15]. The physicochemical properties of the selected cyclic dipeptides—including molecular weight, hydrophobicity (Log P), and net charge at physiological pH (7.4)—were calculated using the MCULE online drug discovery platform, which provides structure-based property predictions for small molecules.

3. Results and Discussion

Molecular docking simulations were conducted to assess the binding affinity of five diketopiperazine (DPK) derivatives to β2-adrenergic receptor (β2-AR). The binding energy and the number of hydrogen bonds formed between each compound and β2-AR were used to evaluate the strength of the receptor–peptide interactions. The 3D structures of DPK derivatives and the results of binding energy are presented in Figure 1 and

Table 1. Physicochemical properties and binding parameters of cyclic dipeptides (DKPs) docked to β2-Adrenergic Receptor (β2-AR).

	Name of DPKs	Binding Energy (Kcal/mol)	No. H Bond	Molecular Weight (g/mol)	Hydrophobicity Log P	Net Charge at pH 7.4
1	2,5 Piperazinedione	−3.00	2	114.10	−1.1100	0
2	Alanine diketopiperazine	−3.70	2	142.1559	−0.3330	0
3	Tryptophan proline diketopiperazine	−5.89	2	283.32	1.4665	0
4	Diketopiperazine	−2.95	2	114.10	−1.1100	0
5	Histidylproline diketopiperazine	−5.13	2	248.2810	0.0984	0

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Among the five DPK derivatives tested, tryptophan-proline diketopiperazine (Compound 3) exhibited the highest binding affinity to β2-AR, with a binding energy of −5.89 kcal/mol. This compound formed two hydrogen bonds with the receptor, which contributed to its stable interaction. Additionally, compound 3 displayed a greater degree of structural rigidity due to the proline residue, which enhances its binding efficiency. The presence of tryptophan in the compound provides aromaticity, promoting π-π stacking interactions that may further stabilize the ligand. Figure 2 depicts the 3D structures of the β2-AR and the Try-Pro DPK. Figure 3 shows a LigPlot+ representation of the β2-Adrenergic Receptor (β2-AR) in complex with Try-Pro DPK. The diagram illustrates two hydrogen bonds formed between Try-Pro DPK and key amino acid residues (Ser203 and Try308) within the receptor’s binding pocket [16].

The docking results revealed significant differences in binding affinities among the DPK derivatives. Compound 3, tryptophan-proline diketopiperazine, demonstrated the strongest binding affinity and interaction efficiency, as indicated by its low binding energy of −5.89 kcal/mol. This can be attributed to several key structural features:

• Aromatic Interactions: The presence of tryptophan in compound 3 introduces aromaticity that may facilitate π-π stacking interactions with residues in the β2-AR binding pocket, thereby enhancing the compound’s binding strength.
• Structural Rigidity: The proline residue in the compound provides rigidity, ensuring a stable conformation within the binding pocket, which further improves the compound’s fit and binding potential.
• Hydrogen Bonding: The two hydrogen bonds formed by compound 3 contribute to the stability of the receptor–ligand complex, suggesting a strong interaction with β2-AR.
• Hydrophobic Contributions: The compound’s hydrophobic features likely contribute to its overall stability in the hydrophobic regions of the receptor’s binding pocket.

Comparative analysis of the docking results revealed that histidylproline diketopiperazine (Compound 5) demonstrated a relatively strong binding affinity (−5.13 kcal/mol), although slightly lower than that of compound 3. In contrast, 2,5-Piperazinedione (compound 1) and Diketopiperazine (compound 4) exhibited noticeably weaker binding affinities, with binding energies of −3.00 kcal/mol and −2.95 kcal/mol, respectively. Overall, the molecular size of the cyclic peptides appears to influence binding performance, as compounds 3 and 5, being slightly larger in structure, showed more favorable interactions within the receptor binding site.

Overall, these findings suggest that the combination of aromaticity, rigidity, and hydrophobicity, as seen in compound 3, plays a key role in optimizing binding efficiency. These properties make compound 3 a promising candidate for further studies aimed at developing peptide-based modulators for β2-AR.

4. Conclusions

This study highlights the potential of diketopiperazines as promising peptide-based modulators for the β2-Adrenergic Receptor. Specifically, tryptophan-proline DPK (compound 3) exhibited superior binding affinity and interaction efficiency, making it a strong candidate for further drug development. The structural advantages of DPKs, including aromaticity, rigidity, and selective binding, support their role as viable alternatives to traditional small-molecule inhibitors. Future research should focus on experimental study through in vitro and in vivo studies to confirm the efficacy and pharmacokinetic properties of these peptide-based compounds for therapeutic applications.