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The potency of blumeatin and luteolin as caspase-1 inhibitor by molecular docking

COVID-19 infection induces inflammation by increasing cytokines such as IL-1β, IL-6, IL-18, IFN-γ, and TNF-α. IL-1β is generated by the involvement of caspase-1. Therefore, caspase-1 inhibitor can be potential for inflammation therapy caused by COVID-19 infection. This study aims to determine the potential of blumeatin and luteolin as anti-inflammatory agents by inhibiting caspase-1 using a molecular docking approach. This study was carried out by caspase-1 (PDB ID: 1RWK) preparation, blumeatin and luteolin structure optimization, docking protocol validation, and docking of blumeatin and luteolin on caspase-1. Bluematin and luteolin had a binding affinity of -5,63 kcal/mol and -5,93 kcal/mol, lower than Q158 native ligand (-3.92 kcal/ mol). Similar amino acid residues in hydrogen bonds interaction were observed between Q158 native ligand, blumeatin, and luteolin with caspase-1 (GLN 283 and ARG 179). Blumeatin and luteolin are potentially anti-inflammation agents through the inhibition of the caspase-1 in silico.


Introduction
COVID-19 infection causes inflammation of the lungs by increasing cytokines [1]. Cytokines have an important role in the immune response, including defense against viral infections. However, COVID-19 infection can result in an overproduction of cytokines (cytokine storm) that can develop into pneumonia. Cytokine storms play a role in causing acute respiratory distress syndrome (ARDS) [2]. ARDS is a critical condition that needs a prompt and appropriate therapeutic intervention to prevent acute lung damage, multi-organ failure, and death [3].
Cytokine storm is a critical, life-threatening condition that requires intensive care. Cytokine storms are characterized by the clinical presentation of excessive systemic inflammation, hyperferritinemia, hemodynamic instability, and multi-organ failure. The trigger for a cytokine storm is an uncontrolled immune response that results in the continuous activation and expansion of immune cells, lymphocytes, and macrophages. Clinical findings of a cytokine storm are caused by pro-inflammatory cytokines such as IL-1, IL-6, IL-18, IFN-γ, and TNF-α [4].
IL-1 is a potent inflammatory cytokine involved in immunological responses to both innate and adaptive immunity. There are two similar molecules of IL-1, IL-1α, and IL-1β [5]. IL-1β is expressed in many tissues, including lung tissues [6]. IL-1β is produced by interleukin-1β converting enzyme (ICE), also known as caspase-1, that can be activated due to OCVID-19 infection [7]. Caspase-1 is a cysteine protease that converts pro-inflammatory IL-1β into active and mature IL-1β [8].
The inhibitory activity of blumeatin and luteolin against caspase-1 can be determined by using in silico molecular docking, a computational simulation used to know the binding between a ligand and protein [13]. This study aims to determine the potential effect of blumeatin and luteolin as anti-inflammation by inhibition of caspase-1 using a molecular docking approach.

Optimization of blumeatin and luteolin
The three-dimensional (3D) structures of blumeatin and luteolin were downloaded from https://pubchem. ncbi.nlm.nih.gov/. The structures were optimized using HyperChem 8 with Austin Model 1 (AM1) semi-empirical computational method and singlepoint calculations and geometry optimization.

Validation of docking method
The molecular docking procedure was validated using the Autodock Tools application (Autodock4 and Autogrid4) through redocking the native ligand Q158 to the prepared caspase-1 protein. The grid box size was set to x = 30 Å, y = 20 Å, z = 30 Å with the x, y, and z coordinate centers were 33.016 Å, 60.302 Å, and 4.934 Å, respectively. The validation parameter of the molecular docking method was the value of root mean square deviation (RMSD), which is valid if the value ≤ 2.0 Å [14].

Blumeatin and luteolin docking to caspase-1
The molecular docking of the prepared blumeatin and luteolin was performed using Autodock 4.2 program. The docking process was conducted with a similar grid box size as validation step. The docking results showed the conformations with the lowest binding energy in complex with caspase-1 protein.

Data analysis
The molecular docking results were binding energy and visualization of the interaction between blumeatin or luteolin and caspase-1 protein. The lower the binding energy, the stronger the interaction of the  compounds with protein, indicating the potential as anti-inflammatory agents.

Caspase-1 preparation
The preparation of caspase-1 protein aims to separate the protein from Q158 native ligand as well as to obtain the Q158 for the docking validation. The prepared caspase-1 and Q158 native ligand are displayed in Figure 2.

Optimization of blumeatin and luteolin
The optimization of blumeatin resulted in total single point energy and geometry optimization of -3996 kcal/mol and -4006 kcal/mol, respectively.
Meanwhile, the total single point energy and geometry optimization obtained of luteolin were -3606 kcal/mol and -3814 kcal/mol (Figure 3).

Validation of molecular docking
The docking method was validated by redocking Q158 native ligand to caspase-1. The validation produced 10 conformations with different RMSD values and binding energy. Conformation 6 had the lowest RMSD value of 1.97 Å ( Table 2), suggesting the docking method is valid.

Docking of blumeatin and luteolin to caspase-1
The optimized blumeatin and luteolin were then docked to the caspase-1 protein. The docking process  produced ten conformations with binding affinity -5.63 kcal/mol and -5.93 kcal/mol for blumeatin and luteolin, respectively (Table 3). Visualization analysis indicated blumeatin and luteolin interacted with caspase-1 by hydrogen bonding through ARG 179 and GLN 283 residues (Figure 4).

Discussion
Our results showed that blumeatin and luteolin have a lower binding affinity (-5.63 kcal/mol and -5.93 kcal/mol) than Q158 native ligand (-3.92 kcal/ mol) ( Table 4). Blumeatin and luteolin have similar hydrogen-bonding interactions with Q158 native ligand through ARG 179 and GLN 283 residues. These results suggested blumeatin and luteolin docked in the same active site of Q158 native ligand in the caspase-1 target protein.
Based on this study, blumeatin and luteolin are predicted to have activity as the anti-inflammation agent. The interaction that occurs between blumeatin and luteolin on the active site of caspase-1 shows inhibitory activity so that it can inhibit the maturation of IL-1β, thereby decreasing the mature IL-1β expression.

Conclusion
The binding energy of blumeatin and luteolin with caspase-1 was lower than Q158 native ligand, indicating these compounds have an affinity for caspase-1. Blumeatin and luteolin are potentially anti-inflammation agents based on the in silico study against caspase-1.