2.2.8. Effect of QTP Binding on the Esterase-Like Activity of HSA

HSA is the most abundant protein in the blood plasma and possesses catalytic functions such as esterase-like activity [47]. Amino acid residues such as Arg-410 Tyr-41 (Sudlow's site II (subdomain IIIA)) of HSA play a predominant function in esterase activity (Watanabe et al., 2000) [48]. However, the effect of QTP binding on the esterase-like activity of HSA is shown in Figure 8. It was observed that upon the addition of QTP (0–75 µM), there is no inhibiting effect on the esterase-like activity of HSA. *Molecules* **2022**, *27*, x FOR PEER REVIEW 11 of 17

**Figure 8.** Estimated esterase activity in HSA (5 μM) in the absence and presence of increasing concentrations of QTP (0–75 μM). **Figure 8.** Estimated esterase activity in HSA (5 µM) in the absence and presence of increasing concentrations of QTP (0–75 µM).

### 2.2.9. Computational Modeling of the HSA-QTP Complex 2.2.9. Computational Modeling of the HSA-QTP Complex

The binding region and amino acid residues involved in the interaction of QTP with HSA were evaluated by molecular docking analysis [27,45,47]. The most suitable confirmation of the HSA-QTP system is given in Figure 9A–C. The molecular docking results suggested the QTP binding region at subdomain IB (Site III) of HSA (Figure 9A). Further, QTP binds to HSA and forms two hydrogen bonds with VAL120 and ASP173 amino acid residues of HSA (Figure 10A). In addition to the two hydrogen bonds, the QTP molecule is surrounded by LEU-179, ARG-117, ALA-176, ASP-121, LEU-179, PRO-118, ALA-172, VAL-120, ASP-173, GLU-119, LYS-174, and ALA-175 through different interactions (Figure 10). The autodock results also showed that the binding affinity of QTP to HSA was −8.2 kcal mol−<sup>1</sup> . Thus, we can conclude that the molecular docking results agree with the The binding region and amino acid residues involved in the interaction of QTP with HSA were evaluated by molecular docking analysis [27,45,47]. The most suitable confirmation of the HSA-QTP system is given in Figure 9A,B. The molecular docking results suggested the QTP binding region at subdomain IB (Site III) of HSA (Figure 9A). Further, QTP binds to HSA and forms two hydrogen bonds with VAL120 and ASP173 amino acid residues of HSA (Figure 10A). In addition to the two hydrogen bonds, the QTP molecule is surrounded by LEU-179, ARG-117, ALA-176, ASP-121, LEU-179, PRO-118, ALA-172, VAL-120, ASP-173, GLU-119, LYS-174, and ALA-175 through different interactions (Figure 10). The autodock results also showed that the binding affinity of QTP to HSA was <sup>−</sup>8.2 kcal mol−<sup>1</sup> . Thus, we can conclude that the molecular docking results agree with the site displacement markers experiments (Figure 5).

site displacement markers experiments (Figure 5).

**Figure 9.** (**A**) Molecular models of HSA complex with QTP. (**B**) Detailed view of the docking poses of the HSA-QTP complex. Selected protein side-chains are shown as ribbons. **Figure 9.** (**A**) Molecular models of HSA complex with QTP. (**B**) Detailed view of the docking poses of the HSA-QTP complex. Selected protein side-chains are shown as ribbons. **Figure 9.** (**A**) Molecular models of HSA complex with QTP. (**B**) Detailed view of the docking poses of the HSA-QTP complex. Selected protein side-chains are shown as ribbons.

**Figure 10.** (**A**) The 2D binding site was magnified to show the surrounding amino acid residue of HSA interacting with QTP. (**B**) Three-dimensional structure of interactions of HSA with QTP. **Figure 10.** (**A**) The 2D binding site was magnified to show the surrounding amino acid residue of HSA interacting with QTP. (**B**) Three-dimensional structure of interactions of HSA with QTP. **Figure 10.** (**A**) The 2D binding site was magnified to show the surrounding amino acid residue of HSA interacting with QTP. (**B**) Three-dimensional structure of interactions of HSA with QTP.

#### **3. Materials and Methods 3. Materials and Methods 3. Materials and Methods**

#### *3.1. Chemical Reagents 3.1. Chemical Reagents 3.1. Chemical Reagents*

HSA (A1887, fatty acid and globulin free) and QTP (purity, 90%) were obtained from Sigma Chemical Co. (St. Louis, Mo, USA) and GLR. Scientific. Co. (Delhi, India), warfarin, ibuprofen through the National Scientific company (Riyadh, KSA) and hemin were obtained from SRL Pvt. Ltd. (Mumbai, India). All other chemicals and reagents for this study were of high analytical grade. HSA (A1887, fatty acid and globulin free) and QTP (purity, 90%) were obtained from Sigma Chemical Co. (St. Louis, Mo, USA) and GLR. Scientific. Co. (Delhi, India), warfarin, ibuprofen through the National Scientific company (Riyadh, KSA) and hemin were obtained from SRL Pvt. Ltd. (Mumbai, India). All other chemicals and reagents for this study were of high analytical grade. HSA (A1887, fatty acid and globulin free) and QTP (purity, 90%) were obtained from Sigma Chemical Co. (St. Louis, Mo, USA) and GLR. Scientific. Co. (Delhi, India), warfarin, ibuprofen through the National Scientific company (Riyadh, KSA) and hemin were obtained from SRL Pvt. Ltd. (Mumbai, India). All other chemicals and reagents for this study were of high analytical grade.

#### *3.2. Sample Preparation 3.2. Sample Preparation 3.2. Sample Preparation*

HSA stock solution (200 µM) was prepared in Tris-HCI buffers (0.2 M) pH 7.4. In addition, the stock of QTP (10 mM) was prepared in methanol and then diluted with Tris-HSA stock solution (200 µM) was prepared in Tris-HCI buffers (0.2 M) pH 7.4. In addition, the stock of QTP (10 mM) was prepared in methanol and then diluted with Tris-HSA stock solution (200 µM) was prepared in Tris-HCI buffers (0.2 M) pH 7.4. In addition, the stock of QTP (10 mM) was prepared in methanol and then diluted with Tris-HCI buffers (0.2 M), pH 7.4, to prepare the working standard samples of QTP. Finally, the buffer was prepared using Type I Millipore water (Burlington, MA, USA).
