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Pirenoxine Synthesis Essay


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1. Introduction

Neoplastic cells need higher concentrations of iron and copper for their growth than normal cells [1,2], so the development of novel Fe and Cu chelators has become a promising anticancer strategy owing to their ability to inhibit cancer cell proliferation [3]. Accordingly, triapine, an iron chelator, is currently in phase II clinical trials [4]. Recently, a Dp44mT analog, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) showed markedly antitumor activity against numerous cancer cell lines. This antitumor mechanism was mediated by its ability to inhibit topoisomerase, leading to cell cycle arrest and induction of DNA fragmentation [5]. Its copper complex, (DPPCAH-Cu) exhibited similar but stronger antitumor activity as indicated by several studies [6,7]. Many anticancer drugs have potent metal chelating ability. These metal chelators, especially copper chelates, possess enhanced biological activity compared to the non-chelating drugs. Mechanistic studies have revealed that the difference is at least partially related to the redox features of the copper complexes [5,8,9]. However, information related to the effect of the interactions of metal chelating agents with biological molecules, such as human serum albumin (HSA) or bovine serum albumin (BSA) and DNA and their contribution to cytotoxicity has received limited attention. DNA and proteins (especially enzymes) are of particular interest as targets for a wide range of anticancer and antibiotic drugs [10]. To understand the nature, structure and behavior of drugs in biological systems, studies related to drug interactions with proteins and DNA are required.

The biological activity of any drug is influenced by drug-protein interactions. Proteins, mainly HSA and bovine serum albumin (BSA) are extensively studied examples, as they are the most important carriers for a broad spectrum of exogenous and endogenous ligands. HSA-drug(s) interactions greatly influence the absorption, distribution, metabolism and excretion of drugs [11,12]. Recently, Merlot et al., reported the cellular uptake of Dp44mT as a novel mechanism facilitated by human serum albumin (HSA) [13]. Accumulation of albumin occurs within the interstitium of solid tumors [14,15,16]. Chemically, BSA is a heart-shaped globular protein, containing three homologous domains (I, II, and III), and each domain includes, two sub-domains (IA and IB) [17]. BSA contains two tryptophan (Trp) residues, one (Trp134) is located on the surface of subdomain IB, and Trp213 located within the hydrophobic binding pocket of subdomain IIA. The interaction of BSA with endogenous and exogenous ligands mostly occurs in these domains.

The objective of the present study was to find the specific binding domains of BSA and DPPCAH by employing computer-aided molecular docking and spectral techniques. Numerous methods are available for investigating protein-ligand binding, such as equilibrium dialysis, fluorescence, calorimetry, and nuclear magnetic resonance [18], yet spectral techniques are widely used in determining the ligand and biomacromolecule interactions, hence our choice. Special emphasis is placed on determining the binding affinity of the DPPCAH and DPPCAH-Cu towards BSA, which could provide additional insight into the differences in antitumor activity detected between these ligands (with metal chelating ability) and their metal complexes.

2. Results and Discussion

2.1. BSA Mediated Cytotoxicity of DPPCAH and Its Copper Complex (DPPCAH-Cu)

It has been reported that HSA could enhance the anti-proliferative activity of Dp44mT [13]. Thus, we examined the effect of BSA on both DPPCAH- and DPPCAH-Cu-mediated inhibition of HepG2 cells in which BSA was the main protein source in the cell culture. As shown in Figure 1, BSA attenuated the growth inhibition of HepG2 cells mediated by DPPCAH, but not DPPCAH-Cu, indicating that the effect of BSA on uptake or cytotoxicity of the drug was drug dependent, which was consistent with earlier reports [13].

Previously, we demonstrated that DPPCAH significantly inhibited the growth inhibition of HepG2 cells (IC50: 4.6 ± 0.2 μM), while DPPCAH-Cu significantly enhanced the antitumor activity [5]. In this regards, the weaker anti-proliferative activity of DPPCAH might have be due to the abatement of BSA. Hence, a study related to their interactions was necessary.

2.2. Interactions of DPPCAH and DPPCAH-Cu with BSA

The two Trp residues of BSA are normally used as intrinsic fluorophores in spectral studies. Fluorescence quenching analyses of these Trp residues provides information regarding the interaction of the drug as quencher with BSA, revealing the mechanism(s) whereby small molecules bind to proteins. Figure 2 shows the fluorescence quenching spectra of BSA with varying concentrations of the DPPCAH and DPPCAH-Cu. The fluorescence intensities of BSA gradually decreased with the addition of the above drugs, suggesting that both DPPCAH and DPPCAH-Cu could associate with BSA. Notably, a bathochromic red shift occurred upon addition of DPPCAH (Figure 2a), unlike the blue shift induced by DPPCAH-Cu (Figure 2b), indicating that the investigated agents had different effects on the environment of Trp residues. The red shift specifies Trp residues exposed to the solvent, whereas the blue shift is a consequence of Trp residues located in a more hydrophobic environment [19].

2.3. Binding Sites and Binding Constants

The observed fluorescence quenching and bathochromic shift (or blue shift) suggested interactions between DPPCAH and BSA or the possibility of formation of non-fluorescent complex(es). To investigate the possible mechanism responsible for fluorescence quenching, Stern–Volmer plots were used to interpret the spectral data. The procedure of fluorescence quenching was first assumed a dynamic quenching process. The dynamic quenching constant Ksv and the apparent biomolecular quenching rate constant Kq were calculated using the Stern–Volmer equation: where, F0 and F are the steady-state fluorescence intensities in the absence and presence of a quencher, respectively; [Q] is the concentration of the quencher; Ksv is the Stern–Volmer dynamic quenching rate constant; τ0 is the fluorescence lifetime of the protein without the quencher, the average life of a fluorescence molecule is 10−8 s; and Kq is the quenching rate constant of biomolecule. Based on the Stern–Volmer Equation (1), plots were generated, as shown in Figure 3. The plots exhibited good linear relationships, suggesting that a single type of quenching phenomenon, either static or dynamic quenching occurred during the formation of BSA-DPPCAH (or BSA-DPPCAH-Cu). To distinguish the quenching model, additional experiments were conducted at different temperatures, as dynamic and static quenching have different temperature dependencies. With an increase in temperature, the Ksv values decrease for static quenching, and increase for dynamic quenching [20]. The data obtained at 303 K and 310 K were plotted (Figure 3a,b), and the slopes decreased (Ksv from 7.0 × 104 to 5.08 × 104 for BSA-DPPCAH, 5.47 × 104 to 4.9 × 104 for BSA-DPPCAH-Cu) with increasing temperature, suggesting that DPPCAH and DPPCAH-Cu quenched the intrinsic fluorescence of BSA mainly by static quenching. Kq values calculated for DPPCAH and DPPCAH-Cu ranged from 5 × 1012~7 × 1012, and >2 × 1010 L·mol−1·s−1, respectively, which further supported the static quenching model [21].

2.4. Binding Sites and Binding Constants

For the static quenching model, important binding parameters including association constant (Ka) and number of binding sites (n) were calculated from the plots [22] using the equation: where, Ka is the binding constant, and n is the number of binding sites per BSA. Ka and n were obtained from the intercept and slope of the curve. As shown in Figure 4a,b, the values of Ka for DPPCAH and DPPCAH-Cu at 288 K were 1.35 ×105 and 6.56 ×104, respectively. The number of binding sites n was 1.0418 for DPPCAH and 1.1053 for DPPCAH-Cu, or approximately 1. Thus, the association of one molecule of quencher with one molecule of protein was confirmed. The value also indicated that a slight decrease of Ka with an increase in temperature, which was in accordance with the aforementioned trend of Ksv. Thus, these results indicate the formation of an unstable complex during the binding. Similar results were reported from studies analyzing interaction of eupatorin with BSA [17].

2.5. Binding Forces

The interaction of small molecules with biomacromolecule involves hydrogen bonds, electrostatic interactions, van der Waals, and hydrophobic forces. In the present study, the nature of the interaction involved in binding was determined based on thermodynamic parameters, namely, Δ𝐻 > 0 and Δ𝑆 > 0 would imply a hydrophobic interaction, Δ𝐻 < 0 and Δ𝑆 < 0 hint imply hydrogen bonding and van der Waals interactions, and Δ𝐻< 0 and Δ𝑆 > 0 imply electrostatic force interactions [23,24]. The thermodynamic parameters were obtained using the van’t Hoff Equation (3): where, 𝐾 is the binding constant; T is the absolute temperature; and 𝑅 is the universal gas constant. ΔH and ΔS were obtained from the slope and intercept of the linear van’t Hoff plot (shown in Figure 5). The free energy change (ΔG) was estimated using the following formula:

Values of ΔH, ΔS, and ΔG for DPPCAH and DPPCAH-Cu binding to BSA are listed in Table 1. The negative value of ΔG reveals that the interaction process was spontaneous.

2.6. Energy Transfer between DPPCAH (DPPCAH-Cu) and BSA

Our experimental data until now proved that DPPCAH and DPPCAH-Cu were bound to BSA in a 1:1 molar ratio. Further, the distance R between the two Trp residues in BSA and the bound DPPCAH (DPPCAH-Cu) was determined using the fluorescence resonance energy transfer (FRET) method [25]. Generally, FRET occurs whenever the emission spectrum of a fluorophore (donor) overlaps with the absorption spectrum of the bound molecule (acceptor). The efficiency of energy transfer could be used to evaluate the distance between the drug and the Trp213 residue in BSA. The relationship between the distance and efficiency of FRET is described by the following equation: where, r is the distance between acceptor and donor and, R0 is the critical distance when the energy transfer efficiency is 50%. The value of R0 is calculated using the following equation:

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