Abstract
The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs.
Highlights
Toad venom, called Chansu in China, is a traditional Chinese medicine (TCM) obtained from the skin and parotid venom glands of the toad, including Bufo bufo gargarizans Cantor and B. melanostictus Schneider
The fluorescence intensities at 300–500 nm revealed a remarkable decrease with the addition of each of these nine bufadienolides. These results indicate that bufadienolide binding with human serum albumin (HSA) causes micro-environmental changes in the carrier protein during the generation of the HSA/bufadienolide complex
To verify that the quenching observed represented complex formation between each bufadienolide of interest and HSA, it is important to calculate the value of Ksv, which showed the rate constant of protein dynamic quenching procedure initiated by drugs
Summary
Toad venom, called Chansu in China, is a traditional Chinese medicine (TCM) obtained from the skin and parotid venom glands of the toad, including Bufo bufo gargarizans Cantor and B. melanostictus Schneider. In China, toad venom is frequently used as an effective clinical TCM preparation (e.g., as cinobufotalin or Chansu injection) to treat malignant tumors [1]. The therapeutic effect of toad venom stems from its major active ingredients, corresponding to assorted bufadienolides [2] (bufalin, cinobufagin and so on). All major bufadienolides in toad venom exhibit significant anti-tumor activity, including the inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance and enhancement of cytotoxic drug activity [3, 4]. Most bufadienolides have short half-lives (the half-life of bufalin is only 0.42 h in rat), and effective drug concentrations cannot be upheld for long periods of time in vivo [5]. The short drug half-lives and toxicity are serious factors limiting their development as chemotherapeutic agents in oncology
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