Binding characteristics of the major kratom alkaloid, mitragynine, towards serum albumin: Spectroscopic, calorimetric, microscopic, and computational investigations.

Mitragynine (MTG) is a prominent indole alkaloid that is present abundantly in Mitragyna speciosa, commonly referred to as kratom. MTG has garnered significant attention due to its selective agonistic characteristics towards opioid receptors and related analgesic effects. In the circulatory system, the in vivo efficacy of MTG is dictated by its interaction with plasma proteins, primarily human serum albumin (HSA). In the present study, we utilized a broad methodology that included spectroscopic, calorimetric, microscopic, and in silico approaches to characterize the interaction between MTG and HSA. Alterations in the UV absorption spectrum of HSA by the presence of MTG demonstrated a ground-state complexation between the protein and the ligand. The K values obtained for the MTG-HSA interaction were in the range 10-10 M based on analysis of fluorescence and ITC data, respectively, indicating an intermediate binding affinity. The binding reaction was thermodynamically favorable as revealed by ΔH, ΔS, and ΔG values of -16.42 kJ mol, 39.97 J mol K, and -28.34 kJ mol, respectively. Furthermore, CD spectroscopy results suggested MTG binding induced minimal effects on the structural integrity of HSA, supported by computational methods. Changes in the dimensions of HSA particles due to aggregation, as observed using atomic force microscopy in the presence of MTG. Competitive drug displacement results seemingly suggested site III of HSA located at subdomain IB as the preferred binding site of MTG, but were in inconclusive. However, docking results showed the clear preference of MTG to bind to site III, facilitated by hydrophobic (alkyl and pi-alkyl) and van der Waals forces, together with carbon hydrogen bonds. Additionally, the MTG-HSA complexation was demonstrated to be stable based on molecular dynamics analysis. The outcomes of this study shed light on the therapeutic potential of MTG and can help in the design of more effective derivatives of the compound.