Understanding the synergistic interaction between a 1,3,4-thiadiazole derivative and amphotericin B using spectroscopic and theoretical studies

Abstract

We present a comprehensive spectroscopic study supported by theoretical quantum chemical calculations conducted on a molecular system (4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1) and the antibiotic Amphotericin B (AmB)) that exhibits highly synergistic properties. We previously reported the strong synergism of this molecular system and now wish to present related stationary measurements of UV-Vis absorption, fluorescence, and fluorescence anisotropy in a polar, aprotic solvent (DMSO and a PBS buffer), followed by time-resolved fluorescence intensity and anisotropy decay studies using different ratios of the selected 1,3,4-thiadiazole derivative to Amphotericin B. Absorption spectra measured for the system revealed discrepancies in terms of the shapes of absorption bands, particularly in PBS. Fluorescence emission spectra revealed that the addition of C1 molecules triggered significant changes in the emission spectra of the system. Measurements of the fluorescence lifetimes and fluorescence anisotropy supported by synchronous spectra clearly showed evidence of disaggregation. The AmB molecular aggregates indicated interaction of C1 with the antibiotic at points responsible for the formation of dimer structures. The spectroscopic results were further corroborated, analyzed, and interpreted using the methods of quantum mechanical modelling. Analyses based on the density functional tight-binding and time-dependent density functional theory confirmed that molecular interactions between "small" molecules and AmB lead to a significant increase in the clinical efficacy of the antibiotic.

Keywords: 1,3,4-thiadiazole (C1); [TD]DFT; Amphotericin B (AmB); DFT; Molecular spectroscopy; Synergism.

Scheme 1

Chemical structures of the C1 (Panel A) and AmB (Panel B) molecules that were considered in this study.

Fig. 1

Panel A – absorption spectra registered for C1, AmB, and the synergistic C1 + AmB system in DMSO. Panel B – absorption spectra registered for the same compounds in PBS buffer. Panel C – fluorescence emission spectra corresponding to the absorption spectra presented in Panel A. Panel D – fluorescence emission spectra corresponding to the absorption spectra in Panel B.

Fig. 2

Juxtaposition of fluorescence lifetimes decay curves for C1, AmB, and the synergistic C1 + AmB composition.

Fig. 3

Fluorescence anisotropy measured in the PBS buffer for C1 (Panel A), AmB (Panel B), and the synergistic composition C1 + AmB (Panel C).

Fig. 4

Panel A –RLS spectra registered for the synergistic C1 + AmB composition in DMSO. Panel B – RLS spectra registered for the synergistic C1 + AmB composition in PBS.

Fig. 5

Fluoresce lifetime decay curves for the C1 + AmB system. AmB was titrated in PBS with different amounts of C1.

Fig. 6

The computed binding energies (ΔE_bind = E[AmB/AmB] – 2*E[AmB]) of all the AmB/AmB dimers that were generated for the “head-to-head” (HtoH, Panel A) and “head-to-tail” (HtoT, Panel B) datasets at the GFN2-xTB level of theory. Each data-point is plotted vs. the distance between the midpoint of the two polyen chains. A particularly stable dimer from each dataset is below each graph as 1 and 2.

Fig. 7

The computed GFN2-xTB binding energies (ΔE_bind = E[AmB/C1] – E[AmB] – E[C1]) of all the AmB/C1 dimers that were generated; the “head-to-head” (HtoH) datasets refer to geometries where the terminal hydroxyl group of C1 is positioned “down” relative to the reference AmB geometry (in the sense that the -OH group is positioned at a lower x coordinate than the thiadiazole ring, as is shown in top panel), whereas the “head-to-tail” (HtoT) datasets refer to geometries where the C1 hydroxyl group is initially positioned “up”. Each data-point is plotted vs. the y coordinate of C1’s central C-C bond, as this indicates whether the C1 is positioned near the polyol (at negative values) or the polyene chain (at positive values). The three most stable characteristic structures are shown as 3, 4, and 5.

Fig. 8

Isosurface plots (isovalue = 0.0005 a.u.) of the CIS-DFT density differences for the first excited singlet states (indicated by λ₁) of a low-energy AmB dimer (model 1_en, panel A) and of a low-energy AmB/C1 dimer (model 5, panel B), computed at the CAM-B3LYP/Def2TZVP TDDFT/TDA level theory. S₀ refers to the optimized ground state GFN2-xTB geometry and S₁ refers to the TDDFT/TDA-optimized geometry for the first singlet excited state.