Advanced Spectroscopic and Theoretical Study and Assessment of Antimycotic Potential in a Synergistic Composition of a 1,3,4-Thiadiazole Derivative and Amphotericin B

Abstract

The paper presents the results of an in-depth spectroscopic, theoretical (quantum chemical), and microbiological study conducted on a promising, synergistic composition of a newly considered 1,3,4-thiadiazole derivative, 1,3,4-thiadiazole: 2,4-dihydroxy-N-(5-methyl-1,3,4-thiadiazol-2-yl)-benzothioamide (TBTA), and the "gold standard" polyene antibiotic, amphotericin B (AmB). The spectroscopic properties of the system were extensively analyzed with a range of spectroscopic measurement techniques, including electronic fluorescence and absorption spectra, resonance light scattering measurements, circular dichroism spectra, dynamic light scattering, and fluorescence anisotropy, which were further complemented with time-resolved measurements of fluorescence lifetimes performed with the single-photon counting method. The samples were prepared in DMSO solutions and/or PBS buffer to facilitate observation of the monomeric, dimeric, and aggregated forms of the antibiotic previously identified in the literature. Absorption and fluorescence emission spectra measured for AmB and the synergistic composition revealed differences that indicated changes in AmB aggregation molecules, particularly in the buffer medium. Together with the results of the other spectroscopic techniques and computations, the effects of AmB disaggregation are clearly observed, and it is seen that TBTA interacts with AmB at the sites where other AmB molecules prefer to interact with it. We also present the first biological analysis of this TBTA/AmB composition, and it confirms the synergistic effects of TBTA. The report provides a detailed description of the synergism observed between a newly synthesized derivative from the group of 1,3,4-thiadiazoles (TBTA) and the antibiotic AmB, an effect that may prove to be very significant in the context of the ongoing efforts to identify new substances with antifungal properties.

1. Chemical Structures of the Molecules: Panel ATBTA and Panel BAmB

1

Electronic absorption spectra for TBTA, AmB, and the synergistic composition of TBTA + AmB, in DMSOPanel A; Panel Belectronic absorption spectra for analogous systems in the PBS buffer medium.

2

Example fluorescence emission spectra for TBTA, AmB, and their synergistic composition in buffer PBS medium, analogous to the spectra in Figure .

3

Example fluorescence anisotropy curves for AmB and the synergistic composition in a buffer PBS medium.

4

CD spectra for: TBTA, AmB, and the synergistic TBTA + AmB composition, in PBS.

5

RLS spectra for: TBTA, AmB, and the synergistic composition of TBTA + AmB, in DMSOPanel A; Panel BRLS spectra recorded for the same systems in the buffer PBS medium.

6

TBTA 0.0750 mM sample (A), AmB 0.0108 mM sample (B), and TBTA + AmB 0.0750 mM + 0.0108 mM sample (C) excited with 365 nm UV light in the buffer PBS medium.

7

Typical fluorescence decay profiles for AmB and the synergistic composition TBTA + AmB, in DMSO (Panel A) and PBS (Panel B). Upper panels show the decays and respective 3-exponential fits. Lower panels show the residual distribution.

8

Mean hydrodynamic sizes (Panel A) and polydispersity indices (Panel B) for AmB particles alone and in the synergistic system of AmB + TBTA, in PBS or DMSO at 22 °C, measured using the technique of DLS, with their corresponding standard deviations.

9

A) Computed binding energies, ΔE _bind, of all the AmB/TBTA dimers that were generated; ΔE _bind is computed at the GFN2-xTB level of theory as the difference in energy between the optimized dimer and each of the optimized reagents. The HtoH (“head-to-head”) data sets refer to geometries where the terminal hydroxyl group of TBTA is positioned “down” relative to the reference AmB geometry (see panel B), whereas the HtoT (“head-to-tail”) data sets refer to geometries where the C1 hydroxyl group is initially positioned “up”. Each ΔE value is plotted vs the y coordinate TBTA’s central N atom, such that a negative value indicates TBTA is positioned near the polyol chain and a positive value implies it is near the polyene chain. (B) Two most stable TBTA/AmB dimers are shown as 1 and 2.

10

Growth curves for S. cerevisiae on RPMI mediaPanel A, showing the impact of TBTA concentrated at 64 μg/mLPanel B, AmB concentrated at 0.5 μg/mLPanel C, 0.25 μg/mLPanel E, 0.125 μg/mLPanel G, as well as synergistic compositions thereof concentrated at AmB 0.5 μg/mL +8 μg/mL TBTAPanel D, AmB 0.25 μg/mL +16 μg/mL TBTAPanel F, and AmB 0.125 μg/mL +64 μg/mL TBTAPanel H.