L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria

Abstract

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy.

Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds.

Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar K_d. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells.

Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

Keywords: Drug repurposing; Flavodoxin; High-throughput screening; Narrow-spectrum antibiotics; Target-oriented drug discovery.

Fig. 1

ITC of Sp-Fld with three potential binders identified in a HTS assay: a) cefotaxime, b)l-thyroxine and c) cefixime.

Fig. 2

Antimicrobial activity of l-thyroxine and 13 chemical analogues against Sp. a) Growth inhibition curves. b) MICs and structures of the tested compounds.

Fig. 3

Antimicrobial activity of 3,3′,5-triiodo thyropropionic acid (Cp4) and 3,5,3′,5′-tetraiodo thyroacetic acid (Cp5) against Sp, S. aureus N305, and E. faecalis FA2-2.

Fig. 4

Best ranked poses obtained with Autodock Vina on pocket F-Po-3 for the four most active compounds (lower MIC values). a) Compound Cp1, b) Cp2, c) Cp3, d) Cp4. At the left side of each panel the best pose (pose 1 in Table S4) is shown (sticks) in its binding site (semi-transparent surface coloured by atom binding affinity). The ligand environment is shown in the form of cartoons (secondary structure) and lines (residues of the side chain). The main ligand-protein interactions are shown in the same way as in the representation on the right side of each panel (red dashed arrows: hydrogen bond acceptor; green dashed arrows: hydrogen bond donor; magenta dashed arrows: halogen bond donor; yellow spheres: hydrophobic contact; red trident: negative ionizable group; blue trident: positive ionizable group). Interacting amino acid residues are indicated close to their partners in grey border boxes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)