Chemically Tuning Resveratrol for the Effective Killing of Gram-Positive Pathogens

Abstract

In the era of antimicrobial resistance, the identification of new compounds with strong antimicrobial activity and the development of alternative therapies to fight drug-resistant bacteria are urgently needed. Here, we have used resveratrol, a safe and well-known plant-derived stilbene with poor antimicrobial properties, as a scaffold to design several new families of antimicrobials by adding different chemical entities at specific positions. We have characterized the mode of action of the most active compounds prepared and have examined their synergistic antibacterial activity in combination with traditional antibiotics. Some alkyl- and silyl-resveratrol derivatives show bactericidal activity against Gram-positive bacteria in the same low micromolar range of traditional antibiotics, with an original mechanism of action that combines membrane permeability activity with ionophore-related activities. No cross-resistance or antagonistic effect was observed with traditional antibiotics. Synergism was observed for some specific general-use antibiotics, such as aminoglycosides and cationic antimicrobial peptide antibiotics. No hemolytic activity was observed at the active concentrations or above, although some low toxicity against an MRC-5 cell line was noted.

Figure 1

Bactericidal/bacteriostatic effect analysis. (A) Killing kinetics for resveratrol (RES) and the different selected RES derivatives, compounds 15, 17, 32, and 33, at 2-fold the MIC concentration. The data represent the average of CFU/mL ± SEM. (B) Bactericidal/bacteriostatic activity of RES and the different selected RES derivatives, compounds 15, 17, 32, and 33. Neg: negative control (nontreated cells). MIC_A indicates the MIC obtained for the bacteria after a first MIC test ± SEM. MIC_B indicates the minimal bactericidal concentration ± SEM. > indicates that the bacteria grew above this concentration for the tested compounds. All the tests were performed in triplicate.

Figure 2

Antimicrobial effect of resveratrol (RES) and the different selected RES derivatives, compounds 15, 17, 32, and 33 on actively growing cells. The % of growth was calculated with respect to the negative control, and it is expressed as the average ± SEM. The concentration of the compounds used is expressed in μM. * indicates compound additions. Gramicidin S (GRA) was used as a positive control. Neg: negative control (nontreated cells).

Figure 3

Propidium iodide membrane permeabilization test for resveratrol (RES) and the different selected RES derivatives, compounds 15, 17, 32, and 33. The data are expressed as the average of random fluorescent units (RFU, fluorescence normalized with the OD₆₀₀) ± SEM. The concentration of the compounds is expressed in μM. Gramicidin S (GRA) was used as a positive control. Neg: negative control (nontreated cells).

Figure 4

DiSC3(5) membrane potential detection for resveratrol (RES) and the different selected RES derivatives, compounds 15, 17, 32, and 33. The data are expressed as the average of normalized RFU ± SEM. The concentration of the compounds is expressed in μM. All the tests were performed in triplicate. Gramicidin S (GRA) was used as a positive control. Neg: negative control (nontreated cells).

Figure 5

(A) Activity of the respiratory chain for RES (resveratrol), CCCP (carbonyl cyanide m-chlorophenyl hydrazone), and compounds 15, 17, 32, and 33, measured by reduction of resazurin (RSC) to resofurin ± SEM. The concentration of the compounds is expressed in μM. (B) Intracellular ATP levels expressed as % with respect to the negative control ± SEM. All the tests were performed in triplicate. Neg: negative control (nontreated cells).

Figure 6

Hemolytic activity of resveratrol (RES) and selected resveratrol derivatives, compounds 15, 16, 17, 20, 31, 32, and 33. Percent of hemolysis was calculated with respect to the positive control (Triton X-100) ± SEM.