The search for new antimicrobial agents is of great concern today, because of the multiple drugs resistance acquired by several pathogens [7
]. Currently, in Latin America, the methicillin resistance rates are higher than 40% among S. aureus
isolates and they are above 70 % among CNS isolates [8
]. These strains present the mecA
gene that encodes a low antibiotic-affinity penicillin-binding protein [7
]. Normally, these multi-drug resistant strains are susceptible only to vancomycin [12
]. However, the large use of this antimicrobial in hospitals has favored the emergence of vancomycin resistant species, including S. aureus
], S. epidermidis
and S. haemolyticus
]. Then, the research of new drugs is interesting.
In this study, bacterial growth inhibition by naphthoquinones showed that the synthetic compounds II (α-lapachone), III (β-lapachone) and IV [(±) 3-hydroxy-β-N
-lapachone] were more effective than their precursor lapachol (compound I), mainly the compounds III and IV. Structural analysis of the compound IV shows that its higher toxic and antimicrobial action could be associated with a hydroxyl group (OH) inserted at furan ring (Fig. ), as well as related to the naphtho 1,2-quinoidal system that is present in both compounds III and IV, making them more effective than the other compounds tested against the MRSA, MRSE and MRSH isolates. The results show that these naphthoquinones have considerable activity against staphylococci (MICs from 4 to 128 μg/mL), as we have previously reported [3
], although the activity presented by the compounds has been bacteriostatic (MBCs > 512 μg/mL). Naphthoquinones activity was observed even against vancomycin hetero-resistant isolates, suggesting that they could be an alternative antimicrobial agent in therapeutic of multi-resistant staphylococcal infections.
Bacterial protein synthesis was not inhibited by naphthoquinones, as it was demonstrated by SDS-PAGE analysis. However, it was observed that some proteins of molecular weight of 100, 70, 60 and 10 KDa, referred as stress proteins, had their levels increased by heat [23
] and by menadione treatment [22
]. As these proteins are associated with bacterial stress, naphthoquinones could have induced an oxidative stress in all microorganisms, since this class of substance is involved with cell toxicity. It is supposed that these substances promote an oxidative stress in the membrane of the bacterial cell when ATP synthesis occurs in the respiratory chain [25
]. Quinones are coenzyme Q analogs (ubiquinone, an electron-transfer substance that carries out the electrons of reduced NAD of the complex I to III), competing with these substances. When the respiratory chain is affected, several reactive-radicals like hydrogen peroxide, hydroxyl radical and superoxide anion have an increase in their concentrations, causing an oxidative stress [25
]. So, the results here presented show that the naphthoquinones analyzed cause a stress reaction in bacterial cell and suggest that it could be related to oxidative stress.
When the compounds were tested in eukaryotic cells (BSC-40), a cytotoxic effect was observed. The data in Table shows that the toxic concentration of all compounds to the eukaryotic cells corresponded to a quarter of their MIC values. These results show that these substances cannot be used by endogenous administration once they could lead to damage to human cells. However, when the substances were applied as a topic preparation in rabbits, no damage was observed, even when it was used at high concentrations, as seen for the compound IV tested in a concentration 100× higher (800 μg/mL) than the MIC observed for it. These results show that it would be possible to propose a topic use for this compound, either in prophylactic procedures or in the treatment of wound infections after major investigation in relation to a long term application on the derm and blood levels of the compounds.
Some authors describe naphthoquinones with a large antitumoral activity [2
]. In this study, although the compounds had presented a high toxic concentration in normal epithelial cells, a therapy with these substances would be possible once the tumor cells have a large metabolic rate. Naphthoquinones would be metabolized firstly by neoplasic cells, acting like other toxic chemotherapeutic agents as base analogs, alquilants, and others used in the antitumoral therapy [2
In this study a relationship between structure and toxicity of the different naphthoquinones was observed. Table shows that the substances with lower MIC (4 – 8 μg/mL) presented the highest toxicity (compounds III and IV), while the compound I with a higher MIC (256 μg/mL) did not show any toxic effect at the tested concentrations. It can be considered that structural modifications had a pronounced effect in the activity of the substances. Further studies involving structural modifications will be necessary to decrease their toxicity to eukaryotic cells, maintaining or increasing the antibacterial activity.