Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

Abstract

Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.

Thioridazine: A Non-Antibiotic Drug Highly Effective, in Combination with First Line Anti-Tuberculosis Drugs, against Any Form of Antibiotic Resistance of Mycobacterium tuberculosis Due to Its Multi-Mechanisms of Action

“…Consequently, we considered the possibility that since TZ inhibits efflux pumps of bacteria [34,35,36] and also acts against efflux pumps of human cells [58,59,60], and phenothiazines in general inhibit Ca 2+ /ion channel transport [25,61], TZ may also inhibit the efflux of ions from the phagolysosomal unit leading to the indirect acidification of the compartment and the activation of hydrolytic enzymes. This possibility is supported by recent studies of Machado et al [26,35] demonstrating that TZ promotes the acidification of the phagolysosomal unit by indirect inhibition of macrophage ion channels. The inhibition of these channels therefore activates the hydrolytic enzymes via the coupling of the vesicular ATPases and consequent killing of the entrapped Mtb organism.…”

“…against XDR Mtb, where TZ showed an excellent synergistic effect with first line drugs [26] (See Figure 1 as an example). In these works TZ was shown to enhance the killing of intracellular antibiotic susceptible and MDR/XDR Mtb by monocyte-derived human macrophages that have little killing action of their own at concentrations in the medium which are equivalent or lower than those present in the plasma of a thioridazine-treated psychotic patient (0.5 mg/L of plasma).…”

“…These TZ ex vivo studies were extended to a large number of TZ derivatives, some of which revealed to be more effective than TZ and all of which expressed no toxicity at their effective ex vivo concentrations [29]. The same rationale and technical approach was further expanded from TZ and CPZ to other ion channel blockers such as verapamil, flupenthixol and haloperidol, with very successful results in enhancing the killing activity of the infected macrophage, regardless of the drug resistance profile of the infectious Mtb, with moderate and acceptable toxicities and excellent synergistic effects with first and second line anti-TB drugs [26]. …”

“…In Figure 3 a model of the putative mechanism of action of thioridazine inside the macrophage is depicted, combining all the contributions made so far to elucidate its remarkable enhancing activity of the macrophage killing activity [26,48,49,50]. …”

“…The initial response of bacteria to an antibiotic or noxious agent is to over-express its efflux pumps [26,33,34,35,78,79,80,81,82,83,84,85,86,87,88]. When the concentration of the agent is progressively increased, the genes that control and code for the efflux pumps of the responding organisms are progressively increased [33,34,35,85,87,88,89,90].…”