The Search for New Agonists to P2X7R for Clinical Use: Tuberculosis as a Possible Target Open Access

Tuberculosis (TB) is a chronic infectious airborne disease. Robert Koch identified its etiologic agent, Mycobacterium tuberculosis (MTB), in 1882 [1]. This disease primarily affects the lungs, which is the first organ to meet and receive the infecting bacilli. Therefore, pulmonary TB is the predominant form of this disease (approximately 70% of the cases). Furthermore, the bacilli may spread and infect other organs, such as the lymph system, bones and meninges, causing extra clinical pulmonary TB manifestations [2].

The Koch bacilli enter the lung parenchyma, infect macrophages and dendritic cells, which phagocytize MTB, and migrate through the lymphatic system to the regional lymph nodes [3]. Phagocytic cells remain in the lung parenchyma and initiate an inflammatory response, which attracts more leukocytes to the region. In this case, the accumulation of inflammatory cells around the bacilli leads to the development of a granuloma, a process that is coordinated by T cells [2]. Once the passage of the bacilli through the airway has begun, the MTB can spread and settle in any organ, either during the initial infection, when the adaptive immunity is not yet fully established, or during a late phase of the infection, which is related to a failure of the host's capacity to maintain the bacilli within the granuloma [3].

TB is most prevalent in low human development index countries or emergent economies [4]. Currently, TB is one of the top three causes of death in women aged 15-44 years in Brazil [5]. Recently, the number of TB cases has increased worldwide due to the emergence of multi-drug-resistant strains, the increase in cases of patients co-infected with both TB and HIV and the difficulties related to treatment. In 2011, 8.7 million people developed this disease, including 1.1 million cases of people co-infected with HIV, and approximately 1.4 million deaths from TB were registered [4].

Another concern involving TB is the increasing number of multi-drug-resistant TB (MDR-TB) strains, with an estimated prevalence of 650,000 cases in 2011 and approximately 150,000 deaths in 2008. MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLDs) [5]. XDR (extremely drug-resistant) is defined as the resistance to at least isoniazid and rifampicin, or any fluoroquinolone, and one of the three second-line drugs (e.g., amikacin, capreomycin and kanamycin) [6]. The number of patients treated for MDR-TB rose to 46,000 in 2011 [1,4]. This demonstrates the necessity of finding new therapeutic approaches. In this context, the search for new medicines and immunomodulators could help reduce the prevalence and incidence of multi-drug-resistant tuberculosis cases [7].

P2XR are receptors that have as physiological ligand ATP. They are ionotropic receptors, forming a non-selective cation channel when activated by agonists, which allows the passage of Na⁺, Ca²⁺ and K⁺ ions across the cell membrane. The activation of these receptors results in a rapid increase in the intracellular calcium concentration, an important cellular messenger, as well as the depolarization of cells in a milliseconds range [8,9]. These receptors consist of only three protein subunits, each one consists of two transmembrane domains (TM1 and TM2), which are separated by an extracellular loop that contains ten cysteine residues that form disulfide bonds, and an intracellular amino residue (C) and a carboxy (C) terminal. [9,10]. Since 1994, seven subtypes of P2XR have been found in mammalian and cloned, which were named P2X1-7 and it is known that these receptors are widely expressed in different species of vertebrates [11]. The P2X1 subunits, 2, 3, 4 and 6 can interact to form homotrimeric or heterotrimeric channels. P2X5 is little active in the homotrimeric form, but appears to be functional in heterotrimeric forms: P2X1 / P2X5, P2X2 / P2X5 and P2X4 / P2X5 [12].

Likewise, P2X7R is the only one that works as a homotrimer and presents some outstanding characteristics. The first is the need for high concentrations of ATP to be activated; the P2X7R has an EC₅₀ ≥ 100 µM to be activated, while other subtypes (P2X1-6) are activated in the range of 0.07 - 10 µM ATP [13]. Another feature is the ability to form a pore membrane when activated by high ATP concentrations (≥ 100 µM) or by prolonged exposure to this agonist. This allows the bidirectional transport of ions through the membrane pore, as well as, the passage of molecules with molecular weight up to 900 Da, except lymphocytes whose molecular weight limit is between 200-300 Da. This pore seems to be related to the cytotoxicity associated with P2X7R, and their formation depends on the intracellular C-terminal domain [8].

P2X receptors are expressed in different cell types, such as neurons, glial cells, epithelial cells, lung cells, muscle cells, endothelial cells and immune cells, including macrophages, monocytes and lymphocytes among other cells [9,12]. Because of this wide expression in many biological systems and the promoted effects reported, the P2XR has been increasingly studied as a potential therapeutic target for several diseases, including chronic inflammatory diseases and pain. In fact, it is already described in the literature that the use of certain selective antagonists for some of these receptors subtypes, such as P2X3R and P2X7R, e.g., in animal models, have shown promising anti-nociceptive and anti-inflammatory effects [11].

There are some effects related to P2X7R activation, including the regulation of the expression and release of some cytokines, inflammatory mediators and inflammatory metabolites, including interleukin (IL)-1β [14], IL-1α [15], IL-2 [16], IL-4, IL-6, IL-13, IL-18 [14,17,18,19], tumor necrosis factor (TNF)-α [14], nitric oxide (NO) [20] and superoxide anions [21,22]. The correlation between P2X7R and the process of IL-1β release is described in the literature, which shows an early involvement of caspase-1 activation through mechanisms dependent on the inflammasome complex [14]. We can also highlight the formation of multinucleated giant cells [23,24], the induction of MTB death [25], cytoskeletal reorganization [26] and caspase activation [27].

Recently, several studies have indicated an important role of P2X7R in MTB infection (Table 1) [25,28]. Macrophages obtained from humans with 1513A → C polymorphism in the P2X7R gene and infected with MTB do not suffer apoptosis and fail to induce mycobacterial death when treated with ATP [29]. Similar results were observed with human macrophages infected with MTB [28,30]. However, one study conducted in 2006 by Placido et al. [31] showed that extracellular ATP induces apoptosis in monocytes/macrophages infected with MTB via the P2X7R and reduces bacilli viability. The immunomodulatory effect of extracellular ATP was also described through P2X7R activation, which increases major histocompatibility complex (MHC) class II expression in macrophages infected with Mycobacterium bovis (BCG) or MTB and contributes to the generation of the antimicrobial T cell response [32]. In addition, a recent work published by Amaral et al. [33] proposed the blocking of P2X7R during hypervirulent and aggressive infections. Since this receptor acts on cellular pathways, which leads to a secretion of pro-inflammatory cytokines, such as the IL-1β. The overstimulation of the immune response promoted by the hypervirulent infection, added to the activation of the P2X7R, could lead to an intense cellular necrosis generating the lung necrosis [33]. It is important to point out that hypervirulent infections are different from MDR and XDR infections. In keeping with the physiological role of the P2X7R in tuberculosis, several case-control studies have shown susceptibility to tuberculosis in single P2X7R polymorphisms of the P2X7R [34]. The most relevant association has been observed with 153A/C mutation; a recent meta-analysis has concluded that it is a risk factor for pulmonary and extra-pulmonary tuberculosis in Asians [35]. Li et al. pointed to the inhibition of the biosynthesis and activity of mycobacterial siderophores as a way to impair the proliferation, virulence and latency of the bacillus [36]. Abate et al. showed that patients with gene mutations for Rifampicin and/or isoniazid resistance showed no correlation with resistance to second line medicines. To second line medicines, although the treatment of these patients with high doses of Rifampicin or Isoniazid was shown to be non-effective [37].

The clinical use of ATP in the treatment of many diseases can be controversial, since the wide distribution of P1 and P2 receptors in the human body, and its degradation by ectonucleotidases, which could result in terrible side effects. Thereto, the first article that showed the use of ATP in humans was published in the 40's, in order to treat rheumatoid arthritis [38], in which the authors claimed an improvement or completely recovery, in a long or short period of time after the treatments, in 66 per cent of the 144 patients studied.

In 1996, Haskell and co-workers returned to evaluate the clinical use of ATP. They described the pharmacodynamics of ATP in humans, based on a phase I study, in which they measured the toxicity, maximum safety tolerated dose (MTD), and pharmacokinetics of intravenous ATP [39]. In this work, the patients received 96-hour infusions of ATP monthly in doses ranging from 50 to 100 micrograms/kg/minute. They concluded that lenghty infusions of ATP are possible, including an acceptable toxicity, and that 50 micrograms/kg/minute is both the MTD and the most appropriate dose rate for a Phase II study within a 96 hour scheme [39]. Since this study, many others have been published, studying the effects of ATP infusions in cancer, cachexia and as it been an energy source [39,40,41,42,43,44]. The majority of these works were cited in different therapeutic protocols as illustrated in Table 2. In addition, the most common side effects was a chest discomfort and the necessity to take a deep breath. In general, these side effects were managed by adjusting the infusion rate and its dosage [40,45]. It is important to point out that patients with previous cardiopulmonary conditions had higher toxicity.

The final plasmatic concentration of ATP is complicated to determinate due to its degradation by ectonucleotidases present in the plasma, or expressed on the cell membrane of leukocytes or on endothelial cells. In addition, the erythrocytes acts as an ATP buffer as shown by Haskell et al. [39 ]and Agteresch et al. [43]. Actually, there are no data in relation to ATP concentration in the plasma or in the interstitial space in human beings. Howsoever, in mice, several groups have found ATP in a concentration ranging a hundred micromolar in inflammation and tumoral sites [46,47]. Recently, a study using the technique of intravascular microdialysis demonstrated interesting results. In this study, Mortensen and cols show the venous and arterial ATP concentration in sixteen healthy young men. The arterial and venous concentrations of ATP were [109 ± 34 and 147 ± 45 nM/L] in rest. In addition, 7 minutes of ATP infusion (0.4, 0.8 and 1.7 umol/min) did not alter the concentration in the venous system. The T1/2 (half-life) found in this study was < 1s [48].

Presently, the oral administration of ATP as a dietary supplement, or medicine, has been applied in several countries [49]. This has generated several concerns about the efficacy and the safe use of ATP in humans; in this context, a recent randomized double-blind study has shown that oral ATP improved the recovery of patients submitted to Total Knee Arthrosplasty [50]. Some tuberculosis patients have a clinical profile similar to a terminal cancer patient [51,52] when infected with untreatable MDR or XDR strains. Thus, in this condition, a clinical trial based on the use of ATP or a selective agonist for P2X7R could be approved, firstly, if they works well in animal models. Unfortunately, BzATP (the most selective agonist for P2X7R) has not been tested in humans to date. There are several studies, describing the action of this molecule in vivo using murine models [53,54,55,56,57].

A recent work published by Xu et al. [58] demonstrates that there are splice variants of the P2X7R with a difference in ATP responses, depending on which cell type expressing these responses. Then, it might be possible to synthesize agonists to act on the P2X7R expressed by macrophages, in this way, decreasing the possibility of serious side effects [58]. Although the research for new selective agonist molecules for P2X7R is scarce [59]. In this context, the screening of new possible selective agonist molecule for its receptor, which they could be resistant to the action of ectonucleotidases avoiding the generation of harmful byproducts, such as ADP, becomes necessary. In the future, they can be useful in the clinical treatment of MDR-TB and XDR-TB cases.

As mentioned previously, the numbers of MDR-TB and XDR-TB cases have increased worldwide. MTB is an intracellular parasite, and chemotherapeutic agents must enter the cytoplasm of the infected cell to kill it. Therefore, novel medicines are essential. We speculate that the synergistic or additive effect of extracellular ATP (or an equivalent analog) in association with bactericidal or bacteriostatic antibiotics could be a treatment for TB. As reviewed above, extracellular ATP activates P2X7R and induces the opening of a membrane pore, which allows the passage of molecules up to 900 Da. This property could be used to increase the delivery of hydrophilic or amphipathic antibiotics to the cellular cytoplasm, such as isoniazid (MW: 137.14 Da), rifampicin (MW: 822.94 Da), ethambutol (MW: 277.2 Da) and ethionamide (MW: 166.2 Da) (Fig. 1).

In addition to the pore mechanism, P2X7R activation could kill infected macrophages and consequently MTB via apoptosis or autophagy [25,29,32]. P2X7R activation stimulates signaling pathways, which lead to the death of the intracellular mycobacteria (Fig. 2). Deng et al. described the influence of the MTB protein Rv1808 on the host immune cell response [60], which could be inhibited through P2X7R activation.

Amaral et al. [61] and Martins et al. [62] reported the inhibition of potassium efflux in MTB-infected macrophages as a survival pathway used by MDR-TB and XDR-TB drug-resistant strains because this efflux could lead to an acidification of the phagolysosome and promote bacillus death. Therefore, the restoration of the potassium efflux could be a therapeutic target to treat infections by resistant MTB strains. In this context, P2X7R emerges as a promising target because it opens a channel that allows potassium efflux when activated by ATP. P2X7R activation by ATP causes the instantaneous opening of a cation channel, which allows for Ca²⁺ and Na⁺ influx and K⁺ efflux. This process initiates a number of downstream signaling events, such as the caspase cascade and activation of the inflammasome complex, and results in apoptosis or phospholipase D activation, which promotes the phagosome-lysosome fusion and causes mycobacterial death [63,64,65]. A recent work published by Santos et al. established an experimental model for in vivo studies in mice to evaluate the P2X7R activity during MTB infections [66].

Infection with multi-drug-resistant strains has become a public health problem in countries with low human development index. These infections cause millions of deaths per year, which signals the need for both improvement in the current treatment and a renewed search for new chemotherapies with greater efficacy than current medicines. This review proposed a therapeutic approach to improve current treatments, focusing on P2X7R activation using ATP, or other selective agonist molecules, as a medicine, which could act sinergically to restore the efficacy of these medicines. In the context of multi-drug-resistant cases and to ameliorate the reduced potency of current medicines via low-dose administrations, which would diminish the occurrence of side effects.

The authors declare no conflicts of interest.