Drug discovery and resistanceAntimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis
Introduction
Tuberculosis (TB) is an infectious disease caused by members of the Mycobacterium tuberculosis (Mtb) complex and comprising one of the most important infectious diseases. Although the antimycobacterial drugs available until present led to controlled TB prevalence rates by several decades, the appearance of resistant cases brought TB to a worrying status [1], [2]. Moreover, the incidence of TB has risen in the last years. In particular, the incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is also increasing, and become the major health concern worldwide [1]. This scenario highlights the importance of design and development of new anti-TB drugs to handle future TB cases.
Nowadays, the treatment of TB is mainly performed in a 6-month regimen. It is widely accepted at present that the physiological state of non-replicating persistence (NRP) subpopulation of mycobacteria is responsible for the antimycobacterial tolerance, as well as the key to shortening the long treatment regimen. It is highly appreciated that new anti-TB agents to be effective against this subpopulation to achieve this goal [3]. The low-oxygen-recovery assay (LORA) [3] is a method considered adequate to identify hits and lead compounds active in this subpopulation. Several examples of bioactive compounds were identified using this approach, such as quinolines [4], quinoxalines [5], arylbenzylpurines, aza- and deaza-purines [6] and aminothiazoles [7].
PZA (Figure 1) is a first-line agent to TB control in several countries (including Brazil). PZA is one of the most efficient agents in killing Mtb inside the granuloma, where slow-growing bacilli are found. PZA interferes with the membrane energy metabolism and function [8] possibly through competitive inhibition of NADPH binding in Mtb fatty acid synthase-I (FAS-I) [9]. Additionally, PZA is more active in acidic conditions [8]. However, its exact mechanism of action remains unknown. The Zhang's hypothesis [8] establishes that PZA actions could be, at least partially, dependent of its conversion to the active acid metabolite, pyrazinoic acid (POA), since some PZA-resistant strains do not express the enzyme responsible for the conversion of PZA into POA (named as nicotinamidase/pyrazinamidase, PZAse). It was verified that the PZA conversion to POA (Figure 1) could play the main role in PZA resistance [10], [11], [12]. Given the characteristics of POA, prodrug approach is a feasible method to transport POA inside Mtb, allowing it to exert its antimycobacterial activity. Several POA esters (Figure 1) were already synthesized and evaluated as prodrugs of POA, which must be activated by mycobacterial esterases. After hydrolysis, these esters (Figure 1) have shown activity even in strains that do not express PZAse, corroborating to Zhang's hypothesis [8].
Although POA esters are important alternatives to be considered in resistant TB strains, they did not achieve the therapeutics yet. Among the problems encountered during their development, poor stability in horse plasma was verified due to rapid hydrolysis by plasmatic esterases [2], [12]. Important pharmacokinetic parameters, as protein binding, were also not evaluated for these molecules, and it is widely known the role of pharmacokinetic to the success of a drug candidate. Moreover, the activity of POA esters in NRP Mtb were not been studied yet. Herein, we report the activity of POA esters in replicating and non-replicating Mtb, the influence of albumin on its activity, and considering POA as the active agent after hydrolysis, two novel duplicate prodrugs of POA were designed and evaluated.
Section snippets
Chemistry
Chemicals were purchased in adequate purity from commercial sources. Six POA prodrugs were synthesized following two methods (A and B). Method A was used to prepare compounds 1–3 and method B to prepare compound 4. The remaining molecules (5–6) were prepared using method C. Some of these methods were already published previously by our group [2], [13]. The final tested compounds (1–6, Figure 2) are presented in Table 1, Table 2, Table 3.
Results and discussion
Although several research groups are engaged in the search for active molecules, only few molecules have reached clinical trials, and in fact, since the introduction of RMP in 1967, only two new chemical entity (NCE) has reached the therapeutics, bedaquiline and delanamid. Considering this, prodrug design is considered a feasible approach to achieve new active molecules, especially chemotherapeutics. This technique consists on designing inactive molecules, which can be activated in vivo in the
Conclusion
In conclusion, the presented POA esters showed moderate anti-Mtb activity, improved in pH 6.0, especially in albumin-free medium. It is noteworthy the activity in LORA, which is an important subpopulation in TB infection, and very low cytotoxicity of the compounds. Novel duplicated prodrugs of POA showed interesting activity, and a new set of duplicated compounds will be synthesized and evaluated considering these results in order to improve the anti-Mtb activity.
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