Investigating the inhibitory potential of 2-Aminopurine metal complexes against serine/threonine protein kinases from Mycobacterium tuberculosis

Abstract

Tuberculosis – a disease caused by Mycobacterium tuberculosis (Mtb), is one of the most devastating disease. The discovery of Ser/Thr protein kinases (STPKs) in Mtb opened a new avenue for developing anti-tubercular inhibitors. The in-vivo inhibitory effects of many metal ions have been demonstrated in literature. But, one of the limitations of metal ions as inhibitors is their inability to traverse the hydrophobic membrane due to polar nature and their propensity for non-specific interactions. To overcome this, we attached a metal ion to 2-A9P – an analog derived from a cell permeable scaffold, 2-Aminopurine (2-AP) which is a known kinase inhibitor. We investigated the inhibitory potential of 2-AP and its analog 2-A9P against protein kinase B (PknB) and showed that both of these can inhibit Mtb STPKs. Next, we evaluated the latent inhibitory activity of metal ions and for the first time showed that they can inhibit the phosphotransfer reaction in PknB, PknG and PknL. Subsequently, 6 different metal complexes (MC) of 2-A9P were used for inhibitory studies and their estimated IC₅₀ values show that most MCs inhibited PknB with low micromolar potency. Further, MIC values determined for the six MCs against Mtb showed that MC-4 and MC-6 exhibit whole cell inhibitory activity. Cytotoxicity studies show that MC-4 and MC-6 do not affect cell viability of A549 cell lines, suggesting that these inhibitors can be further developed as anti-tubercular agents.

Introduction

Despite the development of modern therapy, tuberculosis is of serious concern to mankind - claiming nearly 2 million lives and causing 9.8 million new infections each year [1]. Approximately one-third of the population harbors this disease in its latent or dormant form. In the dormant state, the infection is contained by the immune system within a structure called the granuloma and therefore has 5–10% risk of activation in the entire lifetime [2]. Alarmingly, there has been an emergence of multiple and extensively drug-resistant strains (MDR-TB and XDR-TB) [3], [4]. Mtb has developed resistance against two of the most important first line drugs- isoniazide and rifampicin (MDR-TB). Further, resistance against second line injectable drugs like fluoroquinolones is a growing concern (XDR-TB) [5], [6]. Considering the shortcomings of the available treatment options, there is an immediate need to identify novel targets and develop new scaffolds to fuel the TB drug pipeline.

With the sequencing of Mtb genome, 11 eukaryotic like Serine/Threonine protein kinases (STPKs) have been uniquely annotated in Mtb. They play important roles in a plethora of pathways essential for its survival and pathogenesis [7], [8]. PknA, PknB, PknG and PknL are highly conserved within the Mycobacterium genus, suggesting important physiological roles for these proteins in Mtb. PknB is a trans-membrane kinase known as a regulator of cell-shape and cell-division, and a replication switch in response to hypoxia [9], [10], [11]. It has been shown to be essential for growth and survival of Mtb in host [12]. PknG is reported to play important roles in virulence by inhibiting phagosome-lysosome fusion and in survival inside host during latency [13], [14], [15]. Taken together, STPKs represent an important group of drug targets in Mtb.

Protein kinases are one of the most sought after drug targets in eukaryotes [16]. Several molecules have been identified that exhibit in-vitro as well as in-vivo inhibitory activity against a range of eukaryotic kinases, albeit at millimolar concentrations. Considering the close sequence as well as structural similarity between eukaryotic protein kinases and STPKs, as well as the conserved architecture of ATP binding pocket among these - eukaryotic kinase inhibitors can be evaluated for anti-STPK inhibitory activity. Moreover, for effective anti-tubercular therapies these molecules may need to be further modified for higher specificity and potency against Mtb STPKs. A prominent example of such an inhibitor is 2-Aminopurine (2-AP); an analog of adenine known to act in eukaryotes as an in-vivo inhibitor of RNA dependent protein kinase R (PKR) [17], [18]. Studies have demonstrated that 2-AP has multiple cellular targets and it may inhibit more than one kinase in eukaryotes [19], [20]. In this study, our aim was to evaluate the inhibitory activity of 2-AP against PknB and examine the possibility of re-purposing 2-AP as an inhibitor for mycobacterial STPKs.

Metal ions have been used for centuries as anti-infectives for the treatment of antibacterial and antifungal diseases. Silver ions and complexes were used to prevent bacterial growth and for healing of wounds caused by burns [21]. Zinc ions were used as an antiseptic in many ointments due to their wound healing property. For instance, a complex of zinc (II) acetate with erythromycin has been utilized for acne therapy [22], [23]. Several other metal ions were used without major side effects, highlighting their specificity towards the prokaryotic cells. Hence, there is tremendous scope for rational design of antibacterial inhibitors based on metal ions.

The development of coordination complexes or organometallic inhibitors is gaining prominence since they offer certain advantages over classical organic inhibitors. Variability of oxidation states and achieving different spatial dispositions are interesting properties of coordination compounds, which is lacking in organic molecules. Structurally simple organic molecules exhibit conformational flexibility, which causes undesired binding at nonspecific sites [24]. In metal complexes, compared to the tetrahedral binding mode of carbon, varied coordination geometry of metals offers more structural complexity [25], [26]. Considering these advantages, many metal coordination complexes as inhibitors have been rationally developed, largely in the field of cancer [27], [28], but these were further extended to other disease contexts as well [29], [30].

Based on the above observations, we sought to investigate and develop novel coordination complexes as possible inhibitors of mycobacterial STPKs. As mentioned, competitive inhibition exerted by 2-AP on bacterial kinases has not been evaluated. In this study, we investigated the inhibitory activity of 2-AP and its analog 2-A9P against PknB. We have also evaluated the latent inhibitory activity of metal ions and for the first time showed that metal ions inhibit STPKs at micromolar concentrations.

Although transition metal ions inhibit enzymatic activity, they have certain limitations such as cellular toxicity, lower permeability and non-specificity. Interestingly, Feng et al. developed highly specific kinase inhibitors against GSK3R, PAK1 and PIM1 by introducing ruthenium Ru(II) in the structure of staurosporine, which was otherwise a nonspecific kinase inhibitor [31]. Inspired by this observation, we implemented a similar metal-complex derivatization approach for 2-A9P (an equipotent analog of 2-AP) and various transition metals to develop organometallic inhibitors. These were expected to combine the kinase-specificity of 2-AP with the inhibitory effect of metal ions on kinase activity. These metal complexes were assessed for their inhibitory activity against Mtb kinases as well as for their efficacy against whole cell cultures of Mtb. Further, we also performed cytotoxicity studies on mammalian cells. In summary, the present study demonstrates the inhibitory effect of 2-AP-based organometallics against STPKs PknB, PknG and PknL and underscores the potential these compounds hold, in order to be developed as effective Mtb therapeutics.