MAPs require divalent transition metal ions as cofactors for

MAPs require divalent transition metal ions as cofactors for activity [21]. Previous studies on MAPs from different organisms indicate cobalt ions to be the most preferred divalent metal activator [22]. However, MAPs also exhibit activity with other divalent cations like Mn(II), Ca(II), Ni(II) or Fe(II) [23]. In contrast, Zn(II) is not a very suitable metal cofactor for the amidolytic activity of most MAPs [24]. The lethal phenotypes developed by MAP knockouts in both prokaryotes and eukaryotes highlights the importance of this family of DPQ [25,26]. A recent report on LdMAP2 suggests it to be a key regulator of apoptosis for the human parasite L. donovani [27]. Moreover, given their essentiality [25], MAPs are emerging as potential targets for the discovery of novel chemotherapeutics to combat bacterial, microsporidial and parasitical organisms [13,28,29]. The specific inhibitors of MAP2 like fumagillin and its derivatives have captivated interest in medicine as pro-drugs for diseases like obesity and cancer [[30], [31], [32], [33]]. Even though in clinical trials as a potent inhibitor of MAP2, the use of pro-drug TNP-470 hasn't captivated interest in the recent years due to reports of its toxicity [34,35]. This highlights the need to find newer drugs which are small, potent, highly specific and affordable. We herein report our findings on the expression, purification, and characterization of LdMAP2 as well as its interaction and inhibition with aminopeptidase inhibitor bestatin. We also show recombinant LdMAP2 to be active in the enzymatic assay against fluorogenic substrates. The structural analysis using in silico approach suggests key differences between the human and parasitic orthologs in the MAP2 prosite, catalytic pocket and a different binding mode to broad-spectrum aminopeptidase inhibitor bestatin pointing towards its suitability as a new druggable target. Our results also suggest LdMAP2 to have more affinity towards divalent calcium paving way for its use as a non-toxic biocatalyst.
Materials and methods
Results and discussion
Conclusions In conclusion, we purified LdMAP2 to homogeneity, analyzed its metal dependence, enzymatic characteristics and substrate preferences. Our findings from biochemical assays asserted that LdMAP2 functions optimally at physiological pH and its activity was enhanced by divalent cations with cobalt emerging as the major enhancer for amidolytic activity. We then underscored the need to repurpose bestatin as a prodrug as it potently inhibited LdMAP2. Additionally, CD studies revealed that bestatin and fluorogenic substrates increase the α-helical and β-sheet contents of LdMAP2. Molecular docking suggested that the LdMAP2-bestatin complex was stabilized by the hydrophobic interactions and hydrogen bonding. Furthermore, structural studies indicated that the catalytic pocket of LdMAP2 is different from its human counterpart and suggested different binding modes of two orthologs with bestatin. This substantiates the idea that LdMAP2 can be a potential druggable target to design and develop novel antileishmanials. Our studies provide important insights into the structure of LdMAP2 and critical quantitative and mechanistic data on the binding affinity of bestatin with LdMAP2 which may guide bestatin based drugs against leishmaniasis.
Author contributions
Introduction The analysis of aminopeptidases is of particular interest as they play a major role in the regulation of local tissue peptides, such as neuropeptides or circulatory hormones. Their study is important not only for basic research but also for diagnosis and therapy [38]. Aminopeptidases are involved in the activation of inactive precursors of various proteins, in the biotransformation of active peptides in new ones with different properties and also in their inactivation [38]. A clear example of the importance of aminopeptidases in the regulation of peptides functionality is the renin-angiotensin system (RAS). The trigger mechanism of the enzymatic cascade of the RAS in tissues or plasma depends on the renin activity acting on the precursor angiotensinogen to produce Ang I. However, from this stage, the production and metabolism of subsequent Ang peptides depend on the action of certain aminopeptidases, which possess angiotensinase activity [40]. Briefly, Ang I may be metabolized to Ang II by the action of angiotensin converting enzyme (ACE) or to Ang 2–10 by the activity of aspartyl aminopeptidase (EC 3.4.11.21, AspAP). Subsequently, Ang II may be metabolized to Ang III by the action of glutamyl aminopeptidase (EC 3.4.11.7, GluAP) and Ang III be transformed to Ang IV by alanyl aminopeptidase (EC 3.4.11.2, AlaAP) [40]. Ang IV binds mainly to the AT4 receptor reported to be identical to the insulin-regulated aminopeptidase (IRAP) [1] and equivalent to the enzyme vasopressinase, which can be measured as cystinyl aminopeptidase activity (EC 3.4.11.3, CysAP) [35]. The binding of Ang IV to its receptor (AT4, IRAP, vasopressinase, CysAP) leads to the inhibition of its enzymatic activity, which reduces the metabolism of vasopressin [46]. However, it should be taken into account that because of the broad substrate specificity shown by these enzymes, their activities may also reflect the hydrolysis of other peptides such as cholecystokinin by AspAP and GluAP [24], enkephalins by AlaAP [15] or oxytocin by CysAP [35] (Fig. 1). Therefore, the activity of these enzymes may reflect the functional status of their endogenous respective substrates. The regulation of these enzymes is complex as it depends not only on their synthesis and expression on the plasma membrane but also on the control of their axonal transport [34], on their secretion to plasma through autonomic stimulation [2] or on the influence of the surrounding biochemical environment [38].