# Structure-Based Drug Design for Tuberculosis: Challenges Still Ahead
## 2 Protein Ser/Thr Kinases as Drug Targets
Not only the compound occupies the adenine binding pocket but also makes similar interactions with the main chain atoms of residues Glu233 and Val235 (Figure 1). It was only a few years later that further structural work allowed to elucidate the binding mode of ATP to PknG and suggested a regulatory role of the rubredoxin-like domain by a reversible occlusion of the active site entrance [59]. These initial findings paved the way to further structure-based lead optimization and undoubtedly generated excitement for the potential chemical targeting of this kinase, which would allow preventing the arrest of phagosome maturation, directing _M. tuberculosis_ to lysosomes. Attempts to develop more potent compounds starting from AX20017 yielded sub-micromolar inhibitors, some of which had no activity in macrophage assays [50], and no other structure of a PknG-inhibitor complex has so far been reported. It is, however, worth noting that, more than ten years later, the role of PknG in arresting the phagosome maturation is still elusive. Although progress has been made in identifying the SecA2 system as responsible for the export of PknG outside _M. tuberculosis_[60; 61], and interference by PknG on the host Rab71 signaling pathway has been reported [62], it is largely accepted that several mechanisms, and not a single virulence factor, contribute to the _M. tuberculosis_ capability to escape the phagocytic route and survive into macrophages [22; 63]. Moreover, an increasing amount of evidence has since validated PknG as a key signaling element in the control of central metabolism, as first shown in _Corynebacterium glutamicum_[64; 65], then in mycobacteria [66; 67], where genetic and metabolomic evidence point to a role of PknG in regulating the 2-oxoglutate node according to nutrient availability [68]. Alternative roles of PknG in biofilm formation, adaptation to oxidative stress [69], and hypoxia [70] have also been proposed. Therefore, the observed effects of the lack of PknG on the phagosome-lysosome fusion, and the decreased viability of a _pknG_-deprived _M. tuberculosis_ mutant, both in vitro and in the mouse model [71], may not necessarily indicate a direct interference of the kinase on the host signaling pathways but could be ascribed to metabolic alterations caused by the depletion of PknG [68; 72]. During the last years, further search for PknG inhibitors has allowed identifying scleroorin, an azaphilone derivative isolated from _Penicillium_ sp. ZJ27 (Table 2), a known inhibitor of other unrelated enzymes like lipoxygenase, that, despite showing an IC\({}_{50}\) on PknG around 76 \(\upmu\)M and no inhibition on _M. tuberculosis_ growth in vitro, displays capability to partially reduce the growth of _Mycobacterium bovis_ BCG inside macrophages, and to enhance the effect of rifampicin [73]. On the other hand, the recently described PknG inhibitor NU-6027 (2,6-diamino-4-cyclohexylmethoxy-5-nitrosopyrimidine; Table 2) was identified from phenotypic screening as having an MIC99 value of 1.56 \(\upmu\)M on _M. bovis_ BCG and shown to partially inhibit PknG and PknD autophosphorylation in vitro [74]. Another series of four compounds, all inhibiting PknG in the micromolar range, was also recently reported after the screening of an 80-compound, commercially available kinase inhibitor library (Table 2); three of them were found to promote the transfer of mycobacteria to lysosomes, and two to inhibit _M. bovis_ BCG growth in macrophages [75]. None of the reported compounds, however, issued from a structure-based screening approach.