# Structure-Based Drug Design for Tuberculosis: Challenges Still Ahead
## 2 Protein Ser/Thr Kinases as Drug Targets
The crystal structure of the PknB-mitoxantrone complex was the first showing the kinase catalytic domain in complex with a non-ATP analog, kinase inhibitor. Two crucial, hydrogen bonding interactions were evidenced between the mitoxantrone hydroxyl groups and main chain atoms from the PknB hinge region that connects the two kinase lobes, i.e., the carbonyl oxygen of Glu93 and the amino group of Val95 [48], opening the way to compound optimization (Figure 1A,B). Several other PknB inhibitors were proposed in the course of the following years, starting from hits developed either from known kinase inhibitors like staurosporine analogs [37], 2-aminopurine and its derivatives, including organometallic compounds [49]), or from hits obtained from HTS on public or proprietary libraries using GarA as the substrate [50; 51; 52; 53], or phytocompounds [54] (Table 1). Most of the published work deals with compounds inhibiting the kinase in the micro and sub-micromolar range but with limited activity on mycobacteria. The exception to this trend is IMB-YH-8 (Table 1), a compound that, despite showing an IC\({}_{50}\) (half maximal inhibitory concentration) on PknB in the 20 uM range, shows good selectivity for mycobacterial PknB and PknA and a MIC (minimal inhibitory concentration) in the sub-micromolar range [53]. Compounds highly active on both PknB and PknA have also been reported recently by others in the form of substituted quinazolines, and, for a compound derived from this series (a pyrimidine analog), the crystal structures of the respective complexes with both kinases have been described [55] (Figure 1, Table 1). Both structures underline the common binding mode in the ATP pocket and the crucial interaction with the hinge region (Figure 1B). Despite the inhibition constant for both kinases falls in the nanomolar range, these dual-targeting inhibitors display promising but yet limited antibacterial effect with MIC on _M. tuberculosis_ in the lower micromolar range [55]. A different PknB inhibitor for which a crystal structure in complex with the target is available is GSK690693 (Figure 1C, Table 1), also identified through a virtual screening approach on known kinase inhibitors [56]. This compound, member of the imidazopyridine aminofurazans class, also displays conserved features in its binding mode to the PknB hinge region (Figure 1B), and sub-micromolar affinity to the kinase, but no significant antimycobacterial activity on _Mycobacterium smegmatis_ or _Mycobacterium bovis_ BCG (bacillus Calmette-Guerin). However, the MIC is significantly lowered if the compound is associated with a sub-MIC50 concentration of meropenem, suggesting a synergistic action between PknB inhibitors and \(\beta\)-lactams [56]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Family** & **Name** & **Structure** & **IC\({}_{50}\) (uM)** & **Reference** \\ \hline Quinazolines & Disubstituted series & & \(\leq 1.1\) & [51] \\ Pyrimidines & Disubstituted (-R1 also as -NHR1) & & \(\leq 0.4\) & [51] \\  & Disubstituted (-R1 also as -NHR1) & & \(\leq 0. On the other hand, another _M. tuberculosis_ kinase that attracted as much attention for its potential druggability is PknG, a soluble Ser/Thr kinase that was described, by [PERSON] and coworkers, as a virulence factor secreted into the human macrophage, where it would inhibit the phagosome-lysosome fusion [57]. Most notably, in the same work, the authors showed that chemical inhibition of PknG by the compound AX20017 (a tetrahydrobenzothiophene identified by HTS and found to inhibit PknG with IC\({}_{50}\) in the sub-micromolar range; Table 2) led to the accumulation of _M. tuberculosis_ inside lysosomes [57]. Noteworthy, the 2.4 A resolution crystal structure of an N-terminal truncated form of PknG in complex with AX20017, published later [58], shows a great similarity between the AX20017 binding to PknG and the binding of ATP and mitoxantrone to PknB.