# Structure-Based Drug Design for Tuberculosis: Challenges Still Ahead
## 2 Protein Ser/Thr Kinases as Drug Targets  
Protein phosphorylation is a well-known, widespread mechanism for signal transduction and regulation of several biological functions. Protein kinases have long been known as major drug targets [29], especially for the treatment of cancer, where the deregulation of signaling mechanisms is a hallmark of the disease [30]. Protein kinases are not only pharmaceutical targets in cancer chemotherapy but also for the treatment of parasitic infections, ranging from the ones caused by Trypanosomatids or _Leishmania_[31] to malaria [32]. Since the sequence of _M. tuberculosis_ H37 Rv genome was first reported in 1998 [18], the pathogen has been known to possess eleven genes coding for Hanks-type Ser/Thr kinases [33], named from _pknA_ to _pknL_ (but no _pknC_), providing one of the first challenges to the paradigm of prokaryotic cell signaling as being entirely driven by two-component systems. One gene coding for a transmembrane Ser/Thr phosphatase, _pstP_, was also identified as lying on the same cluster as _pknA_ and _pknB_, forming a putative operon [34], in addition to two genes (_ptpA_ and _ptpB_) coding for Tyr phosphatases [35]. Nine out of eleven Ser/Thr kinases (all but PknG and PknK) were predicted to be integral membrane proteins, all sharing the same topology with an N-terminal catalytic domain in which the archetypal Hanks motifs could be identified [33], a single transmembrane segment and a very variable extracellular, C-terminal domain, hypothesized to be acting as a signal sensing domain. The role of such C-terminal domains is, in most cases, still puzzling, although, in some kinases like PknB, it has been suggested to be involved in the kinase activation process, possibly by controlling the kinase oligomerization state as a function of the external signal [36]. Work from [PERSON]'s group in Berkeley indeed demonstrated the role of the 'back-to-back' dimerization to promote an active kinase conformation, not only for PknB [37; 38] but also for PknD [39], notwithstanding the observation that mutations in the dimerization interface do not abolish kinase activity, as reported by separate groups [37; 40]. PknB was also the first _M. tuberculosis_ kinase for which the crystal structure of the catalytic domain was described in 2003 [41; 42]. Indeed, this first crystal structure confirmed the overall conservation of the bi-lobed protein kinase fold and the initial assumptions about the presence of the known structural features of eukaryotic protein kinases, as predicted by the detection of the Hanks motifs [42], thus underlining the common origin of eukaryotic and prokaryotic kinases. Following these first milestones and the genetic proof of the in vitro essentiality of _pknB_ coming both from transposon mutagenesis [43] and targeted studies [44; 45], PknB then attracted the attention of tuberculosis (TB) research community as an ideal target for structure-based drug design. The community's interest rise when considering the increasing evidence, over the years, of its role in the control of peptidoglycan synthesis and cell division (recently reviewed in [46]). The first description of the potential use of PknB inhibitors as anti-mycobacterial agents reported the compound H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine; Table 1), well before any bacterial protein kinase structure was available [47]. Once the PknB catalytic domain structure was solved, virtual screening carried out on a library of about 40,000 compounds for hits into the PknB adenosine triphosphate-binding (ATP-binding) pocket was performed. The screening led to identifying mitoxantrone, a chemotherapeutic agent known for its DNA intercalating properties, as a sub-micromolar PknB inhibitor [48], similar to staurosporine, K-252-a, and K-252-b (Table 1), who were identified as hits after testing a few commercially available eukaryotic kinase inhibitors [44].