# Structure-Based Drug Design for Tuberculosis: Challenges Still Ahead
## 4 General Remarks  
Several challenges need to be overcome for therapeutic molecules to be active against _M. tuberculosis_. Due to its metabolic adaptability and its known capabilities to occupy different niches inside the human host, from free aerobic bacteria to granulomas, an ideal molecule should be able to act against bacteria in very different, environmentally adapted states. The unique structure and composition of the _M. tuberculosis_ cell wall are also well known to act as a barrier for potentially active compounds. To be active against latent and active infections, an ideal drug candidate should thus be able to reach bacteria in all the tubercular lesions and niches inside the human host [103], permeate the mycobacterial cell wall or be actively internalized, and deliver the desired bioactivity in bacteria under different metabolic states. To overcome these limitations and reduce the risk of further resistance development, TB drugs are delivered in combinations, starting from the standard DOTS (directly observed therapy short-course). Drug combinations, however, have to take in due account the bioavailability and pharmacokinetic properties of the single components and their associations, as extensively treated in this same journal issue [104]. In the course of the last ten years, it has become increasingly clear that some of the criteria commonly used to classify a given _M. tuberculosis_ biological process, in many cases, an enzymatic step in a pathway, as a 'good' target for drug development, needed to be revised. This is particularly true for in vitro genetic essentiality. Even targets confirmed to be essential by the construction of specific conditional mutants do not necessarily show a good 'druggability', even when highly potent hits become available. The case of PknB is an emblematic example: despite the gene was early shown to be essential for _M. tuberculosis_ growth, first by transposon mutagenesis [43], then through attempts to generate a knock-out mutant [44], yet several research groups, including ours, had to face the problem of the lack of correlation between hit potency in vitro and efficacy in vivo [50; 51; 52]. Similar issues were also reported for other _M. tuberculosis_ kinase inhibitors, in addition to the cross-reactivity sometimes reported towards eukaryotic kinases, and the cytotoxicity shown by some compounds on human cell lines [105]. In addition, although the availability of crystal structures of both PknB and PknG in complex with hits issued from medium-throughput screens (mitoxantrone and AX20017, respectively) has looked as a promising 'proof-of-concept' [48; 58], further structure-based work on these kinases has, so far, failed to produce suitable drug candidates. Many reasons might be evoked to explain the lack of _M. tuberculosis_ kinase inhibitors that display significant antibacterial activity, ranging from the poor capacity to penetrate the mycobacterial cell and reach their target to the kinase redundancy in _M. tuberculosis_ and their, relatively poor, substrate specificity [46], which prompted groups to seek for multiple kinase inhibition [105]. Yet, the main causes should perhaps be looked for in our, still limited, knowledge of mycobacterial molecular physiology and the regulatory networks in _M. tuberculosis_, in which Ser/Thr kinases are actors of a complex interplay [46; 106]. Indeed, the activation mechanisms of both PknB and PknG are still a matter of speculation, and changes to _pknB_ expression, either as depletion or overexpression, have been reported to alter the bacterial growth significantly [34]. Given the known _M. tuberculosis_ metabolic plasticity, whose complexity has only recently started to be elucidated, thanks to the development of genetic and 'omics' tools [107], and the adaptability of the pathogen in the course of infection, it is now largely accepted that genetic essentiality of a putative target in laboratory conditions is not necessarily an indication of chemical vulnerability [15], and even more in clinical conditions.