Recent Advances in Antituberculous Drug Development and Novel Drug Targets

Haruaki Tomioka, PhD; Yutaka Tatano, PhD; Ko Yasumoto, PhD; Toshiaki Shimizu, PhD

Disclosures

Expert Rev Resp Med. 2008;2(4):455-471. 

In This Article

Drug Targets Related to Bacterial & Host Cell Signaling

Pathogenic mycobacteria modify host signaling pathways to enable them to survive and replicate in host macrophages, including blocking phagosomal maturation, preventing apoptosis and suppressing the antibacterial immune response.[56] For instance, man-LAM produced by pathogenic mycobacteria inhibits an increase in the Ca2+/calmodulin concentration, preventing phagosome-lysosome fusion. Man-LAM also downregulates MAPK pathways by activating Src-homology 2 domain-containing tyrosine phosphatase 1, thereby resulting in the suppression of macrophage functions, such as the production of proinflammatory cytokines, that are required for the expression of innate immunity against mycobacterial infection.[57] In addition, the MTB genome encodes some proteins, especially protein kinases, that act on and intervene in host cell signaling pathways, thereby yielding an intracellular milieu suitable for bacterial survival and growth in host cells, including macrophages. After the internalization of Mycobacterium bovis (a mycobacterial pathogen belonging to the MTB complex) bacillus Calmette-Guerin (BCG) strain into host macrophages, the BCG serine/threonine protein kinase G (PknG) encoded by the pknG gene is secreted within macrophage phagosomes and inhibits phagosome-lysosome fusion, mediating the intramacrophage survival of the mycobacteria.[58] It is therefore thought that pathogenic mycobacteria, especially the MTB complex, possess eukaryotic cell-like signal-transduction mechanisms capable of modulating host macrophage trafficking pathways. Indeed, MTB has 11 serine/threonine protein kinases, and three of them (PknA, PknB and PknG) are required for mycobacterial growth.[59] Therefore, PknG is an attractive target for a novel type of anti-tuberculous drug. On the basis of this strategy, Szekely et al. screened promising agents exhibiting PknG-inhibitory activity from 1000 compounds listed in the Nested Chemical Library™ (NCM) kinase inhibitory library, containing 124 kinases. They identified AX20017 (tetrahydrobenzothiophene) (Figure ;1B), AX33510 and AX14585 (derivatives of AX20017), potent PknG-inhibitory agents.[60] These agents were active in inducing lysosomal transfer in BCG-infected macrophages.

With respect to new targets of anti-TB drugs, another interesting finding has been reported by Kuijl et al.[61] From experiments using various kinds of kinase inhibitors, especially protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) (Figure 1C) and its analogues, they identified several host kinases capable of inhibiting the intracellular (intramacrophage) growth of facultative intracellular bacteria, including Salmonella typhimurium and MTB. The kinases identified were clustered into one network around AKT1 (also known as PKB). They proposed the following signaling pathways[59]: S. typhimurium-derived effector protein SopB activates AKT1. AKT1 targets PAK4, which phosphorylates GEF-H11, thus controlling RHOA, RAC1 and actin. AKT1 also phosphorylates the RAB14-GTPase activator protein (GAP), AS160. This prevents AS160 binding to the phagosome membrane, thereby causing the activation of RAB14 and consequently resulting in the inhibition of phagosomal maturation. These AKT1-mediated events promoted the intracellular survival and growth of S. typhimurium, especially in macrophages.

Since the intramacrophage growth of MTB, as well as S. typhimurium, is inhibited by the same kinase inhibitors, such as H-89 and its analogue ETB067, it appears that AKT1 plays the same role in the signal-transduction pathways for controlling phagosome-lysosome fusion in the case of MTB-infected macrophages.[59] Thus, a host cell kinase network around PKB/AKT1 is a promising target for new anti-TB drugs. However, it should be noted that kinase inhibitors have generally serious selectivity issues. The employment of appropriate drug-screening systems may solve this problem. In this context, the attributes of proper QSAR analysis should include the ability to predict or deal with important areas in the development of new anti-TB drugs, such as safety/toxicity, oral bioavailability, metabolic stability and drug-drug interactions. In this context, it is important to study not only the drug targets in bacterial cells but also those in human cells, tissues and organs.

Notably, the measurement of test agents regarding their inhibition of intracellular functions of the target proteins, such as PknG and AKT1 as mentioned previously, needs invitro models of macrophage infection, which are not amenable to HTS studies. Moreover, the correlation of such invitro efficacy models with murine in vivo models is still disputable. In addition, the only truly valid corroboration is through invivo studies, which have a very low throughput. Indeed, the activity of these target proteins related to MTB's virulence can be measured or assessed only in animal or macrophage cell culture models and, therefore, early-stage discovery efforts against such targets would be hindered by the need to always test new compounds in animal or macrophage models. However, consequently, it may be possible to identify unique anti-tuberculous drugs that cannot be found by screening methods simply based on the growth inhibition test of extracellularly growing mycobacterial organisms.

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