*3.1. The ESX-1 Secretion System*

Lately, the interest in finding novel lead compounds, which prevent infection and dissemination by inhibiting bacterial virulence factors, has increased. These anti-virulence molecules target one or more proteins in the virulence machinery with one prominent example being the ESX-1 secretion system as ESX-1 deletion mutants show strongly attenuated phenotypes in vitro and in vivo [100]. ESX-1 is a type VII secretion system essential for host cell infection, bacterial spread and macrophage escape but not for bacterial growth in axenic cultures [101]. In a whole-cell-based phenotypic screening assay selecting for compounds that abrogate ESX-1 dependent host cell death, the two ESX-1 inhibitors BTP15 and BBH7 have been found and characterized [102]. BTP15 inhibits the histidine kinase MprB that regulates ESX-1 via the *espA-espC-espD* operon. BBH7 on the other hand disturbs metal-ion homeostasis leading to zinc stress and thus hindering secretion of ESX-1 substrates such as EsxA and EsxB (Figure 1). These inhibitors can also be used to abrogate ESX-1 dependent activation of the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), a main driver of type I interferon (IFN) secretion, thus nicely linking anti-virulence drugs to modulation of the inflammatory response [103].

**Figure 1.** Schematic overview of antivirulence targets in *M. tuberculosis*.

#### *3.2. PhoPR Inhibitors*

The PhoPR two-component system plays a central role in regulating the expression of several proteins relevant for virulence of *M. tuberculosis* as mutants deficient in the effector response regulator PhoP show attenuated growth in infected THP-1 cells and in mice [104]. A microarray-based transcriptional profiling study of *M. tuberculosis* strain H37Rv revealed 110 genes that have been differently expressed in PhoP-deficient mutants [105]. This attenuated strain harbors a single nucleotide polymorphism (S219L) in the DNA-binding domain of PhoP resulting in a reduced DNA-binding capacity [105]. Further studies revealed that PhoP is involved in regulating ESX-1 and in biosynthesis of cell wall components such as sulfolipids, polyacyltrehaloses and diacyltrehaloses [106–108]. Two different approaches identified inhibitors of the PhoPR regulon.
