**3. Disruption of host-pathogen interactions for novel drug discovery**

decrease internalization of not just *L. monocytogenes*, but other bacterial species as well, in‐ cluding *Bacillus subtilis*, *Salmonella typhimurium*, and *E. coli*. Furthermore, pimozide de‐ creased vacuole escape and cell-to-cell spread of *L. monocytogenes* in the host. Thus, pimozide is an example of a small molecule that can be re-purposed to treat infectious dis‐

Small Molecule Screens to Identify Inhibitors of Infectious Disease

http://dx.doi.org/10.5772/52502

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Parasites also employ a life cycle of host cell invasion, replication, and host cell lysis during onset of infection. *Taxoplasma gondii* is the protozoan intracellular human parasite of the phylum Apicomplexa and is related to *Plasmodium* and *Cryptosporidium*, the causative agents of malaria and diarrheal disease, respectively. To discover inhibitors of *T. gondii* invasion, a

microscopy assay was developed to distinguish between extracellular and intracellular para‐ sites in a BS-C-1 epithelial cell model, using differential labeling with fluorescent dyes [16]. Out of a 12,160 structurally-diverse small molecule library, 24 non-cytotoxic inhibitors were identi‐ fied that reduced parasite invasion to <20% compared to control wells. These molecules inhib‐ ited different aspects of the infection process, including gliding motility and secretion of host cell adhesins. One of these inhibitors, tachypleginA, was found to post-translationally modify TgMLC1, a myosin light chain component of the *T. gondii* myosin motor complex, which drives host cell penetration and parasite mobility [17]. TgMLC1 exposed to the small molecule exhib‐ ited a rapid and irreversible change in electrophoretic mobility on SDS-PAGE gels. Although the exact nature of the modification remains unclear, the modification has been mapped to amino acids V46-R59 by mass spectroscopy. These studies provide key mechanistic informa‐ tion on the importance of *T. gondii* motility in pathogenesis and illustrate the potential for small

Many Gram-negative bacteria, including *Pseudomonas* and *Yersinia*, utilize the TTSS as a pri‐ mary mechanism of virulence to inject effector proteins into the host cytosol to downregu‐

ease with potential for broad spectrum anti-microbial applications.

**Table 1** Small molecule screens using host-pathogen systems

molecules to form covalent interactions with target proteins.

*Targeting virulence toxin mechanisms of infection*

high-throughput

Given the innovation gap in the discovery of novel antibiotics post-1960, strategies to inhibit novel targets are greatly needed to combat infectious disease. Multiple studies have identi‐ fied small molecule inhibitors that target gene expression of pathogen TTSS components in *P. aeruginosa*, enteropathogenic *E. coli*, and *Y. pestis* [10, 11, 12]. The small molecule virstatin, 4-[N1,8-naphthalimide)]-n-butyric acid, was identified as an inhibitor of the transcriptional regulator ToxT in *Vibrio cholerae* [13]. The small molecule, 2-imino-5-arylidene thiazolidi‐ none, which blocks TTSS-dependent functions in *S. typhimurium*, was also found to inhibit virulence in *Yersinia*, *Pseudomonas*, and *Francisella* strains, indicating that compounds can be identified that target common processes in multiple pathogens [14].

Research efforts have recently begun to focus on disruption of host-pathogen interactions as a new approach to identify potential targets for drug discovery, rather than solely on specif‐ ic pathogen targets or processes. In particular, the screening of small molecule libraries to identify inhibitors that block pathogen infection of the host, using such phenotypes as pathogen invasion, host morphology, and pathogen replication in the host, is a powerful ap‐ proach for therapeutic development that may uncover fundamental mechanisms of patho‐ genesis and potentially lead to discovery of new classes of anti-infective agents. Here, we describe case studies of the use of small molecules in host infection screens to identify novel inhibitors against infectious disease, including bacterial, viral, parasitic, and fungal infec‐ tions. We will discuss these studies in the context of re-purposing known drugs, inhibitor specificity, and discovery of basic mechanisms of host-pathogen interactions. The screen re‐ sults are summarized in Table 1.
