**4. Whole animal small molecule screens using** *C. elegans*

In vitro high-throughput screens have several limitations for the discovery of therapeutic in‐ hibitors with high efficacy. Synthetic compound libraries often contain toxic compounds with poor pharmacokinetic properties, and many *in vitro* assays are not physiologically-rele‐ vant in the context of which a specific drug is expected to function. In a previous section, we had described the use of a HTS whole organism-based assay based on *Arabidopsis* seedlings as the host system. Here, we detail the use of a whole animal model, the nematode worm *C. elegans*, in chemical screens that permit simultaneous assessment of the immunomodulatory effects, potential toxicity of compounds, and drug efficacy in a host with a functioning im‐ mune system. The results of these screens are summarized in Table 2. Whole animal screens have the distinct advantage of being able to directly discard compounds that induce organ‐ ismal toxicity and can identify compounds that target host-pathogen interactions in a rele‐ vant physiological context.

*C. elegans*, a hermaphroditic nematode normally found in soil, is a versatile, more ethicallyacceptable whole animal system for high-throughput analysis of host response to pathogen infection. *C. elegans* contains a fully sequenced genome that facilitates both genetic and ge‐ nomic analysis, offering an ideal compromise between organismal complexity and experi‐ mental tractability. *C. elegans* offers other experimental advantages, including a rapid 2-3 week life span, simple growth conditions, target-selected gene inactivation, and a relatively low cost of maintenance compared to other whole animal systems. A wealth of experimental data has demonstrated that many developmental, neurological, and biochemical processes have been highly conserved between *C. elegans* and mammals. For example, cellular func‐ tions as diverse as innate immunity, the first line of defense against pathogen infection, and RNA interference to downregulate gene expression via double-stranded RNA, are found in both *C. elegans* and higher eukaryotes, suggesting the existence of a common ancestor of these diverse species. Thus, anti-infective compounds identified using a *C. elegans* infection model may also be translatable in humans.

*C. elegans* as a model host system has been well-studied for numerous bacterial pathogens, in‐ cluding the Gram-positive *S. aureus*, *S. pneumoniae*, and *B. thuringiensis*, and the Gram-nega‐ tive *B. pseudomallei*, *P. aeruginosa*, and *S. marcescens*. In general, different types of bacteria are fed to *C. elegans* in place of their normal *E. coli* food source to provoke detectable symptoms of illness, such as locomotion dysregulation, intestinal cell lysis, and shortened life span.

### *Small molecule inhibitors of bacterial infection*

of histones in the vicinity of the integrated viral genome [42]. Thus, VA was tested as a po‐ tential agent to disrupt HIV-1 latent infection. However, years of VA treatment in combina‐ tion with HAART showed no clearance of the latent HIV reservoir [43]. A more potent HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), approved for treatment of cuta‐ neous T-cell lymphoma, was subsequently tested as a potential agent that could 'flush out' HIV-1 from latently infected cells, based on its superior effect to VA in cell culture models [44, 45]. A substantial effort has also been invested in the design and synthesis of bryostatin chemical analogs, small molecules that activate protein kinase C (PKC) with single nanomo‐ lar concentration [46]. PKC activation leads to phosphorylation of nuclear factor κB (NFκB), a key transcription factor regulator of HIV-1 gene expression [47]. However, modulation of NFκB activity requires great caution, since abnormal NFκB signaling has been related to the

pathophysiology of inflammatory diseases and neurodegenerative disorders [48].

resting CD4+

166 Drug Discovery

verse class of antiviral drugs.

vant physiological context.

A HTS of a small molecule library recently identified novel HIV latency activators [49]. The screen was performed using a lymphoma CD4+ T-cell line (SupT1) harboring latent recombi‐ nant HIV-1 and two reporters that reflect early and late virus gene expression incorporated in the HIV-1 genome [50]. A luminescent assay based on secreted alkaline phosphatase (SEAP) activity, incorporated in the late virus gene transcripts, was applied to screen a chemical library of ~200,000 compounds. Validation of 27 hits with diverse chemical struc‐ tures demonstrated induction of latent virus from various cell models. Compounds with a selective index (CC50/EC50) above 25 were chosen for downstream medicinal chemistry mod‐ ifications. Moreover, the lead compounds were shown to reactivate latent HIV from primary

tent HIV that act in concert using different mechanisms have a better chance of purging the virus out of infected cells [49]. Such pre-clinical data strongly suggests that successful treat‐ ment of HIV infection can be achieved only through combinational therapy consisting of di‐

In vitro high-throughput screens have several limitations for the discovery of therapeutic in‐ hibitors with high efficacy. Synthetic compound libraries often contain toxic compounds with poor pharmacokinetic properties, and many *in vitro* assays are not physiologically-rele‐ vant in the context of which a specific drug is expected to function. In a previous section, we had described the use of a HTS whole organism-based assay based on *Arabidopsis* seedlings as the host system. Here, we detail the use of a whole animal model, the nematode worm *C. elegans*, in chemical screens that permit simultaneous assessment of the immunomodulatory effects, potential toxicity of compounds, and drug efficacy in a host with a functioning im‐ mune system. The results of these screens are summarized in Table 2. Whole animal screens have the distinct advantage of being able to directly discard compounds that induce organ‐ ismal toxicity and can identify compounds that target host-pathogen interactions in a rele‐

**4. Whole animal small molecule screens using** *C. elegans*

T-cells with no induction of cell proliferation. Small molecule activators of la‐

A small manual screen of 6000 synthetic compounds and 1136 natural extracts were ana‐ lyzed in an immunocompromised mutant of *C. elegans* infected with *Enterococcus faecalis* to identify compounds that promoted host survival. [51]. A total of 16 compounds and 9 ex‐ tracts were identified that either modulated bacterial growth *in vitro*, impaired pathogen vir‐ ulence, or boosted host innate immunity. Furthermore, 15 out the 16 compounds did not kill *C. elegans* or mammalian erythrocytes, indicating that the compounds are not toxic.

The development of automated sorting and handling of *C. elegans* rapidly enabled highthroughput screening of small chemical libraries to identify compounds that enhanced sur‐ vival of *C. elegans* in response to bacterial infection. This methodology was enabled by the Complex Object Parametric Analyzer and Sorter (COPAS) BioSort worm sorter (Union Bio‐ metrica) to dispense a defined number of living worms into multi-well plates, which were then imaged using automated microscopy to quantify worm survival. A library of 37,200 compounds and natural product extracts was screened using the same *C. elegans*-*E. faecalis* infection system described above [52]. Twenty-eight compounds and extracts were identi‐ fied that enhanced survival of infected *C. elegans*. Six structural classes of identified com‐ pounds did not affect the growth of *E. faecalis* itself, suggesting that the small molecules inhibited a specific aspect of the host-pathogen interaction. Interestingly, two structural classes are similar to compounds previously identified in a high-throughput screen to iden‐ tify inhibitors of *P. aeruginosa* biofilm development, indicating the presence of common mo‐ lecular targets across multiple bacterial species for drug discovery [53].

A *P. aeruginosa* infection model of *C. elegans* has also been developed to screen for novel antiinfective compounds. The high-throughput assay was based on *P. aeruginosa*-induced slow killing of *C. elegans* in the presence of 1300 bioactive extracts produced by endophytic fungi associated with medicinal plants [54]. The screen identified 36 extracts that promoted the survival of the infected worms, while 4 extracts were found to inhibit *P. aeruginosa* growth using a disc diffusion assay. Given that these extracts contain a mixture of metabolites, the specific compound against *P. aeruginosa* remains to be determined. Nevertheless, this study illustrates the rich reservoir of small molecules in natural symbiotic organisms with antibacterial activity.

such as dequalinium chloride, a potent anti-tumor and protein kinase C inhibitor, and tria‐

Small Molecule Screens to Identify Inhibitors of Infectious Disease

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

169

Chemical library screens are a potent and valuable molecular tool for HTS identification of potential inhibitors of infectious disease. The long-standing paradigm to treat pathogen in‐ fection with small molecules that specifically target pathogen growth or metabolism has led to our current dilemma of microbial drug resistance and re-emergence of once-contained in‐ fectious diseases. Thus, new approaches to target pathogen virulence or host response fac‐ tors rather than essential pathogen functions have become increasingly more attractive strategies that are less likely to induce microbial resistance. Some compounds, such as the FDA-approved anti-psychotic, pimozide, exhibited inhibitory properties against infection by several pathogens, suggesting that small molecules can potentially be developed as broadspectrum anti-infectives. Although the molecular mechanism of inhibition by small mole‐ cules remains unknown in most cases, it may be possible to make an educated guess if targeted pathogens share a common virulence strategy, such as the Type III secretion system in Gram-negative bacteria. In other cases, identification of an inhibitor can lead to a molecu‐ lar understanding of the infection mechanism. For example, the small molecule, tachyplegi‐ nA, was found to post-translationally modify TgMLC1, a myosin light chain component, to

From the various studies detailed in this review, it is apparent that the library screens repre‐ sent a first step on the road of drug discovery. There has been a growing realization that fundamental discovery of biological mechanisms oftentimes reaches a 'valley of death', in which potential translation avenues into clinical therapies and diagnostics for disease treat‐ ment comes to a standstill and is lost. NIH is addressing this widening gap between basic and clinical research with the establishment of Clinical and Translational Science Centers across the country. The research community will have to remain pro-active to move promis‐ ing leads from the initial screen stage into downstream validation and development modes in a timely manner. As with any drug development strategy, there still remain multiple technical challenges that need to be overcome before small molecule inhibitors can success‐ fully transition into the clinic. Researchers will need to assess such parameters as compound toxicity, pharmacokinetics and pharmacodynamics, and validation in animal models. How‐ ever, FDA-approved small molecule libraries can be applied to HTS as a cost-effective meth‐ od to identify existing licensed drugs for repurposing from diseases unrelated to microbial infection. Furthermore, the development of the *C. elegans* whole organism model for small molecule screening provides a novel methodology to simultaneously assess compound tox‐ icity and host response to pathogen infection. It would be informative to determine whether small molecules identified from conventional host cell culture studies can also inhibit patho‐ gen infection in the *C. elegans* model. Future anti-infective treatments will most likely be comprised of combination therapies that produce additive or synergistic effects to target key processes in both the pathogen and the host. The overall promise of discovering novel anti-

dimefon, an inhibitor of ergosterol biosynthesis.

drive host cell penetration by the parasite *T. gondii* [17].

**5. Conclusion**


**Table 2** Small molecule screens using C. *elegans* as host model for infection

#### *Discovery of novel antifungal agents*

The *C. elegans* infection model was also used to screen for compounds that prolonged host survival following infection with the human pathogenic fungus *Candida albicans*. [55]. Given that most compounds that have antifungal activity are also toxic to the human host, highthroughput methods can greatly increase the likelihood of discovering specific antifungal inhibitors. From a screen of 1266 compounds with known pharmaceutical activities, 15 small molecules were identified that increased survival of *C. albicans*-infected nematodes and in‐ hibited *in vivo* filamentation of *C. albicans*, a mechanism of pathogenesis seen during mam‐ malian infection. Two compounds, caffeic acid phenethyl ester (CAPE), a natural component of honeybee propolis, and the fluoroquinolone agent enoxacin, were further shown to exhibit antifungal activity in a mouse model, validating the use of a *C. elegans* model for potential targets in a mammalian system. Interestingly, CAPE is known to inhibit the mammalian transcription factor NF-κΒ and to induce immunomodulatory effects in mice [56, 57]. Since *C. elegans* does not express a NF-κΒ homolog, it may be the case that CAPE affects alternative targets to achieve antifungal activity.

An automated high-throughput screen using the COPAS Biosort was also applied to *C. albi‐ cans* infection of *C. elegans* to assess a library of 3,228 compounds consisting of 1948 bioactive compounds and 1280 small molecules derived from diversity-oriented synthesis [58]. In to‐ tal, 19 compounds were identified that increased *C. elegans* survival in response to *C. albicans* infection, 7 of which are currently used antifungal agents. Several immunosuppressant agents identified in this screen, including ascomycin, cyclosporin A, and FK-506, were previ‐ ously found to exhibit weak antifungal activity against *Cryptococcus* and *Aspergillus*, in addi‐ tion to *C. albicans* [59, 60]. Other hits were predicted to affect an array of biological activities, such as dequalinium chloride, a potent anti-tumor and protein kinase C inhibitor, and tria‐ dimefon, an inhibitor of ergosterol biosynthesis.
