**2. Methodology for high-throughput screens (HTS) using small molecule libraries**

The workhorse platform for anti-bacterial drug discovery is a chemical genetics HTS ap‐ proach using small molecule compound libraries to identify candidates that inhibit bacterial growth or the function of key bacterial enzymes. Small molecules, generally <500 molecular weight, have the potential to enter cells and selectively perturb specific protein activity, thus functioning as therapeutic agents against disease. In general, the precise mechanism of in‐ hibitor activity remains unknown in the initial screen. Subsequent identification of the mo‐ lecular targets of small molecules will have to be performed to implicate the specific bacterial functions that were inactivated in the screen. Thus, HTS can sample a large un‐ biased collection of structurally diverse molecules to select compounds that perturb the de‐ fined cell phenotype of interest. (Fig. 1)

Various chemical compound libraries are now available through commercial and public re‐ sources that include FDA-approved bioactive compounds, therapeutic agents, and natural products. To maximize the structural complexity and diversity of small molecule libraries, scientists have also employed diversity-oriented synthesis,

There is no question that new strategies that target different aspects of pathogen function are urgently needed to combat multi-drug resistant bacteria. However, very few new scaf‐ folds for drug discovery developed after the 1960s have been found to be effective [2]. To date, only four new classes of antibiotics, including mutilins and lipopeptides, have been in‐ troduced, but none of these have proven to be as effective as the panel of classic antibiotics. Instead, established scaffolds have been modified or re-purposed to develop successive gen‐ erations of effective antibiotics. For example, the core structure of cephalosporins have been left intact to preserve activity, but the peripheral chemical groups have been modified to im‐ part the molecule with the ability to penetrate the bacterial membrane more efficiently or be more resistant to β-lactamase [3]. Modifications of four classic antibiotics, cephalosporin, penicillin, quinolone, and macrolide, account for ~73% of the "new" antibiotics filed be‐ tween 1981 and 2005 [4]. It is also important to note that small compounds need to exhibit not only anti-microbial activity, but also minimized cytotoxic properties to widen their ther‐

Although advances in organic synthesis have extended the lifetime of classic antibiotics through synthetic modifications, new scaffolds are also needed. Recent efforts to search for new modalities amongst previously-overlooked natural sources, such as unmined bacterial taxa and ecological niches, have started to bear fruit. The increasingly rapid data acquisition and low cost of ultra high-throughput sequencing has provided rich coverage of bacterial genomes and transcriptomes. For example, genomic analyses of a vancomycin-resistant strain of *Amycolatopsis orientalis* revealed the presence of genetic loci that encode for at least 10 other secondary metabolites. One compound, ECO-0501, exhibited strong anti-bacterial properties against Gram-positive pathogens, including several strains of MRSA [5]. Mass spectroscopy (MS) is another primary methodology used to identify small molecule metabo‐ lites with potential anti-microbial properties. The polycyclic small molecule, abyssomicin C, from the marine actinomycete *Verrucosispora* was characterized as an inhibitor of *p*-amino‐ benzoate biosynthesis by MS and also exhibited antimicrobial properties against MRSA

**2. Methodology for high-throughput screens (HTS) using small molecule**

The workhorse platform for anti-bacterial drug discovery is a chemical genetics HTS ap‐ proach using small molecule compound libraries to identify candidates that inhibit bacterial growth or the function of key bacterial enzymes. Small molecules, generally <500 molecular weight, have the potential to enter cells and selectively perturb specific protein activity, thus functioning as therapeutic agents against disease. In general, the precise mechanism of in‐ hibitor activity remains unknown in the initial screen. Subsequent identification of the mo‐ lecular targets of small molecules will have to be performed to implicate the specific bacterial functions that were inactivated in the screen. Thus, HTS can sample a large un‐ biased collection of structurally diverse molecules to select compounds that perturb the de‐

apeutic window.

158 Drug Discovery

strains [6].

**libraries**

fined cell phenotype of interest. (Fig. 1)

**Figure 1.** General flowchart of high-throughput methodology to screen small molecule libraries for inhibitors of hostpathogen interactions

in which different scaffolds are modified with highly diverse functional groups. [7, 8]. To bolster academic research in chemical biology efforts for HTS-driven identification of bioac‐ tive compounds, the NIH launched the Molecular Libraries Program in 2005 to offer ac‐ cess to ten large-scale automated HTS centers in the Molecular Libraries Probe Production Centers Network, including diverse compound libraries through the Small Molecule Repo‐ sitory and information on biological activities of small molecules in the PubChem BioAs‐ say public database.

A variety of different molecular and cellular methods have been developed for HTS using small molecule libraries. Automated microscopy has been utilized for high-content, imagebased screens of cells exposed to small molecules. Acquired cell images can be analyzed by automated image analysis software to quantitate physiological changes at the single-cell lev‐ el, including phenotypes such as morphology and cell toxicity. Small molecule microarrays, in which ~10,000 small molecules are covalently bound to a glass slide, has been generated to detect high affinity binding to a protein of interest, as a potential inhibitor of function. Binding of the protein of interest to specific compounds on the microarray was then detect‐ ed with fluorescent antibodies [9].
