**1. Introduction**

In the 1940's, the development of penicillin as a potent broad-range antibiotic revolutionized the treatment of infectious disease and ushered in a prolific discovery period of natural small molecules produced by microorganisms that were antagonistic towards the growth of other bacteria. Antibiotics have generally been classified by their mechanism of action. For example, the β-lactam compounds, penicillin and cephalosporins, disrupt the synthesis of the peptidoglycan layer of the bacterial cell wall, whereas protein synthesis inhibitors, such as tetracycline and some aminoglycosides, bind to the 30S ribosomal subunit and block ad‐ dition of amino acids to the growing peptide chain. By the 1960's, the majority of all antibi‐ otics in use today had been isolated and developed for public consumption, leading the U.S. Surgeon General to declare in 1968 that the war on infectious disease had been won.

Unfortunately, nature has found a way to thwart mankind's effort to contain infectious dis‐ ease. Under the selective pressure of antibiotics that target different cell processes, bacteria have evolved to become resistant to the lethal effects of many classes of antibiotics. One stark example that has emerged as a major public health threat is methicillin-resistant *Staph‐ ylococcus aureus* (MRSA), which is estimated to cause ~19,000 deaths in the US annually [1]. MRSA has become resistant to β-lactam antibiotics by acquiring the resistance gene *mecA*, which encodes for a unique penicillin binding protein PBP2A that can function as a surro‐ gate for native staphylococcal PBPs normally inactivated by β-lactam antibiotics. In the last decades of the 20th century, MRSA has continued to evolve in response to a continually changing human environment, from a primary agent of hospital-acquired infections to a multi-drug resistant strain that has also acquired Tn1546 transposon-based vancomycin re‐ sistance. Furthermore, the appearance of MRSA strains in a community setting may be a stepping stone to the evolution of a completely drug-resistant strain.

properly cited.

© 2013 Hong-Geller and Micheva-Viteva; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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‐ apeutic window.

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,

Small Molecule Screens to Identify Inhibitors of Infectious Disease

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

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

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‐

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‐

scientists have also employed diversity-oriented synthesis,

pathogen interactions

say public database.

ed with fluorescent antibodies [9].

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 strains [6].
