Antibiotic resistance is an innate problem involved in the treatment of bacterial infectious diseases and the incidence of these infections is escalating at an alarming rate [1Diekema DJ, BootsMiller BJ, Vaughn TE, et al. Antimicrobial resistance trends and outbreak frequency in United States hospitals Clin Infect Dis 2004; 38(1): 78-85.-3Boucher HW, Talbot GH, Bradley JS, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America Clin Infect Dis 2009; 48(1): 1-12.]. Resistance not only renders current antibiotics useless against dangerous pathogens, but it is also a financial deterrent to efforts towards the discovery of novel antibiotics [4Power E. Impact of antibiotic restrictions: the pharmaceutical perspective Clin Microbiol Infect 2006; 12(Suppl 5): 25-34.]. As a result, many pharmaceutical companies have reduced or eliminated their antibacterial drug discovery efforts [5Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL. Drugs for bad bugs: confronting the challenges of antibacterial discovery Nat Rev Drug Discov 2007; 6(1): 29-40.], and since 1962 only three new classes of antibacterial agents have been introduced [6Lock RL, Harry EJ. Cell-division inhibitors: new insights for future antibiotics Nat Rev Drug Discov 2008; 7(4): 324-8.]. Genomics-driven discovery against targets considered essential has not yielded any clinically approved drugs [5Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL. Drugs for bad bugs: confronting the challenges of antibacterial discovery Nat Rev Drug Discov 2007; 6(1): 29-40.], and one way to avert this looming public health crisis could be to replenish antibiotic drug pipelines with drugs against targets not considered essential for survival. In fact, two-thirds of Escherichia coli genes fall into this population [7Baba T, Ara T, Hasegawa M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection Mol Syst Biol 2006; 2(2006): 0008.], and targeting this previously ignored segment of the bacterial genome may offer possibilities for the discovery of non-traditional pharmaceutical agents that attenuate pathogenicity [8Escaich S. Antivirulence as a new antibacterial approach for chemotherapy Curr Opin Chem Biol 2008; 12(4): 400-8.] or potentiate the pharmacologic effects of known antibacterial agents [9Cottarel G, Wierzbowski J. Combination drugs, an emerging option for antibacterial therapy Trends Biotechnol 2007; 25(12): 547-5.]. In this context, we have focused on the bacterial RecA protein as a prospective target in the treatment of bacterial infectious diseases. RecA’s activities are central to the repair of DNA damage and stalled replication, but are also important for adaptive mutagenesis and horizontal gene transfer. We hypothesize that small molecule inhibitors of RecA may sensitize bacteria to established antibacterial agents and prevent the development and acquisition of genes conferring drug resistance.

In Escherichia coli, a model system to study DNA damage, RecA’s expression is upregulated by events that challenge the integrity of the organism’s genome, and lead to the formation of single-stranded DNA (ssDNA), a hallmark of DNA damage [10Friedberg EC, Walker GC, Siede W. In: SOS responses and DNA damage tolerance in prokaryotes DNA Repair and Mutagenesis. Washington, D.C: ASM Press 1995; pp. 407-64.]. In this event, RecA monomers respond by forming a homopolymer on the exposed ssDNA, initiating a series of programmed events that can be fundamentally associated with two major functions, de-repression of the SOS regulon and recombinational DNA strand exchange [11Haruta N, Yu X, Yang S, Egelman EH, Cox MM. A DNA pairing-enhanced conformation of bacterial RecA proteins J Biol Chem 2003; 278(52): 52710-23., 12VanLoock MS, Yu X, Yang S, et al. ATP-mediated conformational changes in the RecA filament Structure 2003; 11(2): 187-96.]. In the first function, the SOS regulon is initiated when the RecA nucleoprotein filament comes into contact with the LexA repressor, which sits on promoters of up to 40 genes that are involved in repairing DNA damage [13Sassanfar M, Roberts JW. Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication J Mol Biol 1990; 212(1): 79-96.]. Gene products expressed in the early stages of SOS repair the DNA damage by nucleotide excision (e.g., UvrABCD) or recombination (e.g., RecA), while gene products of the late stages of SOS are involved in error-prone DNA synthesis (e.g., PolIV and PolV) that systematically introduces mutations into the bacterial genome and facilitates adaptive evolution [14Kelley WL. Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon Mol Microbiol 2006; 62(5): 1228-38.-17Schlacher K, Pham P, Cox MM, Goodman MF. Roles of DNA polymerase V and RecA protein in SOS damage-induced mutation Chem Rev 2006; 106(2): 406-19.]. In the second function, RecA pairs ssDNA with homologous duplex DNA, and hydrolyzes ATP to induce the conformational rearrangements necessary to catalyze a strand exchange reaction that produces two new DNA molecules [18Cox JM, Tsodikov OV, Cox MM. Organized unidirectional waves of ATP hydrolysis within a RecA filament PLoS Biol 2005; 3(2): e52., 19Cox MM. The bacterial RecA protein as a motor protein Annu Rev Microbiol 2003; 57: 551-77.]. This ATPase-dependent activity is necessary for recombinational DNA repair [20Courcelle J, Ganesan AK, Hanawalt PC. Therefore, what are recombination proteins there for? BioEssays 2001; 23: 463-70., 21Roca AI, Cox MM. RecA protein: structure, function, and role in recombinational DNA repair Prog Nucl Acid Res Mol Biol 1997; 56: 129-223.] and horizontal gene transfer during bacterial transformation, transduction and conjugation [22Radman M, Taddei F, Matic I. DNA repair systems and bacterial evolution Cold Spring Harb Symp Quant Biol 2000; 65: 11-9.].

Recently, the SOS and recombination processes mediated by RecA have been shown to be activated in response to antibiotic treatment [23Beaber JW, Hochhut B, Waldor MK. SOS response promotes horizontal dissemination of antibiotic resistance genes Nature 2004; 427(6969): 72-4.-25Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics Cell 2007; 130(5): 797-810.]. The biological activities controlled by RecA likely enhance a bacterium’s tolerance to antibiotics and can result in the de novo development and horizontal transfer of antibiotic resistance genes. Because RecA and its functions in overcoming antibacterial chemotherapy are highly conserved among bacteria, small molecule modulators of RecA would be valuable for further exploration of its roles in bacterial pathogens and its potential exploitation as a therapeutic target. While ATP-competitive nucleotide analogs [26Lee AM, Ross CT, Zeng BB, Singleton SF. A molecular target for suppression of the evolution of antibiotic resistance: inhibition of the Escherichia coli RecA protein by N(6)-(1-naphthyl)-ADP J Med Chem 2005; 48(17): 5408-11., 27Wigle TJ, Lee AM, Singleton SF. Conformationally selective binding of nucleotide analogues to Escherichia coli RecA: a ligand-based analysis of the RecA ATP binding site Biochemistry 2006; 45(14): 4502-13.], polysulfated naphthyl compounds [28Wigle TJ, Singleton SF. Directed molecular screening for RecA ATPase inhibitors Bioorg Med Chem Lett 2007; 17(12): 3249-53.], short alpha-helical peptides [29Cline DJ, Holt SL, Singleton SF. Inhibition of Escherichia coli RecA by rationally redesigned N-terminal helix Org Biomol Chem 2007; 5(10): 1525-8.] and select transition metal cations [30Lee AM, Singleton SF. Inhibition of the Escherichia coli RecA protein: zinc(II), copper(II) and mercury(II) trap RecA as inactive aggregates J Inorg Biochem 2004; 98(11): 1981-6.] are effective inhibitors of RecA in vitro, none of these molecular classes have demonstrated RecA-specific biological activities in bacterial cell cultures.

Towards the goal of discovering potent cell-permeable inhibitors for the validation of RecA as an important and novel target for the chemotherapeutic treatment of bacterial infections, we carried out a two-tiered HTS campaign against RecA. As ssDNA-dependent ATP hydrolysis is a useful indicator that the RecA protein is active, we elected to screen the compounds using an inexpensive, yet robust and reliable phosphomolybdate blue ATPase assay to identify compounds that inhibit ATP hydrolysis. We initially screened the BRITE diversity set, which is comprised of approximately 10% of our proprietary collection of nearly 350K compounds selected to maximize chemical diversity. From this collection, we identified 40 hits that clustered into four chemotype classes which were then carried into dose-response confirmatory testing. We have also performed Tanimoto-based similarity searching in the entire BRITE library to identify related compounds that will be screened to provide initial insights into structure-activity relationships.

The PMB ATPase screen of the BRITE diversity library resulted in 73 active compounds exhibiting RecA inhibition above a 50% threshold. RecA inhibitors were triaged by selecting the most potent single-point hits and were confirmed with dose-response. The average Z' for this screen was 0.83, with an overall hit rate of 0.22%. Of the 73 hits obtained, 71 of 73 compounds could be classified into one of four chemotype classes based on structural similarity (Fig. 1). The label for chemotype classes D and C are reversed in the lower portion of Fig. (1). “C - 9 compounds” should appear on the lower left, and “D – 2 compounds” should appear on the lower right.

Fig. (1) Four chemotype classes of RecA inhibitors showing the number of compounds discovered for each classes in this PMB ATPase high thoughput screening effort.

The 73 hits identified in the single-point 10 µM screen were carried into dose-response confirmation with 40 compounds exhibiting dose-response behavior, yielding a 55% confirmation rate. A typical dose-response curve for RecA inhibitors is shown in Fig. (2), with characteristic steep dose-response curve likely due to multi-site inhibitor binding and well known positive cooperative binding kinetics [34Mikawa T, Masui R, Kuramitsu S. RecA protein has extremely high cooperativity for substrate in its ATPase activity J Biochem 1998; 123(3): 450., 35Register J, Griffith J. The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange J Biol Chem 1985; 260(22): 12308-.]. The structure and dose-response curve for the most potent RecA inhibitor identified in the BRITE diversity set screen, BRITE-338733, is shown in Fig. (2) and has been analyzed and exhibits a 4.7µM IC₅₀ and a Hill slope of 7.6. The compound BRITE-345133 (chosen as representative of the 2-amino-4,6-diarylpyridine scaffold) has been analyzed by LC/MS and has a confirmed mass of 524.1 m/z (m+1 peak) and is 89% pure. The range of IC₅₀ values for the 40 confirmed compounds is 4.7 µM to 39 µM and the average Hill slope is 7.4. The molecular structures and corresponding IC₅₀ values are shown in Table 1. The 2-amino-4,6-diarylpyridine scaffold is the most potent as shown by BRITE-338733 having an IC₅₀ of 4.7 µM. The 1,2,4-oxadiazole scaffold of BRITE-212537 followed by the quinaxolinone scaffold of BRITE-379089 and the benzimidazole diazepinone scaffold of BRITE-082776 produced IC₅₀ values of 6.2, 11 and 32 µM respectively, and represent the most potent members of their respective classes.

Fig. (2) Dose-response curve for the RecA inhibitor BRITE-338733, exhibiting an IC50 value of 4.7 ± 0.5 µM and characteristic steep Hill-slope (> 5).

Table 1

Chemical Structures of Confirmed RecA Inhibitors

Here we present a focused high throughput screening effort that effectively identified several novel RecA inhibitors for molecular probes to investigate the impact of RecA inhibition on antibacterial drug resistance. Multiple chemical scaffolds were identified but the 2-amino-4,6-diarylpyridine scaffold was the most potent class of inhibitors. We have independently verified biological activity of this chemotype as described in Wigle, 2009 [33Wigle TJ, Sexton JZ, Gromova AV, et al. Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the sos response in Escherichia coli J Biomol Screen 2009; 14(9): 1092-1.].

The PMB ATPase assay, while not a direct measure of nucleoprotein filament formation, provides a valuable means for the identification of RecA inhibitors with excellent screening statistics and normal confirmation rates (see Wigle, 2009) [33Wigle TJ, Sexton JZ, Gromova AV, et al. Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the sos response in Escherichia coli J Biomol Screen 2009; 14(9): 1092-1.]. We found the assay to be very robust; however, the assay window and statistics could vary significantly from RecA protein batch to batch. The cause of this variation was determined to be residual free phosphate from the protein purification process involving phosphate-based buffers. We routinely performed min-max experiments in one 384-well plate (half-min, half-max) to investigate the assay window and signal-to-noise ratio for batch validation and would dialyze the RecA protein in the assay buffer to reduce the amount of free phosphate. Excellent assay statistics were achieved when the UV/Vis absorption assay window (max-minus-min) was above 0.2 for A₆₅₀, and above 1.0 for A₈₂₅. Two changes were made upon reflection on the initial BRITE-diversity set screen prior to the confirmation step; 1) shifting absorption wavelength to 825 nm and 2) with the addition of 10 µg/mL BSA, which resulted in an increase in Z' from 0.83 to 0.90.

Typical IC₅₀ curves for RecA inhibition are steep, with fit Hill slopes typically above five. It is expected that positive cooperative binding (long known in the experimental RecA literature) would result in steep inhibitory Hill slopes. We have performed dynamic light scattering (DLS) experiments (Dynapro-384, Wyatt Tech. Santa Barbara, CA) to independently assess if the 2-amino-4,6-diarylpyridine scaffold (class A, most potent overall) was aggregating RecA protein in the absence of DNA. We found no indication of RecA aggregation in the presence of up to 10 µM BRITE-345133 (chosen as representative of the 2-amino-4,6-diarylpyridine scaffold) with DLS, which is approaching the solubility limit for the compound (data not shown). These DLS experiments confirm that chemotype-A compounds are genuine RecA inhibitors and the kinetics of inhibition adhere to a multi-site kinetic model. The DLS data will be published in a separate manuscript.

The preliminary structure-activity-relationship of the additional 8640 compounds selected from the rest of the 350,000 compound library for the active scaffolds shows a relatively flat profile, with most IC₅₀ values hovering in the low micromolar range (data not shown). We believe that this PMB ATPase assay is not suitable for profiling the SAR because low micromolar concentrations are at the lower edge of the assay dynamic range. We are developing more sensitive assays to adequately profile the SAR for these compounds in the nano- to micromolar range.

We have screened 33,600 compounds selected from a library containing 350,000 compounds. Here we present 40 confirmed inhibitors of RecA ATPase activity. The 40 confirmed hits represented four novel scaffolds for RecA inhibition. These scaffolds will be used for future structure activity relation (SAR) analysis. Among the four scaffolds, the 2-amino-4,6-diarylpyridine compounds are the most potent with IC₅₀ values in the single-digit micromolar range. We are in the process of developing new sensitive assays to further the SAR exploration. This high-throughput screening effort effectively identified a diverse set of active chemical scaffolds to serve as molecular probes for studying the effects of RecA in the development of antimicrobial resistance.