WO2010056914A1 - Bacterial helicase inhibitor compounds and uses thereof - Google Patents

Abstract

Organic compounds are described that inhibit bacterial helicase activity and growth of bacterial cells on surfaces.

Description

BACTERIAL HELICASE INHIBITOR COMPOUNDS AND USES THEREOF

Cross-Reference to Related Applications

This application claims priority to US provisional application No. 61/199,033, filed November 12, 2008. Statement Regarding Federally Sponsored Research

This work was supported by Small Business Technology Transfer (STTR) grant No. AI064974 from the National Institute of Allergy and Infectious Disease (National Institutes of Health). Accordingly, the US Government has certain rights in the invention. Field of the Invention

This invention is in the field of antibacterial compounds. In particular, the invention provides organic compounds that inhibit one or more bacterial helicases and the growth of bacterial cells that possess bacterial helicases that are susceptible to inhibition by such organic compounds. Background of the Invention

Bacterial pathogens pose a continuing and serious threat to public health. The Gram-positive bacterial pathogens Staphylococcus aureus and Enter ococcus faecalis (or the closely related Enterococcus faecium) are primarily nosocomial (hospital-acquired) pathogens, which together account for the majority of nosocomial infections. Streptococcus pneumoniae is a community-acquired pathogen that also causes serious bacterial infections.

Staphylococcus aureus is currently the most frequent cause of nosocomial bacteremia and skin/wound infection and the second most frequent cause of nosocomial lower respiratory infection. Methicillin-resistant S. aureus (MRSA) is now the causative pathogen for the majority of health care-associated, Gram-positive infections (Deresinski, CHn. Infect. Dis., 40: 562-573 (2005)), and the emergence of community-acquired MRSA, such as the USA300 strain, has raised additional concern (Diep et al., Lancet, 367: 731-739 (2006); Tenover et al., J Clin. Microbiol, 44: 108-118 (2006)). Vancomycin has been the mainstay of therapy for MRSA infection, but glycopeptide resistance is emerging (Fridkin et al., Clin. Infect. Dis., 36: 429-439 (2003)). Newer parenteral treatment options are linezolid, daptomycin, and the recently approved tigecycline, as well as dalbavancin, which awaits regulatory approval in the US. A critical need exists for oral antibiotics for effective step-down therapy of nosocomial infections or for initial therapy of community-acquired MRSA, as there are few such antibiotics in phase 2 or later clinical trials (Talbot et al., Clin. Infect. Dis., 42: 657-668 (2006)). Enter ococcus fecalis/E. fecium rank third behind S. aureus and the Gram-negative Escherichia coli as a cause of nosocomial septicemia, endocarditis, and infections of wounds and the urinary tract. Already known for resistance to many standard, clinically- employed antibiotics (such as aminoglycosides, ampicillin, oxacillin, cephalosporin, clindamycin, and trimethoprim sulfamethoxazole), the emergence in nosocomial infections of virulent strains of Enterococcus that are resistant to vancomycin (vancomycin-resistant enterococcus; VRE) is particularly alarming. Streptococcus pneumoniae causes several serious and potentially life-threatening diseases. In the United States, it is estimated that S. pneumoniae accounts annually for 6,000 cases of pneumococcal meningitis, a half million cases of pneumonia, 55,000 cases of bacteremia, and 6 million cases of otitis media. Annual mortality from Streptococcus pneumoniae-induced disease is estimated to be 40,000 in the United States and 3-5 million globally.

Bacteria of the Gram-positive genera Bacillus and Clostridium may persist for many years (even hundreds) in an environment in the form of dormant spores that are significantly more resistant than actively growing cells to various hostile conditions (such as heat, drying, ultraviolet light, harmful chemicals). When conditions improve, such spores may germinate to actively metabolizing and replicating cells. Notable spore-forming, pathogenic bacterial species include Bacillus anthracis, B. cereus, Clostridium tetani, C. perfringens, and C. difficile.

Clearly, humans and other animals are faced with the risk of infection by any of a variety of pathogenic bacterial species in the environment. Moreover, there is a rapidly growing global crisis in the clinical management of life-threatening infectious disease caused by multi-antibiotic-resistant strains of a variety of Gram-negative and Gram-positive bacterial pathogens, including various strains and species of the Gram-positive pathogens Streptococcus, Enterococcus, Bacillus, and Staphylococcus, and various strains and species of pathogenic Gram-negative bacteria such as Escherichia, Salmonella, Pseudomonas, Helicobacter, Legionella, Shigella, Yersinia, and Neisseria.

The above discussion not only illustrates the continuing need for new antibacterial agents to treat an individual (human or other animal) that becomes infected with a pathogenic bacterial species, but also the concurrent and continuing need for antibacterial agents that can be employed to effectively inhibit growth and proliferation of pathogenic bacteria on environmental surfaces as a means to prevent infection of an individual that comes into contact with such surfaces. Summary of the Invention

The invention addresses the above problems by providing compounds that inhibit the activity of one or more species of bacterial helicases and the growth of bacterial cells that possess bacterial helicases that are susceptible to inhibition by such organic compounds. While the level of cytotoxicity of the compounds described herein precludes their use as internally administrable antibacterial agents, the compounds may be used to inhibit bacterial growth on surfaces that would otherwise serve as fomites to transmit infectious pathogens to an individual (human or other animal).

A bacterial helicase inhibitor compound according to the invention has one the following structures:

Compound 1 -[4,8-dimethyl-7-(naphthalen- 1 -ylmethoxy)-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 2 -[7-(4-bromo-2-fluorobenzyloxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 3 -benzyl-7,8-dihydroxy-4-methyl-2H-chromen-2-one

Compound 5 6,7-dihydroxy-4-[(l-methyl-lH-benzimidazol-2-ylthio)methyl]-2H-chromen-2-one

Compound 12

N- {4-[N-(3 ,4-dimethylisoxazol-5-yl)sulfamoyl]phenyl} -2-(5,6-diphenyl- 1 ,2,4-triazin-3- ylthio)acetamide

Compound 16 2-[5-(5-mercapto-l,3,4-oxadiazol-2-yl)furan-2-yl]benzene-l,4-diol

Compound 17 6-ethyl-9-mercapto-6-methyl-5,6-dihydrobenzo[/?][l,2,4]triazolo[3,4-Z)]quinazolin-7(12H)- one

Compound 19

2-[4,8-dimethyl-7-(naphthalen-2-ylmethoxy)-2-oxo-2H-chromen-3-yl]acetic acid, and salts thereof.

Preferably, a bacterial inhibitor compound of the invention has the structure of Compound 1, Compound 2, or Compound 12.

In one embodiment, a bacterial helicase inhibitor compound described herein inhibits the activity of a helicase of one or more Gram-positive bacterial species or strains. Preferably, a helicase inhibitor compound described herein inhibits the activity of a helicase from one or more species or strains of Bacillus or from one or more species or strains of Staphylococcus. Even more preferably, a compound described herein inhibits the activity of a helicase from Bacillus anthracis, a helicase from Staphylococcus aureus, or helicases from both B. anthracis and S. aureus. Still more preferably, a compound described herein inhibits the activity of a helicase from B. anthracis Sterne strain, a helicase from S. aureus Smith strain, or helicases from both B. anthracis Sterne strain and from S. aureus Smith strain.

The invention provides compositions and methods comprising one or more of the helicase inhibitor compounds described above for inhibiting the growth of bacteria on a surface. Preferably, compositions and methods described herein inhibit the growth of cells of a Gram-positive bacterial species or strains. More preferably, compositions and methods described herein inhibit the growth of cells of a species or strain of Bacillus or of a species or strain Staphylococcus. Even more preferably, compositions and methods described herein inhibit the growth of cells of a bacterial species selected from the group consisting of B. anthracis, B. cereus, B. thuringiensis, B. licheniformis, B.subtilis, B. stearothermophilus, B. megaterium, and S. aureus, and combinations thereof.

In another embodiment, the invention provides compositions and methods comprising a helicase inhibitor compound described herein for inhibiting growth of cells of Escherichia coli on a surface. In a preferred embodiment, a composition of the invention comprises a bacterial helicase inhibitor selected from the group consisting of Compound 1, Compound 2, Compound 12, and combinations thereof.

In another embodiment, a composition comprising a bacterial helicase inhibitor described herein also comprises at least one additional compound that provides a desirable property or activity to the composition. Such an additional agent may be, but is not limited to, an antibacterial agent other than a helicase inhibitor described herein, an antifungal agent, an antiviral agent, an anticancer agent, an organic solvent, a surfactant, an emulsifying agent, a dispersing agent, a buffering agent, and combinations thereof. A particularly preferred organic solvent is dimethyl sulfoxide (DMSO). A preferred surfactant is a non-ionic surfactant.

A composition comprising one or more bacterial helicase inhibitor compounds described herein may be applied to a desired surface by any of a variety methods including, but not limited to, coating, immersing, impregnation, and covalent conjugation. Preferably, a composition for applying a helicase inhibitor compound topically on the skin of an individual (human or other animal) does not also significantly enhance absorption of the helicase inhibitor compound through the skin to the underlying tissue or bloodstream of the individual. A bacterial helicase inhibitor compound described herein may also be employed to inhibit bacterial growth on the surfaces of the exterior and lumens of various devices.

In a preferred embodiment, the invention provides a lock solution (i.e., solution or suspension) comprising one or more helicase inhibitors described herein to fill the lumen of a catheter or other medical device to inhibit bacterial growth in the device prior to use or implantation of the device. Brief Description of the Drawings

Figure 1 shows a kinetic analysis of coumarin-type inhibitor Compound 2 versus B. anthracis helicase in a FRET quenching assay. Data are displayed in the following linear transformations: Fig. IA shows a Dixon plot with 0.625 mM (0), 1.25 mM (D), 2.5 niM (Δ), and 5 mM (O) ATP substrate present. Fig. IB shows a Lineweaver-Burk plot with 12.5 μM (D), 6.25 μM (0), 3.13 μM (Δ), and 0 μM (O) inhibitor present. Fig. 1C shows a Dixon plot with 5 nM (0), 10 nM (D), 30 nM (Δ), and 100 nM (O) annealed oligonucleotide substrate present. Lines are drawn based on a linear or polynomial regression analysis of the data. Figure 2 shows viability of B. anthracis Steme cells incubated with Compound 2 in broth culture at multiple concentrations. Compound 2 was added to LB cultures of B. anthracis Sterne cells at 0.5 x MIC (O), 1 x MIC (Δ), 4 x MIC (D), or omitted from the culture (0), and aliquots (ml) were spread on LB agar plates at various times (hours) indicated on the abscissa (x-axis) to determine the number of viable colony-forming units (CFU). Lines are drawn based on an exponential regression analysis of the data.

Figure 3 shows a summary of preliminary SAR results for coumarin type inhibitor, Compound 2. In Fig. 3A, the substructure shared by all five coumarin-type helicase inhibitors in Table 4 is outlined in a box, and approximate effects of specific structural alterations are noted. Fig. 3B shows a pharmacophore representation of the coumarin type helicase inhibitors. Oxygen atoms oversized to indicate polar surface for interaction with hydrophilic region of helicase. Detailed Description of the Invention

In order that the invention may be more clearly understood, the following terms are defined.

Unless indicated otherwise, when the terms "about" and "approximately" are used in combination with an amount, number, or value, then that combination describes the recited amount, number, or value alone as well as the amount, number, or value plus or minus 10% of that amount, number, or value. By way of example, the phrases "about 40%" and "approximately 40%" disclose both "40%" and "from 36% to 44%, inclusive".

With respect to compounds described herein, the cytotoxicity concentration designated "CC₅₀" is the concentration at which a compound kills 50% of the mammalian cells in a culture. Cells of various mammalian cell lines may be employed in determining a CC50. Preferably, the mammalian cells are HeLa cells for determining the CC₅₀ of compounds described herein. Testing the cytotoxicity of a compound of interest in cultures of mammalian cells grown in the presence of fetal bovine serum may result in an inaccurate determination if the compound binds serum proteins and is thereby effectively sequestered and prevented from asserting any affect on the cells in the culture. In that case, the CC₅₀ value could falsely indicate that the compound is less toxic that it actually is toward mammalian cells. Accordingly, to avoid this potential for inaccurate CC₅₀ values and attendant inaccurate view of the cytotoxicity of a compound, it is preferable to determine CC₅₀ values for a compound of interest in cultures of mammalian cells grown in the presence and the absence of fetal bovine serum. The CC₅₀ values from the two cultures can then be compared to determine whether any disparity between the values is likely the result of an artifact, such as binding to serum proteins, which could mask or otherwise interfere with an accurate assessment of the cytotoxicity of the compound of interest. See, Table 4, below.

As used herein, the "minimal inhibitory concentration" or "MIC" is the minimal concentration at which a compound inhibits growth of a bacterial species of interest. MICs of compounds described herein were determined by the broth microdilution method described in the CLSI (formerly NCCLS) guidelines (CLSI Approved Standard Procedures, M7-A7, vol. 26, no. 2 (2006)) against B. anthracis Sterne strain and S. aureus Smith strain. Preferred bacterial helicase inhibitors of the invention have an MIC of less than 100 μM or less than 50 μg/ml for at least one of these bacterial strains.

The "selectivity index" (SI) of a compound described herein is defined as the ratio of the mammalian cell cytotoxicity (CC₅₀) of the compound to its MIC value for B. anthracis (Slβ_a) or S. aureus (SIs_a)- A relatively large SI may be indicative of the potential use of a compound as an internally administrable antibacterial agent. Antibiotics approved for internal administration to humans typically have an SI of 1000 or higher with respect to one or more bacterial species. As the usefulness of an SI value depends on the accuracy of both the CC₅₀ and MIC, it is important to avoid potential artifacts such as discussed above with respect to determining an accurate CC₅₀ value. Accordingly, it is preferable to calculate an SI using CC₅₀ values determined from cultures grown in the presence and the absence of serum so that a difference in cytotoxicity (i.e., between CC₅₀ values) will also be reflected in the SI values. See, Table 4, below.

As used herein, the "half-maximal inhibitory concentration" or "IC₅o" is the concentration of a compound required for 50% inhibition of the maximal activity of an enzyme. For the compounds described herein, the relevant enzyme activity is a bacterial replicative helicase-catalyzed DNA-unwinding reaction and in particular, the DNA- unwinding activity catalyzed by the helicase of B. anthracis Sterne strain or by the helicase of S. aureus Smith strain. An IC₅₀ may be calculated using a preparation of a bacterial helicase or a preparation of permeabilized cells that possess a helicase. See, Table 4, below.

As used herein, the term "fomite" is any surface capable of transmitting infectious pathogenic bacterial cells from one individual to another individual (human or other animal). As used herein, the term "fomite" encompasses inanimate surfaces and any external or exposed surface of an individual, including but not limited to skin, hair, fur, nails, claws, hooves, scales, beaks, and feathers. A fomite receives and possesses (i.e., is contaminated with) a viable inoculum of infectious bacteria by contact with an infected individual. Such contact may be with a contaminated surface of the individual (e.g., skin, hair, fur, nails, claws, hooves, scales, beaks, feathers) or a biological sample of the individual, such as a biological fluid (e.g., blood, lymph, saliva, sputum, urine, perspiration) or feces. The pathogenic bacteria on the fomite are then passed to another human or other animal that comes into contact with the contaminated fomite.

As used herein with respect to bacteria, the terms "pathogen" and "pathogenic" refer to bacterial species and strains that are capable of causing a disease in or on a human or other animal. Accordingly, the terms encompass bacteria that are classified in the art as pathogens (or primary pathogens) as well as bacteria that are classified as "opportunistic" or "potential" pathogens. As the name implies, such opportunistic or potential pathogens may cause disease in or on a human or other animal only under certain conditions, such as, but not limited to, relatively deep wounds, compound fractures, burns, immunodeficiency disease, inflammation of tissue, reduction in protective mucosa, and debilitation of tissue due to a prior infection by a primary bacterial pathogen or other disease-causing agent.

A composition or method described herein as "comprising" one or more named elements or steps is open-ended meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or "comprises") one or more named elements or steps also describes the corresponding, more limited, composition or method "consisting essentially of (or "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and close-ended composition or method "consisting of (or "consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. It is also understood that an element or step "selected from the group consisting of refers to one or more of the elements or steps in the list that follows, including combinations of any two or more of the listed elements or steps.

Unless specifically indicated, a composition or method is not limited by any particular order of the listed elements or steps.

The bacterial replicative DNA helicase is a useful target for new antibiotic discovery because it fulfills an essential role in DNA replication and exhibits no significant homology to mammalian helicases. Through the ATP-dependent unwinding of the DNA duplex, replicative helicases allow access by the rest of the replication machinery to the replication fork and thus permit duplication of the bacterial genome. These enzymes function as hexameric rings, with the DNA occupying the central channel of the hexamer (Bailey et al, Science, 318: 459-463 (2007)). Bacterial replicative helicases have been demonstrated to be essential for bacterial growth, and inhibitors would likely be bactericidal since existing gyrase and polymerase inhibitors are known to kill susceptible bacteria. Bacterial cells contain a variety of putative or actual helicases in addition to the replicative enzyme, but they are not closely related structurally to the replicative helicases and most are involved in DNA repair or plasmid replication and are not essential for bacterial growth.

Prior screening for inhibitors of bacterial replicative helicases has yielded very few potent, selective, and non-cytotoxic inhibitors. In fact, only two compounds have been described as bacterial replicative helicase inhibitors — the natural product flavonoid myricetin inhibits Escherichia coli DnaB helicase (Griep et al., Bioorg. Med. Chem., 15: 7203-7208 (2007)), and a triaminotriazine was identified in a screen for inhibitors of Pseudomonas aeruginosa DnaB helicase (McKay et al, Bioorg. Med. Chem. Lett., 16: 1286- 1290 (2006)). These compounds also exhibit significant cytotoxicity in mammalian cell culture even in the presence of serum and, therefore, have not been considered as desirable lead compounds for drug development.

The invention is based on the results of screening over 186,000 organic compounds for the ability to inhibit a Staphylococcus aureus or Bacillus anthracis helicase-catalyzed DNA strand unwinding reaction. Confirmation of helicase inhibitory activity and further characterization of positive "hits" from the initial screening led to the identification of the following preferred compounds of the invention that inhibit the activity of a helicase from either B. anthracis Sterne strain or S. aureus Smith strain and inhibit the growth of cells of at one of these strains in culture (MIC less than 100 μM or less than 50 μg/ml for at least one of these bacterial strains):

Compound 1 -[4,8-dimethyl-7-(naphthalen-l-ylmethoxy)-2-oxo-2H^r-chromen-3-yl]propanoic acid

Compound 2 -[7-(4-bromo-2-fluorobenzyloxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 3 -benzyl-7, 8-dihydroxy-4-methyl-2H-chromen-2-one

Compound 5 6,7-dihydroxy-4-[(l-methyl-lH-benzimidazol-2-ylthio)methyl]-2H-chromen-2-one

Compound 12 iV-{4-[N-(3,4-dimethylisoxazol-5-yl)sulfamoyl]phenyl}-2-(5,6-diphenyl-l,2,4-triazin-3- ylthio)acetamide

Compound 16 2-[5-(5-mercapto- 1 ,3 ,4-oxadiazol-2-yl)furan-2-yl]benzene- 1 ,4-diol

Compound 17

6-ethyl-9-mercapto-6-methyl-5₅6-dihydrobenzo[/z][l,2,4]triazolo[3,4-ό]quinazolin-7(12H)- one

Compound 19 2-[4,8-dimethyl-7-(naphthalen-2-ylmethoxy)-2-oxo-2H-chromen-3-yl]acetic acid.

A bacterial helicase inhibitor compound useful in the compositions and methods described herein inhibits the activity of one or more species of bacterial helicases. Preferred bacterial helicase inhibitor compounds described herein have an IC₅₀ with respect to a B. anthracis helicase or an S. aureus helicase of 25 μM or less.

Among the helicase inhibitor compounds described herein, Compounds 1, 2, 3, 5, 12, 16, and 17 were discovered as validated helicase inhibitors from the screening protocol and subsequent validation assays (see, Table 4). Compound 19 was determined to be a helicase inhibitor according to the invention based on its IC₅₀ for B. anthracis helicase and a structure-activity-relationship (SAR) analysis compared to that of Compounds 1 and 2 (see, Table 5). Based on SI values, preferred helicase inhibitor compounds of the invention, such as Compounds 1, 2, 3, 5, 12, 16, 17, and 19, are not suitable for internal use. However, these compounds are sufficiently potent inhibitors of the activity of a helicase of B. anthracis and/or that of S. aureus (IC₅₀ of less than or equal to 25 μM, Tables 4 and 5, below) and are sufficiently potent as inhibitors of growth of cells of the B. anthracis Sterne strain and/or of the S. aureus Smith strain (MIC < 100 μM) to find use in compositions and methods for inhibiting growth of bacteria on surfaces that could otherwise serve as fomites for transmission of pathogenic bacteria from one individual or biological sample thereof to another individual. Compounds 1, 2, and 12 are particularly preferred as these bacterial helicase inhibitors exhibit a considerable breadth of antibacterial activity across the Bacillus genus, with some limited activity against S. aureus (see, Table 6, below). Uses and Compositions of Helicase Inhibitors

The compounds described herein inhibit the activity of one or more bacterial helicases present in cells of Bacillus and/or Staphylococcus species and strains. Since helicase activity is critical for growth, a helicase inhibitor compound described herein is also effective in inhibiting growth of bacterial cells that possess a helicase that is inhibited by the helicase inhibitor compound. In a method according to the invention, inhibiting growth of bacteria on a surface comprises bringing a helicase inhibitor described herein into contact with bacterial cells present on the surface. Preferably, a helicase inhibitor described herein is in contact with a surface prior to contact with bacterial cells, however, a helicase inhibitor may also be brought into contact with a surface that already contains bacterial cells to inhibit growth of the bacteria already resident on the surface.

A helicase inhibitor compound described herein may be brought into contact with a solid surface (e.g., by coating, immersing, impregnation, and covalent conjugation) composed of or comprising any of a variety of materials that are capable of retaining and transmitting viable bacterial cells. Such materials include, but are not limited to, plastic, glass, silicon, rubber, metal, stone, cement, nylon, cellulose, polymeric resin, calcium phosphate (for example, as in, but not limited to, hydroxyapatite and bone), calcium carbonate (for example, as in, but not limited to, mollusk shells and mother-of-pearl), keratin (for example, as in, but not limited to, skin, hair, fur, wool, nails, claws, hooves, scales, beaks, and feathers), collagen (for example, as in, but not limited to, animal hides, tendons, and ligaments), chitin (for example, as in, but not limited to, exoskeletons and fungal cell walls), and combinations thereof.

A helicase inhibitor described herein may be incorporated into any of a variety of compositions to provide the benefit of bacterial growth inhibition to the particular composition or to a surface to which the composition may be applied. Compositions comprising a helicase inhibitor described herein include, but are not limited to, solutions, suspensions, dry mixtures, gels, petroleum products, porous membranes, porous filters, microparticles, microspheres, liposomes, micelles, lipid bilayers, resin particles, plastics, paints, glues, pastes, cellulose products, textiles (fiber, yarn, or cloth), and nanoparticles. A helicase inhibitor may also be formulated by standard methods for delivery to a surface in an aerosol of fine solid particles or liquid droplets mixed with a gas.

While in theory a helicase inhibitor compound described herein may be applied to a solid surface as the isolated compound alone (raw compound), it is more likely that the compound will be employed in a composition with at least one other compound. Compositions of the invention may be in any of a variety of forms particularly suited for the intended mode of applying a helicase inhibitor compound to a solid surface to prevent or inhibit growth of bacteria on the surface. A carrier is any compound that provides a medium for using a helicase inhibitor compound described herein. A carrier may be liquid, solid, or semi-solid. To retain its utility, it will be necessary that the carrier (and any other component of a composition) does not totally neutralize the helicase inhibitory activity of the compound(s) of the invention included in the composition. Preferably, the activity of the inhibitory compound will not be affected by the carrier. A suitable carrier for use in the compositions described herein includes, but is not limited to, an organic solvent, an aqueous buffer, water, and a solid dispersing agent. Helicase inhibitor compounds described herein have limited solubility in aqueous solutions (for example, less than 50 - 100 μg/ml). Accordingly, solutions and suspensions comprising a helicase inhibitor compound described herein are preferably prepared using an appropriate organic solvent or emulsifying agent. A preferred organic solvent is dimethyl sulfoxide (DMSO). DMSO-based solutions of a helicase inhibitor compound are particularly useful in providing required concentrations of the compound in various compositions, assays (including growth assays), and procedures. For preparing solutions of coumarin compounds described herein, other organic solvents may also be used, including but not limited to an alcohol, N-methylpyrrolidone (NMP), and N,N-dimethylacetamide (DMA), although for most purposes DMSO is more preferred. As a general guide for using an alcohol as a solvent for a courmarin helicase compound described herein, ethanol is more preferred than isopropanol, which is more preferred than butanol or an aryl alcohol, which are more preferred than methanol.

For solid compositions, conventional solid carriers are preferred and include, but are not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

A composition comprises an effective amount of one or more helicase inhibitors described herein in combination with an acceptable carrier, and, optionally, one or more other active agents, diluents, fillers, and excipients.

An excipient is an inert compound that improves or provides a desirable physical property to a composition. An excipient useful in a composition described herein includes, but is not limited, an emulsifying agent, pH buffering agent, a dispersing agent, co-solvent, a gelling agent, and a drying agent.

An additional active agent provides a desired activity in addition to the bacterial growth inhibitory activity of a helicase inhibitor described herein. An additional active agent useful in compositions and methods described herein may include, without limitation, an antibacterial agent other than a helicase inhibitor compound described herein (e.g., citrate, EDTA, an antibiotic), an antifungal agent, an antiviral agent, an anticancer agent, and combinations thereof.

Antibacterial agents that may be used in combination with the helicase inhibitor compounds described herein include, but are not limited to, penicillins, carbapenems, cephalosporins, macrolides (including erythromycin and ketolides), sulfonamides, aminoglycosides, quinolones (including fluoroquinolones), oxazolidinones, lipopeptides (such as daptomycin), tetracyclines, vancomycin, erythromycin, streptomycin, efflux pump inhibitors, lactoferrins, antibacterial cationic peptides, and combinations thereof.

A composition comprising a helicase inhibitor compound described herein may also comprise a dispersing agent. The dispersing agent may be employed to disperse the helicase inhibitor compound more uniformly in a composition and/or to enhance dispersion of the composition containing a helicase inhibitor over a surface to which the composition is applied. A dispersing agent may be a solid or liquid. Solid dispersing agents may include, without limitation, talc, starch, cellulose, metal oxide (e.g., zinc oxide, titanium oxide), graphite, and combinations thereof. A preferred dispersing agent for liquid compositions is a surfactant, which may be an anionic, cationic, amphoteric, or nonionic surfactant. See, for example, US Patent No. 6,921,745. Preferably, a surfactant is employed at the lowest concentration that provides optimal dispersion of the helicase inhibitor in the composition or optimal dispersion of the composition on a surface while diminishing the growth inhibitory activity of the helicase inhibitor toward a desired bacterial species by no more than 1%; more preferably, by no more than 0.1%; and even more preferably by no more than 0.01%.

Preferred anionic surfactants useful in the compositions and methods described herein include, without limitation, linear alkyl benzene sulfonic acid; alkyl sulfate; polyoxy ethylene alkyl ether sulfate having 1 to 10 moles of ethylene oxide; polyoxyethylene alkyl ether carboxylic acid having 1 to 10 moles ethylene oxide; polyoxy ethylene alkyl amide ether carboxylic or fatty acid having 1 to 10 moles ethylene oxide; and potassium, sodium, magnesium, or alkanolamine salts thereof. Preferably, the alkyl and fatty groups in an anionic surfactant are, independently, 8 to 22 carbon atoms, and more preferably 10 to 18 carbon atoms.

Preferably, a nonionic surfactant useful in the compositions and methods described herein is a nonionic polyoxyethylene ether, including, but not limited to, a polyoxyethylene alkyl ether having an alkyl chain containing 8 to 22 carbon atoms, more preferably 10 to 18 carbon atoms, and having 1 to 30 moles, and more preferably 4 to 20 moles, of ethylene oxide; a polyoxyethylene oxypropylene alkyl ether having 1 to 30 moles, and more preferably 1 to 20 moles, of ethylene oxide, and having 1 to 10 moles, more preferably 1 to 5 moles, of propylene oxide; a fatty acid alkanol amide containing 8 to 22 carbon atoms, and more preferably 10 to 18 carbon atoms to which 1 to 3 moles of ethylene oxide or propylene oxide may be added; and an alkyl polyglucoside having an alkyl chain containing 8 to 22 carbon atoms, and more preferably 10 to 18 carbon atoms, and preferably having 1 to 10 sugars, and more preferably 1 to 2 sugars, condensed therein. A preferred species of nonionic surfactant useful in compositions and methods described herein is (t-octylphen- oxypolyethoxyethanol (e.g., brand name TRITON® X-100 non-ionic surfactant, Sigma- Aldrich, St. Louis, Missouri, US).

Another nonionic surfactant useful in the compositions and methods described herein may be an ester between a fatty acid containing 8 to 22 carbon atoms, and preferably 10 to 18 carbon atoms, and a polyvalent alcohol having a hydrocarbon group containing 2 to 10 carbon atoms and 2 to 8 hydroxy groups. More preferably, the ester is a glycerin fatty acid ester, a polyglycerin fatty acid ester, a sorbitan fatty acid ester, a sucrose fatty acid ester, or a propylene glycol fatty acid ester.

Amphoteric surfactants that may find use in the compositions and methods described herein include, without limitation, those having an alkyl group containing 8 to 22 carbon atoms, such as alkyl amidopropyl-N,N-dimethyl acetate betaine (N-alkanoyl aminopropyl- N,N-dimethyl-N-carboxymethyl ammonium carbobetaine), alkyl amidopropyl-N,N- dimethyl-2-hydroxypropyl sulfobetaine (N-alkanoyl aminopropyl-N,N-dimethyl-N-(2- hydroxy-3-sulfopropyl) ammonium sulfobetaine), alkyl-N,N-dimethyl acetate betaine (N- alkyl-N,N-dimethyl-N-carboxymethyl ammonium carbobetaine), alkyl amidopropyl-N,N- dimethyl-2-propyl sulfobetaine (N-alkanoyl aminopropyl-N,N-dimethyl-N-(2-sulfopropyl) ammonium sulfobetaine), lauryl-N,N-dimethyl-hydroxypropyl sulfobetaine (N-lauryl-N,N- dimethyl-N-(2-hydroxy-3-sulfopropyl) ammonium sulfobetaine), and alkyl amine oxide. Among these, preferred species may include lauric acid amidopropyl-N,N-dimethyl acetate betaine (N-lauroyl aminopropyl-N,N-dimethyl-N-carboxymethyl ammonium carbobetaine), myristic acid amidopropyl-N,N-dimethyl acetate betaine (N-myristyloyl aminopropyl-N,N- dimethyl-N-carboxymethyl ammonium carbobetaine), cocamide amide propyl-N,N- dimethyl acetate betaine (N-coconut composition alkanoyl aminopropyl-N,N-dimethyl-N- carboxymethyl ammonium carbobetaine), lauryl-N,N-dimethyl-2-hydroxypropyl sulfobetaine (N-lauryl-N,N-dimethyl-N-(2-hydroxy-3-sulfopropyl) ammonium sulfobetaine), lauric acid amide propyl-N,N-dimethyl-23-hydroxypropyl betaine (N-lauroyl aminopropyl-N,N-dimethyl-N-(2-hydroxy-3-sulfopropyl) ammonium sulfobetaine), and an alkyl amine oxide having two alkyl groups containing 2 or less carbon atoms and one long- chain alkyl group containing 8 to 22 carbon atoms, which optionally may have an amide linkage.

Cationic surfactants that may be used in compositions and methods described herein include, but are not limited to, a long-chain dialkyl dimethyl ammonium salt, long-chain monoalkyl monobenzyl dimethyl ammonium salt, and monoalkyl trimethyl ammonium salt having a long alkyl chain containing 6 to 24 carbon atoms, and preferably 6 to 18 carbon atoms, which may be interrupted therein with an amide or ester linkage. The counterion of such cationic species is preferably a halogen ion, sulfate ion, or alkyl sulfate containing 1 to 3 carbon atoms. The cationic surfactants of amine type useful in compositions and methods described herein include long-chain dialkyl monomethylamine salts having a long alkyl chain containing 8 to 24 carbon atoms, which optionally may be interrupted therein with an amide or ester linkage. Preferred counterions of such species include hydrochlorides, sulfates, and phosphates thereof.

Compositions comprising a bacterial helicase inhibitor described herein may also be formulated into any of a variety of solid and semi-solid forms including microparticles, microspheres, liposomes, micelles, ointments, creams, gels, jellies, and lotions. Such formulations may be applied directly to a surface (including the skin of an individual) or incorporated into another composition or mixture to provide antibacterial activity to a final product, such as a cosmetic, fabric, or device.

For topical administration to the skin to inhibit bacterial growth, a helicase inhibitor compound is preferably formulated for dispersion over the skin with no or minimal absorption through the lower dermal layers. Such formulations may take the form of microparticles, microspheres, liposomes, micelles, ointments, creams, gels, jellies, and lotions. A compound of the invention may also be incorporated into a dermal patch. Preferably, a topically administrable composition is formulated to prevent or minimize absorption of the helicase inhibitor to the lower dermal layers so that the bacterial growth inhibitory activity of the helicase inhibitor is retained on the epidermal surface.

A surface that is susceptible to contact with or that already contains (i.e., is contaminated with) bacteria may be treated (e.g., coated, immersed, impregnated) with a helicase inhibitor described herein to inhibit growth of bacteria on the surface. Such surfaces are found on a variety of manufactured products including, but not limited to, medical devices (such as central venous catheters (CVCs), implantable pumps, artificial heart valves, and cardiac pacemakers); cardio-pulmonary bypass (CPB) pumps (heart-lung machines); dialysis equipment; artificial respirators; breathing apparatuses (oxygen and air supplies); water pipes; air ducts; air filters; water filters; and plumbing fixtures. The particular composition and properties of a particular surface will determine the preferred method by which the surface is treated to contain a helicase inhibitor described herein.

Medical devices may be stored in or their lumens filled with a composition comprising a helicase inhibitor described herein to inhibit growth of bacteria on the surfaces of the exterior and/or lumens of the devices. Prior to using or implanting the medical device, the composition comprising a helicase inhibitor described herein is thoroughly expelled from the lumens of the device and removed from exterior surfaces under sterile conditions. For example, a helicase inhibitor as described herein may be employed in a "lock solution" (solution or suspension) for use with a central venous catheter (CVC). In standard medical device lock therapies, the lumen(s) of a medical device is filled with a lock solution comprising an anti-bacterial agent (e.g., antiseptic, antibiotic) to prevent bacterial contamination of the device. The lock solution is introduced into the lumen(s) of the device when the device is not in use and then expelled shortly before use. A lock solution according the invention is a solution or suspension comprising a helicase inhibitor described herein at a concentration sufficient to inhibit growth by potentially contaminating bacteria. A lock solution comprising a helicase inhibitor as described herein may further comprise any of a variety of other compounds that enhance the prevention of bacterial contamination and infection in a medical device. Such additional compounds that may be used in preparing a lock solution of the invention include, but are not limited, one or more other antibacterial growth agents (e.g., citrate, EDTA, antibiotic, microbial biocide) at a concentration effective to inhibit growth of (or kill) one or more strains of potentially contaminating bacteria and one or more excipients that provide an additional desirable physical property to the lock solution other than inhibition of bacterial growth. For example, an excipient may provide a density, osmolarity, or viscosity to the lock solution that is similar to the fluid (e.g., blood) that will fill the device lumen when the device is used or implanted. An excipient of a lock solution may also prevent occlusion of the catheter lumen caused by blood clotting and/or formation of a fibrin sheath. To treat plastic surfaces, a helicase inhibitor described herein may be incorporated into a resin prior to polymerization to form the plastic. Alternatively, a plastic surface may be immersed in a solution or suspension of a helicase inhibitor preferably in the presence of one or more swelling agents to adsorb or absorb the helicase inhibitor to the plastic surface (see, e.g,. Schierholz et al., Biomaterials, 18: 839-844 (1997); Schierholz and Pulverer, Biomaterials, 19: 2065-2074 (1998); Schierholz et al., J. Antimicrob. Chemother., 46: 45-50 (2000)). A helicase inhibitor may also be covalently bound to plastic using an appropriate cross-linking agent. Alternatively, a helicase inhibitor may be impregnated into a material, such as a hydrogel or polymer, which would then be used a surface. The use of biodegradable plastic resins, such as poly(D,L-lactic acid) and poly(D,L-lactic acid):coglycolide, combined with an anti-bacterial agent to produce antibacterial device coatings has been described (Gollwitzer et al., J Antimicrob Chemother., 51, 585-591 (2003)). Such technology may be readily adapted for preparing anti-bacterial coatings comprising a helicase inhibitor compound described herein.

Effective amounts of a helicase inhibitor to be applied to a surface or otherwise employed in a method or composition to inhibit growth of bacteria may be determined by the skilled practitioner who is familiar with methods for assessing effective amounts of antibiotics and antiseptics (biocides) on surfaces to meet or exceed standards of authoritative agencies. See, e.g., Guidelines for the prevention of intravascular device- related infections such as those issued by the United States Center for Disease Control (Atlanta, Georgia) (O'Grady et al., Am. J. Infect. Control, 30: 476-489 (2002); examples of biocide and antibiotic impregnated catheters (Potera, Science, 283: 1837, 1839 (1999)); assessment of effectiveness to bacterial challenge by biocide and antibiotic impregnated catheters (Sampath et al., Infect. Control Hosp. Epidemiol, 22: 640-646 (2001)). Such guidelines and procedures are readily adapted to assessing and optimizing the amount and conditions for using a particular helicase inhibitor described herein to inhibit bacterial growth in a particular application (e.g., surface, device, composition, or method).

Preferably, the sum total of all ingredients in a composition comprising a helicase inhibitor compound described herein diminishes the bacterial growth inhibition activity of the helicase inhibitor toward a desired bacterial species or strain by more no more than 10%; more preferably, by no more than 1%; even more preferably, by no more than 0.1%; and still more preferably, by no more than 0.01%. The following example is provided to illustrate various embodiments of the invention and shall not be considered as limiting in scope.

Example. Materials and methods for screening and characterizing compounds as inhibitors of bacterial helicase activity. Bacterial strains and plasmids

The bacterial strains and plasmids used in this study, their sources and relevant genotypes are described in Table 1, below. Table 1. Bacterial Strains and Plasmids

Shatalin et al., FEMS Microbiol. Lett., 245: 315-319 (2005); Green et al, Infect. Immun., 49: 291-297 (1985); ³Love et al., MoI. Gen. Genet., 144: 313-321 (1976); ⁴Ghosh et al., Gene, 176: 249-255 (1996); ⁵Biswas et al., J. BacterioL, 191: 249-260 (2009) QIi gonucleotides

Fluorescence quencher dye BLACK HOLE QUENCHER® 1 ("BHQ®1" fluorescence quencher dye, Biosearch Technologies, Inc., Novato, California, US) and 6- carboxyfluorescein ("FAM") labeled 60-mer oligonucleotides ("Hel-3'BHQ" and "HeI- 5'FAM", see, Table 2, below) were purchased from Eurofins MWG Operon (Huntsville, Alabama, US) and Integrated DNA Technologies, Inc. (Coralville, Iowa, US), respectively, as HPLC purified oligonucleotides. Slight variations in the mass quantitation and in RFU values required a careful calibration of each annealed batch to minimize batch-to-batch variation in the screen. The 30-mer capture strand ("Hel-Cap30"), which is complementary to the 5'-30 nucleotides of the FAM-labeled oligonucleotide, was purchased from Eurofins MWG Operon as desalted, unpurified oligonucleotide. The two labeled oligonucleotide strands were annealed at a 1 :2 (Hel-5'FAM:Hel-3'BHQ) ratio prior to use in the FRET quenching helicase assay. PCR primers used in this study are also shown in Table 2. Table 2. Oligonucleotide Primers and Helicase Substrates.

Table 2. (continued

Cloning, expression, and purification of B. anthracis helicase

The cloning and expression of the B. anthracis dnaBgerxQ and subsequent purification of the helicase has been described (Biswas et al., J Bacteriol., 191 : 249 (2009)). Briefly, the gene was amplified by PCR with primers HCASE45-5'and HCASE45- 3' (Table 2) and B. anthracis genomic DNA. The amplified gene was inserted into a pET30 vector (Novagen Inc., Madison, Wisconsin, US) under the control of a T7 promoter and confirmed by DNA sequencing. B. anthracis dnaB was over-expressed in E. coli strain BL21 (DE3) RlL (Stratagene Corp., La Jolla, California, US) harboring the pET30-DnaB_BA plasmid. Extraction of IPTG-induced cells was done as previously described (Jiang et al., Antimicrob Agents Chemother., 48: 4349-4359 (2004)). Protein was precipitated from the cell extract by addition of 0.25 g/ml (NH₄)₂SO₄, resuspended in buffer A (25 mM Tris-HCl, (pH 7.5), 5 mM MgCl₂, 10% glycerol, 5 mM DTT) and re-precipitated in 0.2 g/ml (NH₄)₂SO₄. The protein pellet was resuspended in buffer A and fractionated by Q- Sepharose chromatography (GE Health Sciences, Piscataway, New Jersey, US), which removed any contaminating endogenous E. coli helicase (Arai et al, J. Biol. Chem., 256: 5247-5252 (1981); Biswas et al., Biochemistry, 38: 10919-10928 (1999)). The flow through fractions were pooled and loaded onto a 6 ml S-Sepharose column equilibrated with buffer A₁₀₀. B. anthracis helicase was eluted with a gradient of buffers A_1Oo and A₅₀₀. The peak fractions were identified by ssDNA dependent ATPase and DNA helicase activities in conjunction with SDS-PAGE. The active fractions were pooled and concentrated by ultrafiltration using a YM-30 membrane. The purified helicase was greater than 98% pure as analyzed by SDS-PAGE (Biswas et al., J. Bacteriol, 191 : 249-260 (2009)). The enzyme was verified to be stable for several hours at room temperature, and for at least 12 weeks at -20⁰C in 20% glycerol storage buffer. B. anthracis helicase activity is dependent on the presence of ATP, with an optimum at 2.5 mM. The pH optimum is broad and centered around pH 7.8. Optimal concentrations of other assay components are as follows: 2 mM

MgCl₂, 0.5 mM dithiothreitol (DTT), 0.01% TRITON® X-100 non-ionic surfactant (t- octylphenoxypolyethoxyethanol, Sigma-Aldrich, St. Louis, Missouri, US), and 25 mM

NaCl.

Cloning, expression, and purification of S. aureus helicase

The genes for the S. aureus replicative DNA helicase idnaC) and helicase loader idnaT) were amplified by PCR from genomic DNA isolated from S. aureus Smith using the primers 5'Sa-dnaC and 3'Sa-dnaC for the helicase gene and primers 5'Sa-dnaI and 3'Sa-dnaI for the loader gene (Table 2). Products were sequence-confirmed and cloned in the dual expression vector pET-Duetl (Novagen Inc., Madison, Wisconsin, US), under control of a T7 promoter/lac operator. The helicase was expressed in native form, while the loader was expressed with an N-terminal hexahistidine tag to facilitate purification. Although the helicase loader proved unnecessary either for solubility or helicase activity, the dual expression clone was used since it produced larger quantities of helicase than did a clone containing the dnaC gene alone in the same vector. Following overnight induction by IPTG at 14⁰C, cells were harvested by centrifugation and lysed in a French press. The lysate was centrifuged, and the resulting cleared supernatant was precipitated by addition of 0.2 grams Of (NFLj)₂SO₄ per ml lysate. The precipate was redissolved in buffer B (50 mM Tris pH 7.5; IM NaCl; 10% glycerol; 2 mM 2-mercaptoethanol) containing 1 mM PMSF, and applied to an IMAC-Ni²⁺ column equilibrated in the same buffer. Following a wash in the same buffer, the column was eluted in a 0-200 mM imidazole gradient. The fractions containing helicase were pooled and applied to a phenyl sepharose column equilibrated in buffer B containing 1 mM EDTA; the column was eluted in gradient of decreasing NaCl (1 M to 0 M) and increasing TRITON® X-100 (t-octylphenoxypolyethoxyethanol, Sigma-Aldrich, St. Louis, Missouri, US) non-ionic surfactant (0% to 1%). The resulting helicase preparation was about 98% pure by SDS-PAGE and essentially free of nuclease activity as judged by minimal ATP-independent activity in the FRET assay. Optimal conditions for the reaction include a pH range of 7.6-8.4, a magnesium concentration of 2 mM, and an ATP concentration of 3 mM. The enzyme was stable at room temperature for at least two hours and at -20°C in 20% glycerol storage buffer for several months. Assays for helicase-catalyzed strand unwinding

Fluorescent resonance energy transfer (FRET) assay

The FRET-based helicase activity assay was performed essentially as previously described (McKay et al, Bioorg. Med. Chem. Lett., 16: 1286-1290 (2006); Zhang et al., Anal.Biochem., 304: 174-179 (2002)) using labeled annealed oligodeoxynucleotides HeI- 5'FAM:Hel-3'BHQ (Table 2). The assay is based on the helicase-mediated dissociation of two annealed oligonucleotides, one with a fluorescent label, the other bearing a quencher moiety. Two complementary annealed 60-mer oligonucleotides with noncomplementary 30-mer ends were labeled, respectively, with 6-carboxyfluorescein (FAM) and BLACK HOLE QUENCHER® 1 (BHQ®1) fluorescence quencher dye. When the two strands are unwound by helicase action, the FAM emits an unquenched signal at 535 nm followed by excitation at 485 nm. To prevent the FAM-labeled strand from reannealing with the quencher strand, an unlabeled 30-mer capture strand, Hel-Cap30, was added in 15-fold molar excess (Table T). The FRET assay demonstrates ATP- and enzyme-dependent helicase activity and is linear in its response for at least 30 minutes at room temperature. It is tolerant of dimethyl sulfoxide (DMSO) concentrations up to at least 2%.

Radiometric assay

Radiometric assays of helicase activity were performed as described previously (Biswas et al., Biochemistry, 38: 10919-10928 (1999); Biswas et al., Biochemistry, 36: 13277-13284 (1997)) utilizing a partial duplex substrate consisting of a radiolabeled 60-mer (SEQ ID NO: 10, Table 2) annealed to a circular Ml 3 single-stranded DNA and possessing 5-nucleotide forks at both the 5' and 3' ends. A standard 20 μl reaction volume contained 25 mM Tris-HCl, (pH 7.5), 10 raM MgCl₂, 10% glycerol, 5 mM DTT, and 0.1 niM ATP, 17 fmol (1-2 x 10⁴ cpm/μl) of substrate and the indicated amount of helicase. The mixtures were incubated at 3O⁰C for 15 minutes, and the reactions were terminated by the addition of 4 μl of 2.5% sodium dodecyl sulfate (SDS), 60 mM EDTA, and 1% bromophenol blue. Displacement was monitored by polyacrylamide gel electrophoresis, followed by autoradiography.

High-throughput screening for inhibitors of helicase activity Two small molecule libraries were screened in this study. The Microbiotix, Inc., collection (MBX) was screened for inhibitors of B. anthracis helicase, and the collection at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Disease (NSRB) at Harvard Medical School (Boston, Massachusetts, US) was screened for inhibitors of S. aureus helicase. Both libraries were comprised of compounds purchased from commercial vendors, and while the overlap between the two libraries has not been calculated, it is likely to be significant because some of the same vendors were used. Compounds in the Microbiotix screening deck were purchased from Chembridge (San Diego, California, US), Timtec (Newark, Delaware, US), and ChemDiv (San Diego, California, US), while the NSRB collection contained compounds from Asinex (Moscow, Russia), Enamine (Kiev, Ukraine), Life Chemicals, Inc. (Burlington, Ontario, Canada), and Maybridge (Fisher Scientific International), as well as Chembridge and ChemDiv. Compounds were selected in the molecular size range of 200 to about 500 daltons (Da) and were filtered to remove unwanted and known cytotoxic fragments, including metal complexes, highly conjugated ring systems, oxime esters, nitroso and strong Michaelson acceptors.

Compounds in the Microbiotix collection were diluted in 96-well master plates at 2.5 mM in DMSO at -20°C. Master plates were thawed at room temperature on the day of the screen, and 1 μl of compound was added to the 384-well screening plates by means of a Sciclone ALH 3000 liquid handling robot (Caliper, Inc.) and a TWISTER® II microplate handler (Caliper Life Sciences, Inc., Hopkinton, Massachusetts, US). Compounds in the NSRB collection were added to assay plates by pin transfer. The first two columns of wells contained the complete reaction without inhibitors (postive control), and the last two columns contained the complete reaction, but no enzyme (negative control). Screening wells contained 30 μl volume consisting of -80 μM compound, 10 nM annealed oligo duplex, 15 x capture strand, 63 ng helicase (12 nM monomer), 30 mM Tris-HCl (pH 7.9), 0.01% TRITON® X-100 non-ionic surfactant, 0.5 mM DTT, 2 mM MgCl₂, and 25 mM NaCl. The reaction was initiated by the addition of ATP to 2.5 mM final concentration using a Wellmate automated microplate dispenser. Plates were incubated at room temperature for 30 min, and fluorescence was read at 530 nm in an Envision 2102 Multilabel HTS Counter (Perkin Elmer) with excitation at 490 nm. Strand unwinding catalyzed by helicase resulted in loss of quenching and a linear increase in fluorescence during the 30 minute incubation at room temperature. The capacity of the screens was 50,000 compounds in singlet per week (7,000 compounds per day). The plate Z' value (Zhang et al., J. Biomol. Screen, 4: 67-73 (1999)) averaged 0.59, and the signahbackground ratio averaged 3.6. All screening data, including the z-score, and confirmation and validation data were stored in one central database (CambridgeSoft ChemOffice 11.0). Validated hits were re-ordered from the vendor and confirmed to be >95% pure and to be of the expected mass by LC-MS analysis. Compounds for SAR analysis (Compounds 19-37 in Table 5) were ordered from Chembridge, Inc. (San Diego, California, US).

Compounds 1, 2, 3, 5, 12, 16, 17, and 19 are preferred helicase inhibitors of the invention. Although these compounds can be ordered from commercial vendors, the compounds may also be synthesized using organic synthesis methods and reagents known in the art. General schemes for synthesizing these preferred bacterial helicase inhibitors of the invention are outlined below. For Compounds 1, 2, and 19:

The coumarin core is constructed by condensation of a β-ketoester and a phenol using acid catalysis (the Pechmann reaction). Once constructed, the core is elaborated by alkylating the remaining phenolic hydroxy group and saponifying the ester to the corresponding acid. For Compound 3 :

The entire structure is elaborated in a single step by condensation of an appropriately elaborated β-ketoester and a phenol using acid catalysis (the Pechmann reaction). For Compound 5 : Nucleophilic displacement

The coumarin core is constructed by condensation of a β-ketoester and a phenol using acid catalysis (the Pechmann reaction). The core structure is elaborated by nucleophilic displacement of the chloro group on the chloromethyl substituent. "Nu" represents a nucleophilic substituent group. For Compound 12:

chlorination ArNH₂ then then displacement nitro reduction

Peptide Coupling

The triazinyl thioacetic acid core is constructed via a nucleophilic displacement of an intermediate chlorotriazene. Simultaneously, an aminophenyl sulfonamide is made from 4-nitrophenylsulfonyl chloride. Peptide coupling of the two halves leads to the final compound. For Compound 16: Suzuki

coupling

The arylfuran if constructed using the Suzuki coupling with a boronic acid and a 2- bromofuroic acid ester. Conversion of the ester to a hydrazide followed by cyclization with carbon disulfide and base provides the target compounds. For Compound 17:

imine acylation

The core system is elaborated stepwise by the addition of a metallated toluene to an α,β-unsaturated ester. Conversion to the acid chloride is followed by formation of an amide imine. The amide imine is then cyclized with carbon disulfide. Amination followed by hydrazine displacement provides a substrate, which is then cyclized to the target compound with carbon disulfide. Secondary assays to confirm and validate inhibitors

Other enzymatic assays

Hits were tested for inhibition of purified P. aeruginosa AmpC β-lactamase in the presence of various concentrations of TRITON® X-IOO non-ionic surfactant to detect compounds acting promiscuously by a colloidal aggregate mechanism as described previously (Feng et al, Nat. Protoc, 1 : 550-553 (2006)). The ability of compounds to inhibit B. subtilis Pol IHC DNA polymerase was assayed by using a 96-well plate format version of the standard DNA polymerase assay as described previously (Barnes et al., Nucleic Acids Res. , 6: 1203-1219 (1979)). Note that since the substrate for the Pol IIIC assay is DNase-activated chromosomal DNA, no helicase function is required for polymerization (Barnes et al., Nucleic Acids Res., 6: 1203-1219 (1979)). Inhibition of B. subtilis DNA gyrase by compounds was measured by a published gel mobility assay (Saiki et al., Antimicrob. Agents Chemother., 43: 1574-1577 (1999)). Inhibition of the ATPase activity of B. anthracis helicase was measured as previously described (Biswas et al., J Bacteriol, 191: 249-260 (2009)). Kinetic measurements

Values for Kj and IC₅₀ for inhibitory compounds were determined by using the FRET-based assay under the same conditions as described for screening except that annealed oligonucleotide substrate or inhibitor concentrations were varied. All IC₅₀ values were determined in duplicate using a 10-point curve consisting of two-fold dilutions of inhibitory compound from 100 μM to 0.2 μM. Substrate and inhibitor concentrations for kinetic experiments are noted in the figures. Rapid assessments of mode of inhibition were done by the method of Wei et al. (J Biomol. Screen, 12: 220-228 (2007)) by determining the variation in IC₅₀ values over a range of substrate and inhibitor concentrations. Ethidium bromide (ΕtBrVdisplacement

Hits were examined in a microplate ethidium bromide displacement assay (Boger et al., J Am. Chem. Soc, 123: 5878-5891 (2001)) to determine whether compounds bind to the DNA duplex substrate.

DNA replication assays in permeabilized cells

Compounds were tested for inhibition of ATP-dependent DNA replication in permeabilized ApolA B. anthracis Sterne cells assay according to a modification of the method of Brown et al. (Nat. New Biol, 237: 72-74 (1972)). This assay was used to confirm whether the compounds are capable of inhibiting an intact bacterial replisome. Minimum inhibitory concentration (MIC) and bactericidal assays Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method described in the CLSI (formerly NCCLS) guidelines (CLSI Approved Standard Procedures, M7-A7, vol. 26, no. 2 (2006)). Unless otherwise indicated, all MICs were expressed in μM to facilitate comparisons with IC₅₀ and CC₅₀ values and were determined in duplicate using a 10-point curve consisting of two-fold dilutions of inhibitory compound from 100 μM to 0.2 μM. For bactericidal tests, inhibitors were examined in a standard method of broth culture of B. anthracis Sterne cells followed by plating on LB agar media and counting colony-forming units (CLSI, Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Guideline (M26-A), vol. 19, no. 18 (National Committee for Clinical Laboratory Standards: Wayne, Pennsylvania, US, 1999)). Determination of mammalian cytotoxicity

The cytotoxic concentration (CC₅₀) of a compound versus cultured mammalian cells (HeLa, ATCC CCL-2, American Type Culture Collection, Manassas, Virginia, US) was determined as the concentration of compound that inhibits 50% of the conversion of MTS to formazan in a colorimetric cell proliferation assay (CELLTITER 96® AQ_ueOus Nonradioactive Cell Proliferation Assay (MTS), Promega, Madison, Wisconsin, US). Briefly, 96-well plates were seeded with HeLa cells at a density of 4 x 10³ per well in minimal essential medium (Catalog No. 0820234DJ, Invitrogen Corp., Carlsbad, California, US) containing 10% fetal bovine serum, or in VP-SFM medium without serum (Catalog No. 11681-020, Invitrogen Corp., Carlsbad, California, US) in the presence or absence of serial dilutions of a compound dissolved in DMSO. Following incubation for 3 days at 37⁰C in VP-SFM, cell viability was measured with the vital tetrazolium salt stain 3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide according to the manufacturer's instructions (Promega, Madison, Wisconsin, US). Values were determined in duplicate using dilutions of inhibitory compound from 100 μM to 0.2 μM.

The "selectivity index" (SI) of a given agent is defined as the ratio of its mammalian cell cytotoxicity (CC₅₀) determined in medium containing 10% fetal bovine serum to its MIC value against B. anthracis (SIβ_a) or S. aureus (SIs_a) or, alternatively, is defined as the ratio of its mammalian cell cytotoxicity determined in medium lacking fetal bovine serum (serum-free medium) to its MIC value against B. anthracis (Sf-SIβa)- Overview of screening results

Genes for the B. anthracis and S. aureus replicative helicases were cloned and expressed in E. coli, the enzymes were purified, and fluorescence-based assays were developed for each and optimized as high throughput screens. Over 186,000 small synthetic molecules were screened to identify inhibitors of the S. aureus or B. anthracis helicase- catalyzed strand unwinding reaction. Primary hits were selected and confirmed by re-assay, requiring over 50% inhibition in at least two of three replicates. The overall confirmed hit rate was about 0.08%, but when calculated separately for each helicase, it was nearly 10- fold higher for the B. anthracis enzyme than for the 5^*. aureus enzyme (Table 3). Table 3. Summary of High Throughput Screens for Inhibitors of Two Helicases

MBX, Microbiotix, Inc.; NSRB, National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Disease

Characterization of confirmed hits

Confirmed inhibitors of each of the helicases were characterized further to eliminate false positives, which act by mechanisms other than direct inhibition of helicase and to measure the concentration-dependence of helicase inhibition. First, hits were examined in an ethidium bromide displacement assay to eliminate compounds that inhibit strand unwinding by binding to the DNA duplex substrate rather than to the helicase. Second, hits were tested in a radiometric assay of helicase activity to ensure that hits block strand unwinding rather than simply quenching FAM fluorescence in the FRET assay. Several hits that resemble known intercalators or minor groove binders or that were strong quenchers were eliminated by secondary assays. Third, hits were tested for inhibition of Pseudomonas aeruginosa AmpC β-lactamase in the presence of various concentrations of TRITON® X- 100 non-ionic surfactant to detect compounds acting promiscuously by a colloidal aggregate mechanism (Feng et al., Nat. Protoc, 1 : 550-553 (2006)). None of the confirmed hits exhibited inhibition of AmpC at 0.01% TRITON® X-100 non-ionic surfactant the concentration used in the FRET helicase assays, indicating that aggregates are not likely to be responsible for the observed helicase inhibition. Finally, hits from each helicase screen were examined for inhibition of the helicase of the other species, and the concentration- dependence of inhibition (IC₅₀) was determined.

Approximately 10% of the 160 confirmed primary hits, a total of 18 compounds, were validated by secondary assays (see, above) and exhibited concentration-dependent inhibition with IC₅₀ values < 25 μM versus at least one of the two helicases (Table 3). These fall into five chemotypes with three additional compounds as singletons (Table 4). With the exception of one chemotype (see below), confirmed hits were demonstrated by LC-MS analysis to be of correct mass and sufficient purity (> 95%) for further evaluation. Selectivity of inhibitors

In order to evaluate the selectivity of the inhibitory effects of these compounds, all confirmed hits were tested for (a) potency of inhibition of DNA replication in permeabilized polA -deficient B. anthracis cells, (b) minimal inhibitory concentration (MIC) versus growth of B. anthracis and S. aureus cells, and counter-screened for (c) potency of inhibition of the replicative DNA polymerase Pol IIIC of B. subtilis, (d) for effects on the viability of mammalian cells (cytotoxicity), and selectivity idex (SI, ratio CCso:MIC for B. anthracis (Ba) or S. aureus (Sa)). Results of assays for the above compounds are shown in Table 4. All data are expressed in μM to facilitate comparisons.

Table 4. Pro erties of Confirmed Helicase Inhibitors from Screenin Protocol

Chemical Class: C = coumarin; B = benzothiazole; R = rhodanine; T = triazine; NP = N-phenyl pyrrole (unstable); S = singleton; ²IC₅₀ versus B. anthracis replicative helicase; ³IC₅₀ versus S. aureus replicative helicase; ⁴MIC versus B. anthracis Sterne; ⁵MIC versus S. aureus Smith; ⁶IC₅O versus ΔpolA B. anthracis Sterne permeabilized cells; ⁷CC₅₀ versus HeLa cells grown in 10% fetal bovine serum-containing medium; ⁸Serum-Selectivity Index = CC₅₀(+S):MIC(Ba); ⁹CC₅O versus HeLa cells grown in serum-free medium; ¹⁰Serum Free-Selectivity Index = CC₅₀(-S):MIC(Ba); ^results were variable due to instability of compound in DMSO (see text for details); n.d. = not done; ind. = indeterminate; Ctl-n = novobiocin control; Ctl-c = coumermycin control Compounds 1-18 in Table 4, above, have the following structures:

Compound 1 (a coumarin) -[4,8-dimethyl-7-(naphthalen-l-ylmethoxy)-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 2 (a coumarin) -[7-(4-bromo-2-fluorobenzyloxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 3 (a coumarin) -benzyl-7, 8-dihydroxy-4-methyl-2/f-chromen-2-one

Compound 4 (a coumarin)

4- { [4-amino-5-(2-fluorobenzyl)-4H- 1 ,2,4-triazol-3 -ylthio]methyl } -6,7-dihydroxy-2H- chromen-2-one

Compound 5 (a coumarin) ,7-dihydroxy-4-[(l-methyl-lH-benzimidazol-2-ylthio)methyl]-2H-chromen-2-one

Compound 6 (a benzothiazole) -ethoxy-N-(6-sulfamoylbenzothiazol-2-yl)benzamide

Compound 7 (a benzothiazole) 3,4,5-trimethoxy-N-(6-(moφholinosulfonyl)benzothiazol-2-yl)benzamide

Compound 8 (a rhodanine) (Z)-3-(3-chlorophenyl)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-2-thioxothiazolidin-4-one

Compound 9 (a rhodanine) (Z)-3-(4-chlorophenyl)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-2-thioxothiazolidin-4-one

Compound 10 (a rhodanine) (Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-3-(4-nitrophenyl)-2-thioxothiazolidin-4-one

Compound 11 (a rhodanine)

(Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-2-thioxo-3-(3-trifluoromethylphenyl) thiazolidin-4-one

Compound 12 (a triazine)

N- {4-[_V-(3 ,4-dimethylisoxazol-5-yl)sulfamoyl]phenyl } -2-(5,6-diphenyl- 1 ,2,4-triazin-3 - ylthio)acetamide

Compound 13 (a triazine) 6-(benzofuran-2-yl)-_J/V²-(4-methoxyphenyl)-l,3,5-triazine-2,4-diamine

Compound 14 (an N-phenyl pyrrole) -(2,5-dimethyl- lH-pyrrol- 1 -yl)-2-hydroxybenzoic acid

Compound 15 (an N-phenyl pyrrole) 2-[4-(2,5-dimethyl- lH-pyrrol- 1 -yl)phenylthio] acetic acid

Compound 16 (singleton structure) 2-[5-(5-mercapto-l,3,4-oxadiazol-2-yl)furan-2-yl]benzene-l,4-diol

Compound 17 (singleton structure)

6-ethyl-9-mercapto-6-methyl-5 ,6-dihydrobenzo[/z] [ 1 ,2,4]triazolo [3 ,4-Z>]quinazolin-7( 12H)- one

Compound 18 (singleton structure) 4-(2-methylthiazol-4-yl)- 1 H-pyrrole-2-carboxylic acid Inhibitors with a coumarin core structure

Three hits from the B. anthracis helicase screen and two hits from the S. aureus helicase screen share a coumarin chemotype (Compounds 1-5, Table 4). These coumarin- type compounds are potent screening hits, with IC₅₀ values ranging from 5-10 μM versus B. anthracis helicase. All five compounds exhibit significant inhibition of both B. anthracis and 5^*. aureus helicases, but they are all less potent versus S. aureus helicase, with IC₅₀ values ranging from about 2- to 20-fold higher than those for B. anthracis helicase. They are poor inhibitors of another DNA replication enzyme, B. subtilis replicative DNA polymerase Pol HIC (IC₅₀ values >100 μM) confirming their selectivity for helicase. As expected for helicase inhibitors, they inhibit DNA replication in permeabilized /?øL4- deficeint B. anthracis Sterne cells, but the potency of Compounds 1 , 4, and 5 is less than that exhibited in the in vitro FRET-based assay. Coumarins 2 and 4 are not cytotoxic to mammalian cells when tested up to 100 μM. Furthermore, Compound 2 inhibits the growth of B. anthracis cells with an MIC of 6 μM, resulting in an overall selectivity index (SI, CC₅₀/MIC) > 16. Inhibitors with a benzothiazole core structure

Two hits from the B. anthracis helicase screen share a benzothiazole chemotype (Compounds 6 and 7, Table 4). They both exhibit IC₅₀ values versus B. anthracis helicase of 12 μM but are slightly less potent versus S. aureus helicase. These two compounds exhibit no detectable inhibition of B. subtilis DNA polymerase Pol IIIC, but also fail to demonstrate detectable inhibition of DNA replication in permeabilized B. anthracis cells. Neither of the benzothiazoles has a detectable MIC, but some cytotoxicity is observed, with CC₅₀ values of 10 μM and 70 μM for Compounds 6 and 7, respectively. Inhibitors with a rhodanine core structure

Four confirmed hits from the B. anthracis helicase screen share a rhodanine chemotype (Compounds 8-11, Table 4). This class of hits is unique for two reasons — they are the most potent helicase inhibitors identified, and all are approximately equipotent against both B. anthracis and S. aureus helicase, with IC₅₀ values of 1-4 μM. Three members of this class exhibit no detectable cytotoxicity (CC₅₀ >100 μM), but the replacement of the 4-chloro substituent with a nitro-group (Compound 10) resulted in pronounced cytotoxicity (CC₅₀ = 11 μM). The three non-cytotoxic rhodanines (Compounds 8, 9, and 11) inhibit DNA replication in permeabilized B. anthracis cells, but with potencies considerably poorer than those demonstrated in direct helicase assays. None exhibited a detectable MIC. Inhibitors with a triazine core structure

Two confirmed hits from the B. anthracis helicase screen share a triazine chemotype (Compounds 12 and 13, Table 4), and they bear some resemblance to the triaminotriazine inhibitor of P. aeruginosa replicative helicase described previously (McKay et al., Bioorg. Med. Chem. Lett, 16: 1286 (2006)). They are moderately potent inhibitors of B. anthracis helicase, with IC₅₀ values of 12 and 24 μM, for Compounds 12 and 13, respectively, and are about half as potent versus S. aureus helicase. Compound 13 fails to inhibit DNA replication in permeabilized B. anthracis cells and is among the most cytotoxic of the hits. Compound 12 inhibits DNA replication in permeabilized B. anthracis cells and displays about equal potency for bacterial and mammalian cell growth inhibition. Inhibitors with an N-phenyl pyrrole core structure

One confirmed hit from the B. anthracis helicase screen and one confirmed hit from the S. aureus helicase screen (Compounds 14 and 15, respectively, in Table 4) share an N- phenyl pyrrole chemotype. While they appeared to be quite potent, they were the only confirmed hits that failed to exhibit the expected mass in LC-MS analysis. Further studies demonstrated that fresh DMSO solutions of re-ordered compounds exhibited no detectable helicase inhibitory activity, but solutions aged for several days in DMSO, did exhibit potent activity. This class of inhibitors was considered artifactual due to instability and not pursued further. They have also been described previously as inhibitors of other targets (Schepetkin et al., J Med. Chem., 49: 5232 (2006); Jiang et al., Antimicrob. Agents Chemother., 48: 4349 (2004)), and thus, appear to be promiscuous inhibitors as well. Inhibitors with unique structures (singletons)

Three confirmed B. anthracis helicase inhibitors (Compounds 16, 17, and 18, Table 4) represent singleton chemotypes. Only Compound 17 inhibited helicase in vitro as well as DNA replication in permeabilized cells. While not active against S. aureus helicase, this inhibitor exhibited an MIC against B. anthracis Sterne cells consistent with its IC₅₀ and displayed low cytotoxicity, yielding a selectivity index (CC₅₀/MIC) of -2.7. Singleton 18 was about equally potent at inhibiting both B. anthracis and S. aureus helicases, but failed to produce a detectable MIC versus either species. Computational searches of the screening library database revealed no analogs with 90% or better similarity to any of the three singletons. Further characterization of the coumarin-tvpe helicase inhibitors

Based on results from these secondary assays, three of the validated helicase inhibitors, Compounds 2, 12, and 17, were studied further. However, the coumarin-type inhibitor, Compound 2, is clearly the most potent and selective (CC_5O > 100 μM; helicase IC₅₀ -8 μM; MIC -6-12 μM, or -3-6 μg/ml, Table 4). Further characterization of this inhibitor, including mode of inhibition, determination of an inhibition constant (Kj), cidality, antibacterial spectrum, and preliminary SAR studies are described below. Mode of inhibition and K, determination

Since the aminocoumarins coumermycin and novobiocin are known to inhibit the DNA gyrase function in DNA replication by competing with ATP binding (Sugino et al., Proc. Natl. Acad. Sci. USA, 75: 4838 (1978)), the mode of inhibition of the coumarin-type helicase inhibitor Compound 2 was examined. Assays of the single-stranded (ss) DNA- stimulated ATPase activity of B. anthracis replicative helicase in the presence of Compound 2 revealed only weak inhibition (16% inhibition in the presence of 100 μM Compound 2), indicating that this coumarin-type helicase inhibitor has little or no effect on ATP binding or hydrolysis by helicase in the absence of its duplex DNA substrate. Helicase kinetic studies with varying concentrations of ATP substrate and inhibitor suggested that Compound 2 is uncompetitive with ATP at low inhibitor concentrations (Fig. IA). However, a Dixon plot analysis (1/V versus [I]) of the data was non-linear at inhibitor concentrations above the K₁ at the two lower ATP concentrations tested, suggesting a possible second mode of inhibition at high Compound 2 concentrations. Helicase kinetic studies with varying oligonucleotide substrate and inhibitor concentrations demonstrated that Compound 2 exhibits a mixed competitive-uncompetitive mode of inhibition with the DNA substrate (Fig. IB), and a Dixon plot indicates a K₁ inhibition constant of ~8 μM (Fig. 1C). In addition, two coumarins (Compounds 1 and 2) as well as one triazine (Compound 12) helicase inhibitor were tested and failed to inhibit gyrase at concentrations of up to 50 μM (data not shown). This is not surprising since they lack the noviosyl sugar linkage, which is important for gyrase-inhibiting coumarins (Hooper et al., Antimicrob. Agents Chemother., 22: 662-671 (1982); Pi et al., Proc. Natl. Acad. Sci. USA, 101: 10036-10041 (2004)). By contrast, two known gyrase-inhibiting coumarins, coumermycin and novobiocin, were potent inhibitors of gyrase in the assay (IC₅₀ values < 1 μM) but did not inhibit B. anthracis helicase (data not shown). Thus, while these helicase inhibitors share a portion of the coumarin scaffold, they do not share a common biological mechanism. Bactericidality

Inhibitors of DNA replication such as the fluoroquinolones and the anilinouracils are typically bactericidal (Daly et al., kntimicrob. Agents Chemother., 44: 2217-2221 (2000); Hooper, Clin. Infect. Dis., 32 Suppl. 1: S9-S15 (2001)) because successful replication and segregation of the chromosome into each cell is essential for viability. The coumarin-type helicase inhibitor Compound 2 was examined and proved to be rapidly bactericidal within 5.5 hr at 4 x MIC (Fig. 2). Initial structure-activity-relationship (SAR) of the coumarin chemotype

To probe the structure-activity relationship for Compounds 1 and 2, nineteen additional structurally related compounds were ordered from the vendor Chembridge, Inc., and examined against B. anthracis helicase (Table 5).

aIC5o versus B. anthracis replicative helicase

The core bicyclic ring, which is common to all five coumarin-type helicase inhibitors identified in screening, was not altered but some changes to all of the substituents were examined. The SAR results are summarized schematically in Fig 3A. Only one alteration increased the helicase inhibitory activity of Compound 2, replacement of the substituted phenyl ring for an unsubstituted naphthyl ring. However, this change was also associated with increased cytotoxicity (Compound 1, Table 4). Loss of the 8 position methyl group or shortening of the 3 position chain by one methylene group modestly reduced activity, but esterification of the 3 position carboxylic acid eliminated activity. These results suggest a pharmacophore representation as shown in Fig. 3B in which the acidic group and the oxygens interact with a hydrophilic region while the 4, 7, and 8 position substituents interact with a hydrophobic region.

As shown in Table 5, above, Compound 19 has an IC₅₀ for the B. anthracis helicase of 25 μM. Compound 19 also has an MIC of 40 μM (16 μg/ml) versus cells of B. anthracis Sterne strain and an MIC of 90 μM (35 μg/ml) versus cells of S. aureus Smith strain. These values are similar to those for other preferred helicase inhibitor compounds of the invention (see, e.g., Compound 12, in Table 4). Accordingly, Compound 19 is also considered a preferred bacterial helicase inhibitor of the invention. Spectrum of antibacterial activity

The antibacterial spectra of the two related coumarin inhibitors, Compounds 1 and 2, as well as that of the triazine inhibitor, Compound 12, were examined. See, Table 6, below. Table 6. Average MICs (μg/ml) for Helicase Inhibitor Compounds 1, 2, and 12.

aMIC (μg/ml) for helicase inhibitor Compound 1 versus indicated bacterial strain; MIC (μg/ml) for helicase inhibitor Compound 2 versus indicated bacterial strain; ⁰MIC (μg/ml) for helicase inhibitor Compound 12 versus indicated bacterial strain

The results in Table 6 reveal considerable breadth of activity by the helicase inhibitors across the Bacillus genus, with some limited activity against S. aureus. Only the triazine Compound 12 displayed any activity against E. coli, and that was only in the tolC efflux- deficient strain. The Gram-negative outer membrane barrier or efflux may rescue E. coli cells from inhibition by these compounds. These results indicate that the helicase inhibitor compounds are effective for inhibiting growth of a variety of important bacterial species, including species of important pathogenic bacteria.

All publications, patent applications, patents, and other documents cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Obvious variations to the disclosed compounds and alternative embodiments of the invention will be apparent to those skilled in the art in view of the foregoing disclosure. All such obvious variants and alternatives are considered to be within the scope of the invention as described herein.

Claims

What is claimed is:

1. A method for inhibiting growth of bacterial cells on a surface comprising the step of applying to said surface a composition comprising at least one bacterial helicase inhibitor compound selected from:

Compound 1 -[4,8-dimethyl-7-(naphthalen- 1 -ylmethoxy)-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 2 -[7-(4-bromo-2-fluorobenzyloxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 3 -benzyl-7,8-dihydroxy-4-methyl-2H-chromen-2-one

Compound 5 6,7-dihydroxy-4-[(l-methyl-l/i-benzimidazol-2-ylthio)methyl]-2H-chromen-2-one

Compound 12

N-{4-[N-(3 ,4-dimethylisoxazol-5-yl)sulfamoyl]phenyl} -2-(5,6-diphenyl- 1 ,2,4-triazin-3- ylthio)acetamide

Compound 16 2-[5-(5-mercapto-l,3,4-oxadiazol-2-yl)furan-2-yl]benzene-l,4-diol

Compound 17 6-ethyl-9-mercapto-6-methyl-5,6-dihydrobenzo[/?][l,2,4]triazolo[3,4-ό]quinazolin-7(12H)- one

Compound 19

2-[4,8-dimethyl-7-(naphthalen-2-ylmethoxy)-2-oxo-2H-chromen-3-yl]acetic acid, salts thereof, and combinations thereof.

2. The method according to Claim 1, wherein said bacterial cells are cells of Bacillus, Staphylococcus, or a combination thereof.

3. A composition for inhibiting bacterial growth on a surface comprising at least one bacterial helicase inhibitor compound selected from:

Compound 1 -[4,8-dimethyl-7-(naphthalen- 1 -ylmethoxy)-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 2 -[7-(4-bromo-2-fluorobenzyloxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl]propanoic acid

Compound 3 3-benzyl-7,8-dihydroxy-4-methyl-2H-chromen-2-one

Compound 5 6,7-dihydroxy-4-[(l-methyl-lH-benzimidazol-2-ylthio)methyl]-2H-chromen-2-one

Compound 12 iV-{4-[jV-(3,4-dimethylisoxazol-5-yl)sulfamoyl]phenyl}-2-(5,6-diphenyl-l,2,4-triazin-3- ylthio)acetamide

Compound 16 2-[5-(5-mercapto- 1 ,3,4-oxadiazol-2-yl)furan-2-yl]benzene- 1 ,4-diol

Compound 17 6-ethyl-9-mercapto-6-methyl-5,6-dihydrobenzo[Λ][l,2,4]triazolo[3,4-Z?]quinazolin-7(12H)- one

Compound 19

2-[4,8-dimethyl-7-(naphthalen-2-ylmethoxy)-2-oxo-2H-chromen-3-yl]acetic acid, salts thereof, and combinations thereof.

4. The composition according to Claim 3, wherein said composition comprises one, two, or three of the listed bacterial helicase inhibitor compounds to the exclusion of the other compounds listed therein.

5. The composition according to Claim 3, wherein said composition comprises a bacterial helicase inhibitor compound selected from the group consisting of Compound 1, Compound 2, Compound 12, and combinations thereof.

6. The composition according to Claim 3, wherein said composition comprises bacterial helicase inhibitor Compound 1 to the exclusion of the other compounds listed therein.

7. The composition according to Claim 3, wherein said composition comprises bacterial helicase inhibitor Compound 2 to the exclusion of the other compounds listed therein.

8. The composition according to Claim 3, wherein said composition comprises bacterial helicase inhibitor Compound 12 to the exclusion of the other compounds listed therein.

9. The composition according to any one of Claims 3-8, wherein said composition further comprises a carrier.

10. The composition according to Claim 9, wherein said carrier is an organic solvent.

11. The composition according to Claim 10, wherein said organic solvent is selected from the group consisting dimethyl sulfoxide (DMSO), an alcohol, N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), and combinations thereof.

12. The composition according to Claim 9, further comprising a dispersing agent.

13. The composition according to Claim 12, wherein said dispersing agent is a surfactant.

14. The composition according to Claim 9, further comprising an additional active agent.

15. The composition according to Claim 14, wherein said additional active agent is selected from the group consisting of an antibacterial agent other than said helicase inhibitor compound, an antifungal agent, an antiviral agent, an anticancer agent, and combinations thereof.