WO2010056914A1 - Composés inhibiteurs de l'hélicase bactérienne et leurs utilisations - Google Patents

Composés inhibiteurs de l'hélicase bactérienne et leurs utilisations Download PDF

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WO2010056914A1
WO2010056914A1 PCT/US2009/064270 US2009064270W WO2010056914A1 WO 2010056914 A1 WO2010056914 A1 WO 2010056914A1 US 2009064270 W US2009064270 W US 2009064270W WO 2010056914 A1 WO2010056914 A1 WO 2010056914A1
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compound
helicase
bacterial
compounds
chromen
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PCT/US2009/064270
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WO2010056914A8 (fr
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Donald T. Moir
Daniel Aiello
Marjorie H. Barnes
John D. Williams
Subhasis B. Biswas
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Microbiotix, Inc.
University Of Medicine And Dentistry Of New Jersey
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Publication of WO2010056914A1 publication Critical patent/WO2010056914A1/fr
Publication of WO2010056914A8 publication Critical patent/WO2010056914A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/50—1,3-Diazoles; Hydrogenated 1,3-diazoles
    • A01N43/52—1,3-Diazoles; Hydrogenated 1,3-diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
    • A01N43/707—1,2,3- or 1,2,4-triazines; Hydrogenated 1,2,3- or 1,2,4-triazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/82—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with three ring hetero atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • This invention is in the field of antibacterial compounds.
  • 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.
  • 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.
  • 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.
  • 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:
  • a bacterial inhibitor compound of the invention has the structure of Compound 1, Compound 2, or Compound 12.
  • a bacterial helicase inhibitor compound described herein inhibits the activity of a helicase of one or more Gram-positive bacterial species or strains.
  • 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.
  • 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.
  • 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.
  • compositions and methods comprising one or more of the helicase inhibitor compounds described above for inhibiting the growth of bacteria on a surface.
  • 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.
  • compositions and methods comprising a helicase inhibitor compound described herein for inhibiting growth of cells of Escherichia coli on a surface.
  • 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.
  • 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.
  • 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).
  • DMSO dimethyl sulfoxide
  • 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.
  • a composition for applying a helicase inhibitor compound topically on the skin of an individual 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.
  • 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.
  • a lock solution i.e., solution or suspension
  • 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.
  • 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 ( ⁇ ), and 5 mM (O) ATP substrate present. Fig. IB shows a Lineweaver-Burk plot with 12.5 ⁇ M (D), 6.25 ⁇ M (0), 3.13 ⁇ M ( ⁇ ), and 0 ⁇ M (O) inhibitor present. Fig.
  • FIG. 1C shows a Dixon plot with 5 nM (0), 10 nM (D), 30 nM ( ⁇ ), 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 ( ⁇ ), 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.
  • 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.
  • the cytotoxicity concentration designated “CC 50" 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.
  • the mammalian cells are HeLa cells for determining the CC 50 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 CC 50 value could falsely indicate that the compound is less toxic that it actually is toward mammalian cells.
  • CC 50 values for a compound of interest in cultures of mammalian cells grown in the presence and the absence of fetal bovine serum.
  • the CC 50 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.
  • 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 ⁇ M or less than 50 ⁇ g/ml for at least one of these bacterial strains.
  • SI selectivity index
  • CC 50 mammalian cell cytotoxicity
  • SIs a B. anthracis
  • SIs a S. aureus
  • 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 CC 50 and MIC, it is important to avoid potential artifacts such as discussed above with respect to determining an accurate CC 50 value.
  • CC 50 values determined from cultures grown in the presence and the absence of serum so that a difference in cytotoxicity (i.e., between CC 50 values) will also be reflected in the SI values. See, Table 4, below.
  • the "half-maximal inhibitory concentration" or "IC 5 o" is the concentration of a compound required for 50% inhibition of the maximal activity of an enzyme.
  • 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 IC 50 may be calculated using a preparation of a bacterial helicase or a preparation of permeabilized cells that possess a helicase. See, Table 4, below.
  • 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.
  • a biological fluid e.g., blood, lymph, saliva, sputum, urine, perspiration
  • the pathogenic bacteria on the fomite are then passed to another human or other animal that comes into contact with the contaminated fomite.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ M or less than 50 ⁇ g/ml for at least one of these bacterial strains):
  • 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 IC 50 with respect to a B. anthracis helicase or an S. aureus helicase of 25 ⁇ M or less.
  • 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 IC 50 for B. anthracis helicase and a structure-activity-relationship (SAR) analysis compared to that of Compounds 1 and 2 (see, Table 5).
  • 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 (IC 50 of less than or equal to 25 ⁇ M, 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 ⁇ M) 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).
  • 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.
  • inhibiting growth of bacteria on a surface comprises bringing a helicase inhibitor described herein into contact with bacterial cells present on the surface.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 ⁇ g/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.
  • 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.
  • NMP N-methylpyrrolidone
  • DMA N,N-dimethylacetamide
  • ethanol is more preferred than isopropanol, which is more preferred than butanol or an aryl alcohol, which are more preferred than methanol.
  • conventional solid carriers 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.
  • 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.
  • the alkyl and fatty groups in an anionic surfactant are, independently, 8 to 22 carbon atoms, and more preferably 10 to 18 carbon atoms.
  • 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
  • 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).
  • 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-alkano
  • 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
  • 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.
  • 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.
  • 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.
  • a helicase inhibitor described herein to inhibit growth of bacteria on the surface.
  • 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.
  • a composition comprising a helicase inhibitor described herein 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.
  • a helicase inhibitor as described herein may be employed in a "lock solution" (solution or suspension) for use with a central venous catheter (CVC).
  • a lock solution solution or suspension
  • CVC central venous catheter
  • 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.
  • an anti-bacterial agent e.g., antiseptic, antibiotic
  • 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.
  • 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.
  • a helicase inhibitor described herein may be incorporated into a resin prior to polymerization to form the plastic.
  • 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.
  • a helicase inhibitor may also be covalently bound to plastic using an appropriate cross-linking agent.
  • a helicase inhibitor may be impregnated into a material, such as a hydrogel or polymer, which would then be used a surface.
  • 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.
  • 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%.
  • 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%.
  • 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.
  • B. anthracis dnaBgerxQ 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.
  • Protein was precipitated from the cell extract by addition of 0.25 g/ml (NH 4 ) 2 SO 4 , resuspended in buffer A (25 mM Tris-HCl, (pH 7.5), 5 mM MgCl 2 , 10% glycerol, 5 mM DTT) and re-precipitated in 0.2 g/ml (NH 4 ) 2 SO 4 .
  • buffer A 25 mM Tris-HCl, (pH 7.5), 5 mM MgCl 2 , 10% glycerol, 5 mM DTT
  • 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.
  • MgCl 2 0.5 mM dithiothreitol (DTT), 0.01% TRITON® X-100 non-ionic surfactant (t- octylphenoxypolyethoxyethanol, Sigma-Aldrich, St. Louis, Missouri, US), and 25 mM
  • 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.
  • 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-Ni 2+ column equilibrated in the same buffer. Following a wash in the same buffer, the column was eluted in a 0-200 mM imidazole gradient.
  • buffer B 50 mM Tris pH 7.5; IM NaCl; 10% glycerol; 2 mM 2-mercaptoethanol
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 10 radiolabeled 60-mer
  • a standard 20 ⁇ l reaction volume contained 25 mM Tris-HCl, (pH 7.5), 10 raM MgCl 2 , 10% glycerol, 5 mM DTT, and 0.1 niM ATP, 17 fmol (1-2 x 10 4 cpm/ ⁇ l) of substrate and the indicated amount of helicase.
  • the mixtures were incubated at 3O 0 C for 15 minutes, and the reactions were terminated by the addition of 4 ⁇ l of 2.5% sodium dodecyl sulfate (SDS), 60 mM EDTA, and 1% bromophenol blue. Displacement was monitored by polyacrylamide gel electrophoresis, followed by autoradiography.
  • Screening wells contained 30 ⁇ l volume consisting of -80 ⁇ M 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 MgCl 2 , 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.
  • 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 ⁇ -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.
  • the core is elaborated by alkylating the remaining phenolic hydroxy group and saponifying the ester to the corresponding acid.
  • the coumarin core is constructed by condensation of a ⁇ -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.
  • the triazinyl thioacetic acid core is constructed via a nucleophilic displacement of an intermediate chlorotriazene.
  • an aminophenyl sulfonamide is made from 4-nitrophenylsulfonyl chloride. Peptide coupling of the two halves leads to the final compound.
  • Suzuki coupling
  • the core system is elaborated stepwise by the addition of a metallated toluene to an ⁇ , ⁇ -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
  • Kj and IC 50 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 IC 50 values were determined in duplicate using a 10-point curve consisting of two-fold dilutions of inhibitory compound from 100 ⁇ M to 0.2 ⁇ M. 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 IC 50 values over a range of substrate and inhibitor concentrations. Ethidium bromide ( ⁇ tBrVdisplacement
  • MICs were expressed in ⁇ M to facilitate comparisons with IC 50 and CC 50 values and were determined in duplicate using a 10-point curve consisting of two-fold dilutions of inhibitory compound from 100 ⁇ M to 0.2 ⁇ M.
  • 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
  • cytotoxic concentration (CC 50 ) of a compound versus cultured mammalian cells 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 ueO us Nonradioactive Cell Proliferation Assay (MTS), Promega, Madison, Wisconsin, US). Briefly, 96-well plates were seeded with HeLa cells at a density of 4 x 10 3 per well in minimal essential medium (Catalog No.
  • the "selectivity index" (SI) of a given agent is defined as the ratio of its mammalian cell cytotoxicity (CC 50 ) determined in medium containing 10% fetal bovine serum to its MIC value against B. anthracis (SI ⁇ 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-SI ⁇ a)- Overview of screening results
  • MBX Microbiotix, Inc.
  • NSRB National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Disease
  • C coumarin
  • B benzothiazole
  • R rhodanine
  • T triazine
  • NP N-phenyl pyrrole (unstable)
  • S singleton
  • subtilis replicative DNA polymerase Pol HIC (IC 50 values >100 ⁇ M) confirming their selectivity for helicase.
  • helicase inhibitors they inhibit DNA replication in permeabilized /? ⁇ L4- 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 ⁇ M.
  • Compound 2 inhibits the growth of B. anthracis cells with an MIC of 6 ⁇ M, resulting in an overall selectivity index (SI, CC 50 /MIC) > 16.
  • Inhibitors with a benzothiazole core structure As expected for helicase inhibitors, they inhibit DNA replication in permeabilized /? ⁇ L4- deficeint B. anthracis Sterne cells, but the potency of Compounds 1 , 4, and 5 is less than that exhibited in the in vitro
  • B. anthracis helicase inhibitors 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 IC 50 and displayed low cytotoxicity, yielding a selectivity index (CC 50 /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
  • 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).
  • SAR structure-activity-relationship
  • 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.
  • Compound 19 has an IC 50 for the B. anthracis helicase of 25 ⁇ M.
  • Compound 19 also has an MIC of 40 ⁇ M (16 ⁇ g/ml) versus cells of B. anthracis Sterne strain and an MIC of 90 ⁇ M (35 ⁇ g/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
  • aMIC ( ⁇ g/ml) for helicase inhibitor Compound 1 versus indicated bacterial strain MIC ( ⁇ g/ml) for helicase inhibitor Compound 2 versus indicated bacterial strain

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Abstract

La présente invention concerne des composés organiques qui inhibent l'activité hélicase bactérienne et la croissance de cellules bactériennes sur des surfaces.
PCT/US2009/064270 2008-11-12 2009-11-12 Composés inhibiteurs de l'hélicase bactérienne et leurs utilisations WO2010056914A1 (fr)

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CN109320483A (zh) * 2017-08-01 2019-02-12 南京大学 香豆素衍生物、其制备方法及其作为药物的用途
WO2019210103A3 (fr) * 2018-04-25 2020-01-16 The Regents Of The University Of California Inhibiteurs de slc26a3 et utilisation associée
WO2020115009A1 (fr) * 2018-12-03 2020-06-11 Universität Bern INHIBITEURS DE LPAAT-β POUR LE TRAITEMENT DU CANCER
WO2020205683A1 (fr) * 2019-03-29 2020-10-08 The Scripps Research Institute Dérivés de stabilisation de benzopyrane et d'imidazole utilisés pour la stabilisation de chaînes légères d'immunoglobulines amyloïdogéniques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109320483A (zh) * 2017-08-01 2019-02-12 南京大学 香豆素衍生物、其制备方法及其作为药物的用途
WO2019210103A3 (fr) * 2018-04-25 2020-01-16 The Regents Of The University Of California Inhibiteurs de slc26a3 et utilisation associée
US11591304B2 (en) 2018-04-25 2023-02-28 The Regents Of The University Of California SLC26A3 inhibitors and use thereof
US20230339878A1 (en) * 2018-04-25 2023-10-26 The Regents Of The University Of California Slc26a3 inhibitors and use thereof
WO2020115009A1 (fr) * 2018-12-03 2020-06-11 Universität Bern INHIBITEURS DE LPAAT-β POUR LE TRAITEMENT DU CANCER
WO2020205683A1 (fr) * 2019-03-29 2020-10-08 The Scripps Research Institute Dérivés de stabilisation de benzopyrane et d'imidazole utilisés pour la stabilisation de chaînes légères d'immunoglobulines amyloïdogéniques
US11945806B2 (en) 2019-03-29 2024-04-02 The Scripps Research Institute Stabilization of amyloidogenic immunoglobulin light chains

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