Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
  • C07D239/48—Two nitrogen atoms
  • C07D239/49—Two nitrogen atoms with an aralkyl radical, or substituted aralkyl radical, attached in position 5, e.g. trimethoprim
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/06—Antibacterial agents for tuberculosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
  • C07D239/48—Two nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

• methotrexate has an MIC 50 of 20 ⁇ g/mL or an MIC 90 of 100 ⁇ g/mL.
• the weakly basic, non-classical antifolate trimethoprim (FIG. 1) with weaker DHFR inhibition is in fact a potent antibacterial against both MRSA and E.
• the Ar ring is a phenyl, pyridyl, or pyrimidinyl ring substituted with at least one -COOH or -OCOOH group and optionally substituted with one or more substituents independently chosen from halogen, hydroxyl, amino, Ci-C 6 alkyl, Ci-C 6 alkoxy, Ci- C 2 haloalkyl, and Ci-C 2 haloalkoxy.
• FIGURE 1 Antibacterial agents effective against Gram-positive or Gram- negative bacteria with relevant physiological properties.
• a previous PLA, UCP1021, is compared to a COOH-PLA.
• substituted means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded.
• an oxo group substitutes a heteroaromatic moiety the resulting molecule can sometimes adopt tautomeric forms.
• a pyridyl group substituted by oxo at the 2- or 4-position can sometimes be written as a pyridine or hydroxypyridine.
• a "dosage form” means a unit of administration of an active agent.
• dosage forms include tablets, capsules, injections, suspensions, liquids, emulsions, creams, ointments, suppositories, inhalable forms, transdermal forms, and the like.
• a "patient” is a human or non-human animal in need of medical treatment. In some embodiments the patient is a human patient.
• Certain PLAs of this disclosure are active against Myobacterium tuberculosis (Mtb) DHFR. DHFR is not currently invoked for TB therapy. Methotrexate, pyrimethamine and trimetrexate, clinically approved antifolates, are potent inhibitors of the MtbDHFR enzyme but fail to inhibit the growth of Mtb, most likely due to an inability to permeate the lipid-rich cell wall. Certain PLAs of this disclosure inhibit the MtbDHFR enzyme activity and also inhibit the growth of live Mtb. Several of the compounds potently inhibit the growth of Mtb with MIC values less than 1 ⁇ g/mL. Certain compounds of the disclosure are very potent inhibitors of the growth of MDR- and XDR-TB strains and are not subject to cross-resistance with other known mechanisms.
• Trimethoprim inhibits dihydrofolate reductase while sulfamethoxazole inhibits dihydropteroate synthase. Both of these essential enzymes are involved in the folate biosynthetic pathway, which is critical for the creation of one-carbon donors in metabolism. While this combination has had wide success, resistant strains have become common.
• Mutation of the dfrB chromosomal gene is a principal mode of trimethoprim resistance.
• Additional mechanisms of resistance include the acquisition of plasmid-encoded
• Point mutations in dfrB confer resistance with MIC values ⁇ 256 ⁇ g/mL; acquisition of S I DHFR confers greater levels of resistance with MIC values > 512 ⁇ g/mL.
• variables in formula I can carry the definitions set forth in the SUMMARY section. Additionally these variables can carry any of the definitions set forth below.
• J and M are independently selected from H, halogen, hydroxyl, Ci-C 6 alkyl, Ci-C 6 alkoxy, Ci-C 2 haloalkyl, and Ci-C 2 haloalkoxy.
• the disclosure includes a pharmaceutical composition containing at least one compound of formula I as the active agent together with a pharmaceutically acceptable carrier.
• compositions of the disclosure include ocular, oral, nasal, transdermal, topical with or without occlusion, intravenous (both bolus and infusion), inhalable, and injection (intraperitoneally, subcutaneously, intramuscularly or parenterally) formulations.
• the composition may be in a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, sterile ocular solution, parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto-injector device, or suppository; for administration ocularly, orally, intranasally, sublingually, parenterally, or rectally, or by inhalation or insufflation.
• the dosage form containing the composition of the disclosure contains an effective amount of the active agent necessary to provide a therapeutic effect by the chosen route of administration.
• the composition may contain from about 5,000 mg to about 0.5 mg (preferably, from about 1,000 mg to about 0.5 mg) of a compound of the disclosure or salt form thereof and may be constituted into any form suitable for the selected mode of administration.
• the dosage form may be formulated for immediate release or controlled release, including delayed release or sustained release.
• the pharmaceutical composition may include a compound for formula I as the only active agent or may be combined with one or more additional active agents.
• the pharmaceutical composition includes a compound of formula I and at least one direct acting antibiotic (a compound efficacious for killing pathogenic bacteria in vivo).
• the disclosure includes a method of treating a bacterial infection in a subject by administering an effective amount of one or more compounds of the disclosure to a subject at risk for a bacterial infection or suffering from a bacterial infection.
• the disclosure includes a method of treatment in which a compound of formula I is used to treat a bacterial infection and methods in which a compound is used to sensitize bacteria to an antibacterial agent.
• a compound of formula I is administered to a patient having a bacterial infection, simultaneously or sequentially, with a therapeutically effective amount of the antibacterial agent.
• the compound of formula I increases the efficacy, often by lowering the MIC, of the other antibacterial agent.
• the bacterial infection or antibiotic-tolerant or antibiotic -resistant infection is caused by a Gram-positive bacterium.
• the bacterial infection or antibiotic-tolerant or antibiotic -resistant infection is caused by a Gram-negative bacterium.
• the bacterial infection is a Mycobacterium tuberculosis (Mtb).
• Mtb Mycobacterium tuberculosis
• the Mycobacterium tuberculosis infection is a strain that is multi-drug resistant, for example a strain resistant to amikacin, kanamycin, and/or capreomycin. Certain compounds disclosed herein inhibit Mtb with MIC values less than ⁇ igl mL.
• Acinetobacter Agrobacterium, Bacillus, Bacteroides, Bartonella, Bordetella, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella, Listeria, Morganella, Moraxella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas, Vibrio, and Yersinia.
• Such bacteria include Vibrio harveyi, Vibrio cholerae, Vibrio parahemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum, Pseudomonas aeruginosa, Yersinia enterocolitica, Escherichia coli,
• Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, Burkholderia cepacia, Acinetobacter baumannii, Staphylococcus epidermidis, and Staphylococcus aureus.
• the infection is a yeast infection, such as Candida albicans.
• Klebsiella e.g., Klebsiella pneumoniae
• Acinetobacter e.g., A.
• cephalosporins cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, quinolones including fluoroquinolones and similar DNA synthesis inhibitors, tetracyclines,
• aminoglycosides macrolides, glycopeptides, chloramphenicols, glycylcyclines, lincosamides, lipopeptides, lipodepsipeptides, such as daptomycin, and oxazolidinones.
• the subject is a mammal, e.g., a human or non-human mammal.
• the methods include treating one or more cells, e.g., cells in a culture dish.
• the present disclosure features a method of treating a Gram- negative infection in a subject, the method comprising administering to said subject in need of such treatment a therapeutically effective amount of a compound described herein.
• the Gram-negative infection is caused by Pseudomonas aeruginosa.
• the disclosure includes treating an infection caused by Gram- positive bacteria, such as Staphylococcus epidermidis and Staphylococcus aureus.
• the subject is a trauma patient or a burn patient suffering from a burn or skin wound.
• the present disclosure features a method of reducing bacterial tolerance in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound described herein.
• the method further includes identifying said subject suffering from an infection with bacteria resistant to antimicrobial therapy.
• the disclosure includes methods of treatment in which a compound or composition of the disclosure is administered orally, topically, intravenously, or parenterally, or is inhaled.
• a compound of the disclosure may be administered about 1 to about 5 times per day. Daily administration or post-periodic dosing may be employed. Frequency of dosage may also vary depending on the compound used, the particular disease treated and the bacteria causing the disease. It will be understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
• Inventors have established DHFR inhibition as a mechanism for cell growth inhibition. Certain compounds of the disclosure were also tested for antibacterial activity in two M. smegmatis strains that overexpress DHFR under the control of two different promoters. All compounds, with the exception of UCP 1066, have significantly increased MIC values of at least 32 ⁇ g/mL in both strains, indicating that the antibacterial activity is likely due to DHFR inhibition. This compound has an MIC value of 4 ⁇ g/mL in the overexpressing strains, which is the same as its value against the Erdmann strain. [0088] Inventors expressed and purified M.
• tuberculosis DHFR tuberculosis DHFR (MtbDHFR) protein and measured enzyme inhibition (See Table 6, Example 10) using standard procedures that spectroscopic ally follow the oxidation of the cofactor NADPH at 340 nm. Experiments were performed in triplicate and verified on two different dates. Many of the inhibitors tested in the assay are moderately potent against the MtbDHFR enzyme with IC 50 values in the range 70-600 nM. The range of IC 50 values is relatively narrower than the range of MIC values, again suggesting that activity against the enzyme is only one of the factors that drives antibacterial potency. Table 6 shows that most of the compounds have very similar IC 50 values against the human enzyme.
• PLAs of the disclosure have also been assessed: five multi-drug resistant TB strains, Mtb 365, Mtb 276, MTb 352, Mtb 56 and Mtb C-31 and one extensively drug- resistant (XDR) strain, Mtb 5. See Table 7, Example 10. Overall, each of the strains are resistant to INH with MIC values between 0.25-4 ⁇ g/mL. Three of the strains, Mtb 5, Mtb 365 and Mtb 56, are also highly resistant to rifampin with MIC values of 8, 64 and 32 ⁇ g/mL, respectively. The strains are also resistant to a variety of other agents including ethambutol, streptomycin and moxifloxacin.
• the PLAs are active against the majority of the MDR strains, albeit at slightly decreased levels. The compounds did not show activity against strain C-31. Excitingly, compounds 58 and 59 are very active against the MDR strain (Mtb 352) and XDR strains (Mtb 5) with MIC values of 0.06 or 0.5 ⁇ g/mL, respectively, against Mtb 352 and 0.25 and 2 ⁇ g/mL against Mtb 5.
• Protein was desalted using a PD-10 column (GE Healthcare) into buffer containing 20 mM Tris pH 7.0, 20 % glycerol, 0.1 mM EDTA, 2 mM DTT and stored in aliquots at -80 °C.
• Inhibitor in DMSO was added to the enzyme/NADPH mixture and incubated for 5 minutes prior to the addition of 0.1 mM dihydrofolate in 50 mM TES.
• the number provided in parenthesis in the SaDHFR and EcDHFR IC50 columns represents the fold selectivity over inhibition of the human DHFR enzyme.
• Synchrotron Radiation Lightsource SLAC National Accelerator Laboratory. Data were indexed and scaled using HKL2000. Phaser crystallographic software (McCoy, A.J. et al., J. Appl. Cryst., (2007) 40:658-674) was used to identify molecular replacement solutions using PDB ID: 3F0Q as a probe. Coot and Phenix were used for structure refinement until acceptable Rwork and R Fre e were achieved.
• Purified SaDHFR was co-crystallized with 2 mM NADPH and ImM 16 in DMSO via hanging drop method. Crystallization details were similar to those used above except for a change in buffer to 0.1M MES, pH 5.0. Data were collected on the Rigaku Highflux Homelab system at the University of Connecticut's Protein X-Ray Crystallography Facility. Data were indexed and scaled using Structure Studio (d*Trek). Similar to above, Phaser was used for molecular replacement; Coot (Emsley, P. and Cowtan, K., Acta Crysta., (2004) D50: 2126-2132) and Phenix (Adams, P.D., Acta Cryst, (2010) D66: 213-221) were used for structure refinement.
• COOH-PLA compounds of the disclosure To examine the drug-like potential of the COOH-PLA compounds of the disclosure, a series of in vitro assays probed their effects on human cells, their inhibition of critical CYP isoforms and their lifetime in microsomal stability assays.
• the COOH-PLAs have no measurable cytotoxicity (IC 50 > 500 ⁇ ) toward HepG2 and MCF-10 and cells. Coupling the potent activity against Gram-positive bacterial cells with low cytotoxicity yields a high therapeutic index (>500,000).
• Applicants have also measured cytochrome P450 inhibition for certain compounds of the disclosure.
• Isoniazid (INH) was purchased from Sigma Chemical Co., St. Louis, MO. and dissolved in 100 % dimethyl sulfoxide (DMSO) to a concentration of 1 mg/ml prior to freezing at -20°C.
• DMSO dimethyl sulfoxide
• the DHFR inhibitors were dissolved in 100 % DMSO to a concentration of 20 mg/ml.
• M. tuberculosis ATCC 35801 (strain Erdman) was obtained from the
• the mycobacterial isolates were grown in modified Middlebrook 7H10 broth (pH6.6; 7H10 agar formulation with agar and malachite green omitted) supplemented with 10% Middlebrook oleic acid-albumin-dextrose-catalase enrichment (Difco Laboratories, Detroit, MI) and 0.05% Tween 80 on a rotary shaker at 37°C for 5-10 days.
• the organisms were diluted in 7H10 broth to 1 Klett unit (equivalent to about 5 X 10 5 CFU/ml) using a Photoelectric Colorimeter (Manostat Corp., New York, NY) for use in the broth dilution assay.
• Polystyrene 96-well round-bottom microtiter plates (Corning Inc., Corning, NY) were filled with 50 ⁇ of modified 7H10 broth. The compounds were prepared at 4 times the maximum concentration at which they were to be tested and then were added to the first well prior to being serially diluted 2-fold. INH was tested using a range of concentrations from 8 ⁇ g/ml- 0.008 ⁇ g/ml. The DHFR inhibitors were tested using a range of 32 ⁇ g/ml to 0.03 ⁇ g/ml. The inocula used for each strain were measured by titration and plated on 7H10 agar plates to determine the actual inocula.
• the 7H10 agar plates were incubated at 37 °C for 4 weeks. Fifty microliters of the inocula was added to each well containing compound to yield an initial concentration of about 2.5 x 10 5 CFU/ml (range for various isolates tested was 1.25 X 10 6 CFU/ml - 8 X 10 4 CFU/ml).
• the microtiter plates were covered with SealPlate adhesive sealing film (Exel Scientific, Wrightwood, CA) and were incubated at 37 °C in ambient air for 14-21 days prior to reading. Each isolate was tested in duplicate. The MIC was defined as the lowest concentration of antimicrobial agent yielding no visible turbidity. Expression and purification ofM. tuberculosis and human DHFR
• BL21(DE3) competent E. coli cells (New England BioLabs) were transformed with recombinant pET-41a(+) plasmid harboring the dfrA gene constructed by GenScript. Transformed cells were grown in LB medium supplemented with 30 ⁇ g/mL kanamycin at 37°C until the OD 60 o reached 0.6-0.7. The cells were induced with ImM IPTG for 20 h at 20°C and spun down at 8000 rpm for 15 minutes.
• Enzyme activity and inhibition assays were performed by monitoring the NADPH-dependent reduction of dihydrofolate catalyzed by the DHFR enzyme.
• the rate of NADPH oxidation was measured spectrophotometrically at 340 nm in assay buffer containing 20 mM TES pH 7.0, 50 mM KCl, 10 mM 2-mercaptoethanol, 0.5 mM EDTA and 1 mg/mL bovine serum albumin. All measurements were performed at room temperature by adding pure enzyme (2mg/mL), 100 ⁇ NADPH and 100 ⁇ DHF to the buffer.
• inhibitors dissolved in 100% DMSO, were added to the mixture and incubated for 5 minutes before the addition of DHF. Average IC 50 values and standard deviations were measured in triplicate.
• IC 5 o's for certain PLA compounds at Mtb and human DHFR as well as Mtb MIC's is provided in Table 6.
• Trimethoprim (TMP) an non-DHFR inhibiting antibiotic was used as a negative control.
• DHFR inhibitors Isoniazid (INH) and trimetrexate were used as positive controls.
• Resistant strains were selected by plating 100 ⁇ ⁇ of overnight culture (approx. 1012 CFU/mL) of progenitor strain on Isosensitest (Oxoid) agar plates containing 6x MIC of 1 and incubated at 37 °C for 18 hours. Single colonies were isolated and the dfrB gene was identified by directly sequencing the colony PCR product. For colony PCR, cells were lysed using 1 mg/mL lysostaphin and 20 ⁇ g/mL proteinase K in 0.1 M Tris, pH 7.5.
• the gene was amplified using sense primer (5 ' - ATGACTTTATCCATTCTAGTTGC-3 ' ) , anti-sense primer (5 ' -TTATTTTTTACGAATTAAATGTAG-3 ' ) and rTaq Polymerase (Takara) following standard PCR conditions. PCR products were purified using Promega Wizard SV Gel and PCR Clean Up system and sequenced using the sense primer.
• the mutational frequency of 1 was determined by the number of resulting colonies divided by the total inoculum (lxlO 11 CFU/mL) for each progenitor strain.
• the frequency of the specific mutations was determined by multiplying the mutational frequency for the inhibitor- strain pair by the frequency of sequenced colonies containing the specific mutation.
• MICs Minimum Inhibitory Concentrations
• Minimum inhibitory concentrations were determined according to Clinical and Laboratory Standards Institute's guideline for Standard Micro-dilution broth20 assay using a final inoculum of 5 x 105 CFU/mL in Isosensitest Broth (Oxoid). The MIC was defined as the lowest concentration of inhibitor to visually inhibit growth. Growth was monitored at A 6 oo after 18 h of incubation at 37 °C. MICs were confirmed, calorimetrically, using Presto Blue (Life Technologies). Isosensitest broth supplemented with 10 ⁇ g/mL thymidine was used to determine any off-target antibacterial activity.
• LB media 50 mL was inoculated with 1 mL of overnight culture. Growth was monitored at A 6 oo every 30 minutes. The doubling time was determined from the linear portion of the growth curve by the following equation:
• the inhibitory effect of the Table 10 compounds was determined using the following DHFR binding assay. Enzyme inhibition assays were performed by monitoring the rate of NADPH oxidation by DHFR via absorbance at 340 nm at room temperature in assay buffer containing 20 mM TES, pH 7.0, 50 mM KC1, 0.5 mM EDTA, 10 mM beta- mercaptoethanol, and 1 mg/mL BSA using 0.1 mM NADPH and 2 ⁇ g/mL enzyme. Inhibitor, in DMSO, was added to enzyme:NADPH mixture and allowed to incubate for 5 minutes before the addition of 0.1 mM DHF in 50 mM TES, pH 7.0. The inhibitor concentration and volume are based on the conditions that result in a 50% reduction in enzyme activity.
• Enzyme kinetics were determined by Lineweaver-Burke plots generated by enzyme activity assays using 12.5-100 ⁇ DHF with 20 ⁇ NADPH for DHF K M and V max or 12.5-100 ⁇ NADPH with 50 ⁇ DHF for NADPH K M .
• K M values were determined by non-linear regression analysis using GraphPad.
• Table 11 discloses the inhibitory constants, Ki (nM), of PLA compounds against WT and mutant DHFR enzymes. TABLE 11
• PLAs A-F, 60, 15, and 29 were more potent against the wild-type strain than TMP and many were more potent against the single mutants (MIC values between 0.078 and 5 ⁇ g/mL). However, PLAs A-F, 60, 15, and 29 also suffered significant losses against the strains with double mutations in DHFR. PLAs 16, 31, and 32 have superior activity against the wild-type strain as well as strains with both single and double mutants.

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