Substituted diphenyl ethers as a novel chemotherapeutic platform against 1

24 Identification of a novel class of anti-Burkholderia compounds is key in 25 addressing antimicrobial resistance to current therapies as well as naturally 26 occurring resistance. The FabI enoyl-ACP reductase in Burkholderia is an 27 underexploited target that presents an opportunity for development of a new 28 class of inhibitors. A library of substituted diphenyl ethers were used to identify 29 fabI1 specific inhibitors for assessment in B. pseudomallei ex vivo and murine 30 efficacy models. Active FabI1 inhibitors were identified in a two-stage format 31 consisting of percent inhibition screening and minimum inhibitory concentration 32 (MIC) determination by microbroth dilution method. Each compound was 33 evaluated against the B. pseudomallei 1026b (efflux-proficient) and Bp400 34 (efflux-compromised) strains. In vitro screening identified candidate substituted 35 diphenyl ethers that exhibited MIC less than 1μg/ml and enzyme kinetic assays 36 were used to assess potency and specificity against FabI1 enzyme. These 37 compounds demonstrated activity in a Burkholderia ex vivo efficacy model and 38 two demonstrated efficacy in an acute B. pseudomallei mouse infection model. 39 This work establishes substituted diphenyl ethers as a suitable platform for 40 development of novel anti-Burkholderia compounds that can be used for 41 treatment of melioidosis. 42 43 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom INTRODUCTION 44 Burkholderia pseudomallei is a gram-negative bacteria endemic to Southeast 45 Asia and Northern Australia and is the causative agent of melioidosis. This 46 disease is difficult to manage and often fatal in humans if not aggressively and 47 appropriately treated (1-5). While there are current effective treatment options for 48 Burkholderia infections, there has been increasing concern regarding drug 49 resistant and natural resistant strains that render the current treatment regimen 50 ineffective and negatively impact the ability to treat and manage the disease (2, 51 5-7). Also, mounting evidence indicates B. pseudomallei as an emerging 52 pathogen in other areas of the world and demand for treatment of melioidosis will 53 increase as a result (8-12). These reasons along with the ability of Burkholderia 54 to be easily spread via aerosol and ease of dissemination provide the need to 55 develop novel compounds that inhibit underexploited drug targets. 56 The enoyl-ACP reductase enzyme FabI1 (encoded on chromosome 1) 57 involved in fatty acid elongation in the bacterial fatty acid biosynthesis type II 58 (FASII) pathway is an underexploited drug target in Burkholderia. Our own 59 antibacterial discovery program has demonstrated that substituted diphenyl 60 ethers inhibit the FabI enzyme of a variety of organisms leading to in vivo efficacy 61 (13, 14). The ability of this compound class to exhibit broad-spectrum activity and 62 demonstrate efficacy in vivo make it attractive for development of novel 63 Burkholderia chemotherapeutics. 64 Using our library of substituted diphenyl ethers, our drug discovery efforts 65 have been expanded to assess whether inhibition of the B. pseudomallei enoyl66 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom ACP reductase FabI1 demonstrates efficacy in ex vivo and animal models of 67 infection. To assess the activity of our library of substituted diphenyl ethers we 68 first identified lead inhibitors by whole bacterial screening against B. 69 thailandensis strains E264 and Bt38 (E264 Δ[amrAB-oprA] Δ[bpeAB-oprB]) and 70 followed up with MIC determination using B. pseudomallei strains 1026b (15) and 71 Bp400 (1026b Δ[amrAB-oprA] Δ[bpeAB-oprB]) (16). We then assessed the most 72 potent FabI1 inhibitors in both the Burkholderia ex vivo efficacy model and our 73 rapid animal efficacy model. The use of selective FabI1 inhibitors, efflux mutant 74 strains, and the ex vivo and animal models of efficacy allow us to directly assess 75 whether or not targeted inhibition of FabI1 could be used as a suitable platform 76 for novel anti-Burkholderia therapies. 77 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom MATERIALS AND METHODS 78 79 Kinetics and inhibition of FabI1. FabI1 and octenoyl-CoA (Oct-CoA) were 80 prepared as described previously (17). Inhibition constants (Ki) were analyzed 81 using the standard equation for uncompetitive inhibition. Initial velocities were 82 determined at a fixed substrate concentration and the data were fit to eq 1 using 83 a value of Km (Oct-CoA) = 160 μM. 84 vi/v0 = (Km + S)/(Km + S[1 + I/ Ki]) (1) 85 The parameters vi and v0 are the initial velocities in the presence and absence of 86 inhibitor. The substrate concentration was fixed at 30 μM, and the inhibitor 87 concentration was varied from 0-8000 nM. Data fitting was performed using 88 Grafit 4.0 (Erithacus Software Ltd.). 89 Progress curve analysis was performed as described previously to identify 90 slow-onset inhibitors of FabI1 (13). Assays were performed by adding enzyme (5 91 nM) to solutions containing glycerol (8%, v/v), BSA (0.1 mg/v), DMSO (2%, v/v), 92 Oct-CoA (300 μM), NADH (250 μM), NAD (200 μM) and inhibitor (50 nM). 93 Reactions were monitored until the progress curve became linear, indicating that 94 the steady-state had been reached. In this protocol, the low enzyme 95 concentration and high substrate concentration ensure that substrate depletion 96 was minimized so that the progress curves were approximately linear over a 97 period of 30 min in the absence of inhibitors. 98 Preincubation assays were performed to obtain the true inhibition 99 constants and to determine the preference of slow-onset inhibitors for the 100 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom different cofactor-bound forms of FabI1. Enzyme (10 nM) was preincubated in 101 the presence of a fixed concentration of DMSO (2% v/v), NAD (20–400 μM), 102 NADH (250 μM) and inhibitors (0–2000 nM) for 5 h at 4°C. After warming to 103 25°C, assays were initiated by the addition of Oct-CoA (30 μM). The inhibition 104 constant was calculated as previously described (18). 105 106 Bacteria and screening and minimum inhibitory concentration 107 determination. B. thailandensis E264 (efflux-proficient) (19), B. thailandensis 108 Bt38 (E264 Δ[amrAB-oprA] Δ[bpeAB-oprB]) (kindly provided by H. P. Schweizer 109 Colorado State University), B. pseudomallei 1026b (efflux-proficient) (15), and B. 110 pseudomallei Bp400 (1026b Δ[amrAB-oprA] Δ[bpeAB-oprB]) (16) were grown to 111 an OD600 of ~0.6, frozen at -80C in 10% glycerol and were used as standard 112 bacterial stocks for these studies. For each evaluation bacteria were prepared 113 fresh by growth from the standard stocks on Luria-Bertani (LB) Agar, Miller (BD) 114 grown at 37C for 48-72 h. Bacteria recovered from the LB plates were 115 inoculated in 50 mL LB Broth. Broth cultures were then incubated for 24hrs at 116 37C passed 1:100 and incubated for an additional 6 h at 37C. Bacteria were 117 then diluted in to a concentration of 1x10 colony forming units (CFU)/mL in 118 cation-adjusted Mueller-Hinton broth (caMHB; BD, Franklin Lakes, NJ) and 50 μL 119 of sample was added to each well of the test plate. Substituted diphenyl ethers 120 were screened in a percent inhibition high-throughput fashion against B. 121 thailandensis E264 and B. thailandensis Bt38 in a 96-well plate format to identify 122 lead compounds. All compounds were diluted in caMHB to concentrations of 80 123 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom and 40 μg/mL in a 50 μL volume/well. MIC of candidate compounds was 124 determined against B. pseudomallei 1026b and B. pseudomallei Bp400 by broth 125 microdilution method. For MIC determination, compounds were added to the 96126 well plate starting at either 512 μg/mL in the first column and serially diluted 1:2 127 to column 12 for a final concentration of 0.12 μg/mL caMHB. MIC plates were 128 incubated at 37C for 18 h at which time 10 μL of Alamar Blue (Invitrogen, 129 Carlsbad, California) was added to each well and plates were incubated for an 130 additional 4 h. Reduction of Alamar Blue was determined by absorbance 131 readings at wavelengths of 570 and 600 nm using a microplate reader (Biotek, 132 Winooski, VT). Percent growth reduction was calculated from 133 spectrophotometric readings over the drug concentration series. MIC was 134 determined as the lowest drug concentration to inhibit visibal growth. 135 136 Cytotoxicity assay. A Vero cell line (African green monkey kidney cells) was 137 used to assess potential cytotoxicity similar to previously described (20). Vero 138 cells were maintained in Leibovitz’s (L15) growth media without phenol and 139 supplemented with heat inactivated Newborn Calf serum and Penicillin140 Streptomycin. The cells were grown at 37°C and subcultured weekly. Cell 141 suspensions containing 1.3 X 10 cells per ml and resazurin (21) were added to 142 the 96 well plates. The compound was added and 2 fold serial dilutions were 143 completed with the final volume in the wells 100uL. The plates were incubated 144 for 72 hours at 37°C in the dark. The plates were read at 570nm and 600nm in a 145 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom plate reader and the LD50 (lethal dose causing 50% loss of cell viability) of the 146 compound was calculated using standard procedures. 147 148 Burkholderia ex vivo model of efficacy. Infected RAW 264.7 mouse 149 macrophages (American Type Tissue Collection, Manassas, VA) were used to 150 assess intracellular efficacy of our compounds. RAW cells were cultured in 151 complete medium that consisted of minimal essential medium (Invitrogen), 10% 152 fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine (Invitrogen), 1x 153 nonessential amino acids (Invitrogen), and 0.075% sodium bicarbonate (EMD 154 Science, Gibbstown, NJ). Bacteria were added to 2.5x10 RAW cells per well in 155 a 24-well tissue culture plate at a multiplicity of infection of 5 CFU per cell in a 0.5 156 mL media. The plates were then centrifuged at 2,400 X g for 2 min and placed at 157 37C/5% CO2 to incubate for 1 h. Supernatant was then removed; plate washed 158 once with 2 mL PBS and 1 mL of either 0.35 mg/mL kanamycin (Sigma-Aldrich, 159 St. Louis, MO) for wild-type strains or 0.1 mg/mL kanamycin (Sigma-Aldrich) for 160 double efflux mutant strains in complete media. Plates were then incubated for 1 161 h and washed 2 times with 2 mL PBS. 162 Selected compounds were then added to each well at either 10, 1, 0.1 163 μg/mL in complete media in triplicate. Untreated, 1 μg/mL doxycycline (Sigma164 Aldrich), and 10 μg/mL ceftazidime were included as controls. Plates were 165 incubated for 18 h and cells observed for signs of infection. Cells were washed 3 166 times with 2 mL PBS and 1 mL sterile ddH2O added to each well. Each well was 167 thoroughly mixed/scraped to insure complete cell lysis. Lysates were then 168 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom serially diluted 1:10 and inoculum plated from the neat, 10, 10, 10 and 10 169 onto LB agar plates and plates were incubated for 48 h at 37C. Colonies from 170 each plate was counted and Log10 CFU/mL lysate was calculated. 171 172 Acute B. pseudomallei mouse model of infection for evaluation of efficacy. 173 5-6 week old BALB/c female mice (Charles River Laboratories, Wilmington, MA) 174 were challenged by intranasal infection with 5,000 CFU/mouse B. pseudomallei 175 Bp400. Animals were anesthetized with a mixture of 100 mg/kg ketamine and 10 176 mg/kg xylazine delivered intraperitoneally. The bacteria were diluted to the 177 appropriate concentration in PBS to achieve an inoculum concentration of 178 2.5x10 CFU/mL. The inoculum was then delivered in a 20 μL volume dropwise 179 in alternating nostrils. Ceftazidime was formulated for injection in a 0.1 % BSA 180 solution in PBS (pH 7.4) at a concentration of 40 mg/ml. PT52 and PT68 were 181 formulated for injection in a lipid-based delivery system of a combined lipid and 182 surfactant mixture consisting of 40% Captex 200 (Abitec Corp., Janesville, WI): 183 40% Solutol HS15 (BASF, Mount Olive, NJ): 20% Capmul MCM (Abitec Corp., 184 Janesville, WI). The mixture was gently heated at 50C to homogenize 185 components. The resulting homogenous isotropic mixture was then diluted with 186 PBS to yield a fine dispersion at a concentration of 40 mg/mL. 200 mg/kg 187 ceftazidime, PT52, or PT68 were delivered intraperitoneally with treatment 188 beginning at time of infection and repeated doses delivered every 12 h. The 189 number of viable bacteria in lung and spleen was determined at 60 h post190 exposure by plating serial 10-fold dilutions of homogenates onto LB agar and 191 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom incubating for 48 h at 37C. Bacterial burden was assessed using a one-way 192 analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test 193 with significance determined by a P value < 0.05. Outliers were determined by 194 Grubb’s test. 195 196 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom RESULTS 197 198 Substituted diphenyl ethers are potent inhibitors of Burkholderia. 199 A library of diphenyl ethers were screened for potency in an established 200 high throughput percent growth inhibition assay and confirmed using the 201 microbroth dilution method. The substituted diphenyl ethers were tested against 202 the efflux mutant strain B. thailandensis Bt38 and efflux-proficient strain B. 203 thailandensis E264 to assess each candidate as a pump substrate as well as to 204 determine inhibitory activity against whole bacteria. Thirty-two compounds out of 205 the library tested exhibited greater than 50% growth reduction and twenty-two 206 showed greater than 90% growth reduction at 20 μg/mL against B. thailandensis 207 Bt38. Only PT52 inhibited greater than 50% growth against B. thailandensis 208 E264 at 40 μg/mL. PT51, PT52, PT55, and PT68 were further investigated due to 209 previous SAR studies (17) and to evaluate the effect of A-ring substitutions on 210 efficacy (22). 211 MIC values were determined for the efflux-compromised B. pseudomallei 212 Bp400 mutant strain and efflux-proficient strain B. pseudomallei 1026b. PT52, 213 PT55, and PT68 had MIC values less than 1 μg/mL against B. pseudomallei 214 Bp400 (Table 1). Evaluation of PT51, PT52, PT55, and PT68 against the efflux215 proficient strain B. pseudomallei 1026b resulted in increased MIC values due to 216 drug efflux (Table 1). While the majority of substituted diphenyl ethers 217 demonstrated potency against Burkholderia, they were also readily effluxed. 218 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom The cytotoxicicty of each compound was evaluated using a standard Vero 219 cell assay (Table 1). Each diphenyl ether compound exhibited LD50 values 100220 fold greater than MIC, which is consistent with our previous reports for 221 compounds in this drug series (20). There was no toxicity observed in the ex 222 vivo RAW264.7 macrophage assay for the clinical drugs ceftazidime and 223 doxycline, which is consistent with published reports (23). 224 225 Diphenyl ethers with whole bacteria activity have potency against FabI1 226 enoyl-ACP reductase enzyme. 227 Based on the analysis of MIC values, PT51, PT52, PT55 and PT68 were 228 selected as lead compounds and their binding affinities and mode of inhibition 229 were determined against FabI1 (Table 2). Compounds PT51 and PT55 were 230 rapid reversible inhibitors displayed moderate binding affinity to FabI1 with IC50 231 values of 2699 ± 287 nM and 695 ± 132 nM. 232 The addition of a fluoro substituent to the A-ring (PT51 vs. PT55) resulted 233 in only a four-fold improvement in the binding affinity. However, replacement of 234 the fluoro substituent with a larger functional group such as a chlorine (PT52) or 235 a propenyl group (PT68), dramatically enhanced binding affinity to the FabI1236 NAD binary complex with K1 values of 3.69 ± 0.35 nM and 6.12 ± 0.31 nM, 237 respectively. Progress curve analysis also indicated that compounds PT52 and 238 PT68 were time-dependent inhibitors, displaying a slow-onset inhibition 239 mechanism against FabI1. It is not unusual for some inhibitors to exhibit this 240 kinetic behavior since triclosan and selected diphenyl ethers were shown to be 241 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom slow-onset inhibitors of FabI1 (17). Preincubation experiments allowed us to 242 evaluate the dependence of enzyme inhibition on NADH or NAD, which provides 243 the true thermodynamic affinity of the inhibitor for E-NAD (K1) and E-NADH (K2). 244 For both PT52 and PT68, the dependence of K′i on [NAD] was best described 245 by the equation that includes the inhibitor binding to both E-NAD and E-NADH 246 forms of FabI1, albeit based on the K1 and K2 values of these two compounds 247 there is a 100-fold preference for E-NAD. 248 249 Substituted diphenyl ethers demonstrate efficacy in a Burkholderia ex vivo 250 model of efficacy and in the Burkholderia rapid animal model of efficacy. 251 PT51, PT52, PT55 and PT68 were further evaluated in a Burkholderia ex 252 vivo model of efficacy based on their whole bacterial activity. Evaluation of 253 activity in the ex vivo model was conducted against B. pseudomallei 1026b at 10 254 μg/mL and at multiple concentrations against B. pseudomallei Bp400 (Figure 255 1AB). PT51, PT52, PT55 and PT68 all inhibited the growth of the efflux pump 256 mutant B. pseudomallei Bp400 >90% at 10μg/ml, and growth of B. pseudomallei 257 1026b was not inhibited by PT51, PT52, PT55, and PT68. 258 To assess whether PT51, PT52, PT55 and PT68 demonstrated inhibition 259 in a concentration dependent manner, they were further evaluated for activity 260 against B. pseudomallei Bp400 in the ex vivo model. Each of the compounds 261 inhibited growth of B. pseudomallei Bp400 in a dose dependent fashion (Figure 262 1B), and the precise 90% growth reduction values for each compound in the ex 263 vivo model were determined (Table 3). Importantly, PT51, PT52, PT55 and 264 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom PT68 had MIC values in the ex vivo model comparable to those determined in 265 artificial medium. 266 PT52 and PT68 were tested in the acute B. pseudomallei animal model of 267 infection to determine efficacy. These compounds were chosen due to their 268 slow-onset inhibitor characteristics observed in enzyme kinetic assays, in 269 addition to inhibitory activity in the ex vivo efficacy model. Mice were challenged 270 intranasally with 5,000 CFU B. pseudomallei Bp400. Bacterial burden was 271 assessed at 60 h post infection and treated groups were compared to the 272 untreated control group. PT52 treated mice exhibited a 0.6 Log10 CFU/mL 273 reduction (P<0.05) in the lung and a 1.1 Log10 CFU/mL reduction (P<0.01) in the 274 spleen (Figure 3AB). PT68 showed reduction only in the spleen at 1.3 Log10 275 CFU/ml (Figure 3B). Mice treated with 200 mg/kg ceftazidime were included as a 276 positive control and was determined to have a bacterial burden at the level of 277 detection in both lung and spleen. These studies substantiated that these 278 substituted diphenyl ethers can subterfuge and gain access to the efflux-deficient 279 bacteria under host conditions and inhibit growth through inhibition of FabI1 (24). 280 281 Discussion 282 Discovery of new drug targets and chemotherapeutics is key in managing 283 emerging pathogens such as B. pseudomallei. B. pseudomallei can be 284 particularly challenging to treat due to intrinsic and emerging antimicrobial 285 resistance and the ability to efflux antibiotics. The enoyl-ACP reductase 286 enzymes, particularly FabI in the FASII pathway, are under-exploited new targets 287 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom for the development of anti-Burkholderia drugs. Our compounds were developed 288 around the diphenyl ether pharmacophore and in previous studies have exhibited 289 whole bacteria activity and in vivo efficacy against F. tularensis Schu4 (13, 14). 290 In this study we have shown this same class of compounds to be 291 efficacious for in vitro and ex vivo activity against efflux-compromised B. 292 pseudomallei Bp400. Unfortunately, the majority of compounds in this class are 293 efflux pump substrates, and while they are effluxed similar to existing clinically 294 used drugs to treat melioidosis, for instance doxycycline, modifications to existing 295 compounds will be made to address this issue or current compounds will need to 296 be formulated to deliver the higher dose requirement that exceeds the higher 297 MIC value. 298 Importantly, PT52 and PT68 were shown to significantly reduce bacterial 299 burden in the spleen and only PT52 significantly reduced bacterial burden in the 300 lung when compared to untreated mice. This observation, along with the ability 301 of these compounds to display a low nanomolar binding affinity to FabI1 in vitro, 302 suggests that the use of substituted diphenyl ethers to inhibit FabI1 enoyl-ACP 303 reductase in Burkholderia can be exploited to control infection. Steady-state 304 kinetic analysis showed that PT52 and PT68 were promising inhibitors due to 305 favorable optimization of thermodynamic parameters, specifically inhibitor binding 306 to the FabI1-NAD complex represented by K1. This final stable ground state was 307 coupled with a slow-onset inhibitor mechanism, which was lacking in compounds 308 PT51 and PT55 that displayed higher K1 values. The loss of slow-onset inhibitor 309 mechanism of PT51 and PT55 against FabI1 may arise from no A-ring 310 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom substituent and the smaller van der Waals radius of fluorine compared with that 311 of chlorine (PT52), respectively. Also, it has been demonstrated that A-ring 312 substituents play a key role in hydrophobic interaction between diphenyl ether 313 inhibitors and FabI from F. tularensis (13), which leads to slow onset inhibition 314 and high binding affinity in this system. 315 By using the diphenyl ethers as tool compounds we were able to show 316 that inhibition of B. pseudomallei FabI1 results in efficacy in vitro and in the 317 animal model of infection. Further optimization of this class, or similar classes of 318 FabI inhibitors, could open up the possibility for the development of a novel 319 compound that could be used to treat Melioidosis. 320 321 322 Acknowledgements 323 This work was supported by RP006 of the Rocky Mountain Regional Center of 324 Excellence funded by NIH, NIAID grant AI065357 (R.A.S. & P.J.T.).325326onJuly9,2017bygesthttpaac.asm.rg/Downladedfom References3271. Cheng AC, Currie BJ. 2005. Melioidosis: epidemiology, pathophysiology, and328management. 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Targeting fatty acid396biosynthesis for the development of novel chemotherapeutics against397Mycobacterium tuberculosis: evaluation of A-ring-modified diphenyl ethers as398high-affinity InhA inhibitors. Antimicrobial agents and chemotherapy 51:3562-3993567.40023. Judy BM, Whitlock GC, Torres AG, Estes DM. 2009. Comparison of the in vitro401and in vivo susceptibilities of Burkholderia mallei to Ceftazidime and Levofloxacin.402BMC Microbiol 9:88.40324. Cummings JE, Kingry, L.C., Rholl, D.A., Schweizer, H.P., Tonge, P.J., and404Slayden, R.A. 2013. The Burkholderia pseudomallei enoyl-ACP reductase FabI1405is essential for in vivo growth and is the target of a novel chemotherapeutic with406efficacy. Antimicrob Agents Chemother 2013 Nov 25. [Epub ahead of print]407PMID: 24277048.408409410onJuly9,2017bygesthttpaac.asm.rg/Downladedfom Tables411412Table 1. MIC values of fabI1 inhibitors against wild type and efflux-compromised B.413pseudomallei strains. MIC values were determined using the microbroth dilution414method.415416Table 2. Compounds further evaluated for activity against B. pseudomallei417Bp400 ex vivo. Drug concentration that inhibits 90% growth was calculated from418 linear regression analysis of data obtained at 10, 1 and 0.1 μg/ml.419420Table 3. Structure activity relationship studies performed show A-ring421substitutions are critical in determining type of inhibition and binding affinity for422both E-NAD (K1) and E-NADH (K2).423424425onJuly9,2017bygesthttpaac.asm.rg/Downladedfom Figures.426Figure 1. RAW264.6 macrophages were infected with B. pseudomallei at a427multiplicity of infection (MOI) of 5 and treated overnight with either ceftazidime or428select substituted diphenyl ethers. B. pseudomallei 1026b was treated with429 10μg/ml compounds for 18hr, macrophages lysed, and lysate plated on solid430 media for bacterial enumeration (A). B. pseudomallei Bp400 was treated at the431same concentration with ceftazidime and at 10, 1, and 0.1mg/ml with substituted432diphenyl ethers to observe concentration dependent inhibition. Again433macrophages were lysed and lysate plated onto solid media for bacterial434 enumeration (B).435436Figure 2. Bacterial burden in mouse lung (A) and spleen (B) at 60 h post437 infection. The mean of each group was plotted, error bars indicating +/standard438deviation, and dashed line indicates level of detection. Significance was439 determined by one-way ANOVA and a P-value <0.05. This data is representative440of two independent experiments.441onJuly9,2017bygesthttpaac.asm.rg/Downladedfom Table 1In vitro Activity of FabI Inhibitors Against B. pseudomalleiB. pseudomallei 1026b B. pseudomallei Bp400 Vero CytotoxicityFabI Inhibitor MIC mg/LMIC mg/LLD50 mg/LPT5122190.2PT5210.12589.8PT5540.5164.9PT6840.5152.9Doxycycline0.50.125NDCeftazidime21NDND – Not determined in this study onJuly9,2017bygesthttpaac.asm.rg/Downladedfom Table 2: inhibition of bpFabI-1 by substituted diphenyl ethersInhibitorK1 (nM) K2 (nM) Type of InhibitionPT523.69±0.35 359±10 Slow-onset PT686.12±0.31 776±17 Slow-onset InhibitorIC50 (nM) Type of InhibitionPT512699±287 Rapid equilibrium PT55695±132 Rapid equilibriumonJuly9,2017bygesthttpaac.asm.rg/Downladedfom Table 3Ex vivo Activity of FabI Inhibitors Against B. pseudomallei Bp400FabI Inhibitor 90% GrowthReduction mg/LFabI Inhibitor 90% GrowthReduction mg/LPT51 6.1 +/2.6 PT68 2.0 +/2.0PT52 2.5 +/2.5 Ceftazidime >10PT556 +/4.3 Doxycycline <1 onJuly9,2017bygesthttpaac.asm.rg/Downladedfom