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Mechanistic Studies of Inhibitors of DNA Replication Restart Pathways in Neisseria Gonorrhoeae Chaitanya DRK Aduri and Matthew E

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Mechanistic Studies of Inhibitors of DNA Replication Restart Pathways in Neisseria Gonorrhoeae Chaitanya DRK Aduri and Matthew E Mechanistic studies of inhibitors of DNA replication restart pathways in Neisseria gonorrhoeae Chaitanya DRK Aduri and Matthew E. Lopper, Ph.D. Department of Chemistry, University of Dayton, Dayton, OH Abstract Effect of 0039 0207 Affects PriA-catalyzed ATP hydrolysis Complete and faithful replication of a cell’s genetic information is an essential process. Many enzymes are involved in the successful duplication of cells genetic information and the Effect of 0039 on PriA Effect of 0039 on PriA and PriB • A coupled spectrophotometric assay was used to measure steady-state rates of ATP hydrolysis 0.9 integrity of these enzymes can be compromised when they encounter DNA damage. 0.9 catalyzed by PriA in the presence and absence of 0207 0.8 0.8 Bacterial cells use a pathway called “DNA replication restart” to resume DNA replication 0.7 0.7 average following a disruptive encounter of the DNA replication enzymes with DNA damage. This 0.6 0.6 pathway is catalyzed by primosome proteins, including PriA, PriB, PriC, DnaT, DnaB, DnaC, 0.5 0.5 0.4 and DnaG. The importance of DNA replication restart for bacterial cell survival is demonstrated 0.4 Fraction unwound 0.3 by the inability of strains that carry mutations in key primosome genes to grow and resist DNA Fraction unwound 0.3 0.2 0.2 damaging agents. Furthermore, this pathway is specific for bacterial cells: human cells don’t use 0.1 300 ATP Titrations the same replication restart pathway and they don’t encode genes for the primosome proteins 0 0.1 -50 0 50 100 150 200 250 300 350 250 -0207 0 [Inh] that function in bacteria. Since DNA replication restart pathways are essential for bacterial cell -50 0 50 100 150 200 250 300 350 [Inh] 200 growth and survival and are notably absent in human cells, we seek to answer the following question: can bacterial DNA replication restart pathways be targeted with novel antibacterial 150 +0207 This is a weak inhibitor and is not dependent on PriB. compounds? 100 In order to answer this question, we have developed an enzyme based assay for high-throughput ATP hydrolysednm/s 50 inhibitor screening to identify compounds that block the function of the primosome proteins Effect of 0046 0 Effect of 0046 on PriA Effect of 0046 on PriA and PriA and PriB. Several interesting lead compounds have already been identified from the -200 0 200 400 600 800 1000 1200 PriB 0.9 preliminary screening. In this study, the lead compounds have been validated as legitimate 0.9 -50 0.8 [ATP] inhibitors and characterized with respect to their potency and mechanism of action. 0.8 0.7 0.7 The effects of 0207 on Vmax and Km suggest a mixed mode of inhibition with respect to both DNA and 0.6 CGS 15943 inhibits PriA:PriB-catalyzed duplex DNA unwinding 0.6 ATP. DNA replication and DNA replication restart 0.5 0.5 DNA Titrations:0207 Km Vmax DNA Titrations: No 0207 Km Vmax 0.4 0.4 Trial 1 110.87 243.49 Trial 1 16.82 264.59 Fraction unwound 0.3 Fractionunwound The restart pathways follow two trends with respect to the origin, origin dependent and origin 0.3 Trial 2 80.03 202.87 Trial 2 18.77 286.97 0.2 independent, differing mainly in the mechanistic features. In E. coli, one of the several major 0.2 0.1 Trial 3 76.15 170.68 Trial 3 20.08 284.34 pathways to activate stalled replication forks is DnaA catalyzed origin-dependent initiation of 0.1 0 Average 89.01 205.6 Average 18.54 278.63 DNA replication. This is very carefully regulated sequence specific event which is catalyzed by -50 0 50 100 150 200 250 0 [Inh] -50 0 50 100 150 200 250 the initiator protein DnaA. DnaA recognizes and binds to a replication origin site and recruits [Inh] ATP Titrations : 0207 Km Vmax ATP Titrations : No 0207 Km Vmax the replicative helicase and the helicase loader protein, DnaB and DnaC respectively. The samples ranging from 300um – 800um concentration has shown precipitation. Trial 1 18.25 119.9 Trial 1 31.5 226.69 This inhibitor turned out to be a weak inhibitor, and is also not dependent on the PriB. Trial 2 26.1 127.38 Trial 2 37.6 252.04 Origin-independent initiation of DNA replication (known as DNA replication restart) requires a Trial 3 34.7 107.51 Trial 3 37.8 245.37 distinct cellular machinery to reload the replication machinery (replisome) at a repaired DNA Average 26.35 118.25 Average 35.33 241.36 replication fork. This replication process is initiated by an assembly of the primosome proteins, Effect of 0441 which collectively include PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, and Rep proteins. Effect of 0441 on PriA Effect of 0207 on E. Coli PriA 1 0.5 0 -10 0 10 20 30 40 50 60 -0.5 Fraction unwound -1 -1.5 -2 [Inh] PriA is the target of CGS 15943 inhibition This inhibitor had a solubility issue, thus the maximum concentration used in the assay was 50uM. The inhibitor can be regarded as a weak inhibitor , this can be taken into account as the one which might Origin dependent and origin independent DNA replication. be affecting the DNA by itself irrespective of the PriA. 0207 also shows its effect on E. coli PriA and the IC50 value was calculated to be 1.72uM. Identification of inhibitors that selectively inhibit DNA Effect of 0207 replication restart primosome proteins Conclusions Effect of 0207 on PriA Effect of 0207 on PriA and PriB 0207 was proved to be a genuine inhibitor. 1 0.8 0207 is not a species specific inhibitor, it also shows its effects on E. coli PriA. 0.9 average 0.7 0.8 0207 shows mixed mode of inhibition. 0.6 0.7 0.6 0.5 0.5 0.4 fraction unwound unwound Fraction 0.4 0.3 References We developed an enzyme-based assay to use in high-throughput screening to identify inhibitors 0.3 1. Marians KJ. (2000) PriA directed replication fork restart in E.coli. Trends in Biochemical Sciences. 25( 4): 185-189. 0.2 of PriA:PriB function. 0.2 2. Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill 0.1 0.1 0 Synthetic DNA oligonucleotides were used to construct a forked DNA substrate in which the 0.1 1 10 100 0 0.1 1 10 100 [inh] nascent lagging strand arm is fluorescently labeled. [inh] Fluorescence polarization spectroscopy was used to report PriA:PriB-catalyzed unwinding of This inhibitor looks genuine and the IC50 value was calculated to be 1.64uM. the forked DNA substrate by monitoring changes in fluorescence anisotropy (FA). This indeed is not dependent on PriB. Precipitation was found in the samples ranging from 100uM and 200uM concentrations. The Life chemicals 2 library was screened to identify inhibitors of PriA:PriB-catalyzed DNA unwinding. .
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