In Vivo Validation of Newly Discovered LpxC-Targeting Inhibitors .

1. In Vivo Validation of Newly Discovered LpxC-Targeting Inhibitors. Hilliary Johnson, Supervised by Dr. Pei Zhou, Mentored by Chiljin and Daina Department of Biochemistry Northampton County High School-West, 27832 ABSTRACT RESULTS DISCUSSION The dacetylation of UDP-3-0-acyl-GlcNAc by LpxC is the committed reaction of lipid A biosynthesis. LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising antibiotic target. A previous study identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeroginosa and therefore were not developed further as antibacterial drugs. The inactivity of the LpxC inhibitors for P. aeroginosa raised the possibility that LpxC activity might not be essential for all gram-negative bacteria. By placing the lpxc gene of P. aeroginosa under tight control of an arabinose-inducible promoter, we demonstrated the essentiality of LpxC activity for P. aeroginosa. It was found that compound L-161,240, was active against a P. aeroginosa construct in which the endogenous lpxc gene was inactivated in and in which LpxC activity was supplied by the lpxc gene from E. coli. Conversly, an E. coli construct in which growth was dependent on the P. aeroginosa lpxc gene was resistant to the compound. These data validate P. aeroginosa LpxC as a target for novel antibiotic drugs and should help direct the design of inhibitors against clinically important gram-negative bacteria. The results of our experiments lead us to conclude that our hypothesis is not fully supported, yet not refuted. There are numerous noticeable trends that agree with our hypothesis. However, it is in the statistical analysis that the basis for doubt is formed. Most of the trends that appear to be present are not statistically significant. The fact that the trends are there, however, is the basis for partially accepting the hypothesis. Further study would need to be done in order to be certain that our hypothesis is fully supported, or whether it needs to be amended. While it is presumed that length or size of a compound does indeed have an effect it’s ability to inhibit cell development, a direct relationship between the two factors cannot be established using only the data produced from this study. If a direct relationship were to exist, then the inhibition of LpxC using XC-2-31 would produce better results than using XC-12-19 produced. Results generated from this study failed to validate such a relationship, and therefore we can only propose that although XC-2-31 was unsuccessful in inhibiting E. coli-P.A cells that it may be more effective at higher levels of concentration or with a different strain of E. coli. Title 1 Title 2 Title 2 Title 2 Figure 1: First MIC Results Collected Figure 2: Second MIC Results Collected. INTRODUCTION CONCLUSION The amount of antibiotic-resistant bacteria increases everyday. This emergence of antibiotic-resistant bacteria is especially a problem in hospitals and clinics. Many of theses microorganisms have survived for thousands of years by being able to adapt to antimicrobial agents. Bacteria become drug resistant either through spontaneous mutation, changes in the nucleotide sequence of the genetic material, or DNA transfer, the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign genetic material(DNA). This has lead to a need for novel antibiotics that are potent enough to inhibit such bacteria and cells. • If we extend the binding area of existing compounds by introducing various functioning groups into the deacetylene scaffold, then we will have found rational design of LpxC inhibitors with the potency and broad spectrum antibiotic properties. • Refuted ACKNOWLEDGEMENTS • GOD • My friends and family • The HamnerInstitutes of Health Sciences • Project SEED • Duke University • The Zhou lab • The Raetz lab • North Carolina Local Section of the American Chemical Society, Blue Cross and Blue Shield, AT&T, BiogenIdecs, and the Burroughs Wellcome Fund Grant #1006597 HYPOTHESES If we extend the binding area of existing compounds by introducing various functioning groups into the deacetylene scaffold, then we will have found rational design of LpxC inhibitors with the potency and broad spectrum antibiotic properties. Figure 3: E. coli Wild Type REFERENCES • Raetz C. R. H. & Dowhan W (1990) Biosynthesis and function of phospholipids in Escherichia coli. (Translated from eng) J BiolChem265(3): 1235-1238 (in eng). • Raetz, C. R. H. and Whitfield, C. Lipopolysaccharideendotoxins. Annu Rev Biochem2002; 71: 635-700 • Levy, S. B. (2005) Antibiotic resistance-the problem intendifies, Adv. Drug Deliv. Rev. 57 1446-1450 MATERIALS & METHODS In my research, I used such materials as a Multi-channel pipette, An incubator, A shaker incubator, Spectrometer, Petri dishes, Toothpicks, Bunsen Burner, 96-Well Plate, and the Refrigerator Figure 6: Inhibitor XFL-2-29C Figure 7: Inhibitor XFL-2-31 Figure 5: Inhibitor XFL-2-28C Figure 4: Inhibitor XC-12-19