DEVELOPMENT OF BROAD-SPECTRUM ANTIBIOTICS AGAINST BACTERIAL PATHOGENS

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0207398
• Project Start Date: Jul 1, 2006
• Project End Date: Jun 30, 2008
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
VETERINARY BIOMEDICAL SCIENCE

Non Technical Summary
Antimicrobial agents (antibiotics) continue to play an essential role in the fight against infectious diseases of human and veterinary importance. In human disease, the AIDS pandemic, migration, and the globalization of the world economy are some underlying causes sustaining this trend. Moreover, the potential use of biological agents as terrorist weapons of mass destruction could expose people to agents of acute infectious. In all of these cases, rapid interventions to treat infected and/or diseased individuals become paramount. Likewise in veterinary medicine, the widespread use of antibiotics has resulted in the emergence of drug resistance. Nonetheless, antibiotics still provide an essential means to accomplish these rapid intervention strategies. The expected outcome of this research effort will be the identification of candidate broad-spectrum antimicrobial agents that minimize the emergence of drug resistance. By targeting multiple proteins, the probability of acquiring and propagating resistance becomes negligible. Thus, the discovery of novel drugs that target the mechanisms of resistance will inherently prolong the usefulness of existing antibiotics and provide new antimicrobial agents.

Animal Health Component

33%

Research Effort Categories

Basic

34%

Applied

33%

Developmental

33%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1100	50%
722	4010	1100	50%

Knowledge Area
311 - Animal Diseases; 722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

broad-spectrum antibiotics

drug resistance

mur proteins

mycobacterium bovis

mycobacterium tuberculosis

nuclear magnetic resonance

peptidoglycan biosynthesis

Goals / Objectives
To be effective, the next generation of antibiotics can not use old approaches by simply focusing on targeting another essential protein or trying to modify the antibiotic to overcome the acquired bacterial changes. This effort is daunting since the resistance mechanisms are generic, rapid and applicable to any protein target or modification that can be made to the antibiotic. Available antibiotics tend to target a single enzyme for inhibition. This increases the likelihood that a key mutation in the active-site will render the antibiotic inactive with a minimal negative impact on the enzyme function. An alternative approach would target multiple related enzymes in an essential pathway with either a broad-spectrum inhibitor or multiple inhibitors. The peptidoglycan biosynthesis pathway is an ideal target for developing such a broad-spectrum inhibitor. The pathway includes numerous related enzymes (Mur) with similar active-site characteristics. These Mur enzymes are responsible for amino-acid ligation during the elongation of the peptidoglycan chain. Since each Mur protein binds the product of the previous Mur protein in this simple process, there is significant overlap in the sequence and structure of the active-site for these Mur proteins. Emergence of resistance would then be unlikely requiring mutations in each of the genes encoding the Mur enzymes. Project Objectives. We will test this approach using the Mur enzymes of Mycobacterium tuberculosis, a pathogen of utmost importance in human health. This is also applicable to Mycobacterium bovis, a zoonotic agent that can cause tuberculosis in both animals and humans. In this context, we propose to (1) overexpress and purify M. tuberculosis Mur enzymes in recombinant E. coli and (2) combine NMR and combinatorial chemistry to identify lead inhibitors.

Project Methods
Research Procedures: (1) Overexpression and purification of Mur enzymes. To overproduce and purify the Mur proteins, the commercially available pET system will be used. Primers for the M. tuberculosis mur genes will be designed and used with thermostable high-fidelity DNA polymerase to amplify the mur genes from genomic DNA. The resulting PCR product will be purified, digested with the appropriate restriction enzymes, and cloned into pET15b following established procedures. Restriction digestion analysis and DNA sequencing will be performed to ensure that the correct in-frame fusion has been made. After verification, the plasmid will be transformed into E. coli BL21(DE3) overproduction host. Protein expression will be induced by adding 1.0 mM IPTG to cultures of recombinant E. coli when the OD600 is between 0.5 and 1.0. Cells will be harvested at various times after induction and whole-cell crude extracts will be analyzed by SDS-PAGE to detect the expression of the Mur proteins. For large scale preparations, E. coli cells will be disrupted with a French Press and the resulting cell lysate passed through a Ni affinity column. Recombinant proteins with the His tag bound to the column will be eluted and the process repeated until the protein is purified close to homogeneity. The His tag in the recombinant protein will be cleaved by thrombin prior to analysis. The purity and identity of the recombinant protein will be confirmed by 2D SDS-PAGE, mass spectrometry and N-terminal sequencing. We have experience in these procedures and have large amounts of two M. tuberculosis enzymes involved in peptidoglycan biosynthesis. (2) Functional NMR Screen. An automated and high-throughput NMR screening protocol will be used to identify ligands with known functional activities that bind the target proteins. The nature of the bound ligands, the identity of the active-site, and the details of the protein-ligand conformation will be used to assign biological function from comparison with comparable data for proteins of known function. (3) NMR co-structures. After a ligand has been identified, a rapid co-structure of the protein ligand complex will be determined by NMR. To significantly shorten the time requirement, we will develop protocols to determine NMR co-structures based on a hybrid approach that combines prior structural data with molecular modeling and a minimal set of NMR data. (4) Combinatorial chemistry. The identified functional ligand and subsequent co-structure will provide the basis to design and synthesis a directed chemical library as part of a drug design effort. (5) Lead Discovery. The combinatorial chemistry library will be screened by NMR to identify binders, where binders that compete with the functional ligand would be identified as lead compounds and subject to further development and analysis.

Progress 07/01/06 to 06/30/08

Outputs
OUTPUTS: (3) Determine binding and kinetics constants for substrates and IC50 values for representative inhibitors of the Mtb Ddl. We carried out kinetic analysis and performed preliminary binding assays. The kinetics of the uninhibited enzyme displayed properties consistent with cooperative effects at both the D-alanine (K' = 5.83 mM; n = 0.8) and ATP (K' = 1.0 mM; n= 2.3) binding sites. IC50 values for representative compounds were also determined. (4) Synthesize additional derivatives of DCS and test their inhibitory activity against Alr and Ddl purified enzymes. (5) For lead compounds showing a reasonable activity against the purified enzymes, perform further chemical synthesis to obtain more active derivatives. For these new compounds, determine the IC50 and the minimal inhibitory concentration (MIC) for Mtb. Dr. Takacs Laboratory is currently working on synthesizing 13C-DCS. As potential inhibitors of Alr and Ddl, three additional derivatives of N-octyl-D-cycloserine were synthesized: 1) N-octyl-D-cycloserine + 2-pyridine carboxaldehyde = 2-pyridinyl- N-octyl-D-cycloserine 2) N-octyl-D-cycloserine + 4-pyridine carboxaldehyde = 4-pyridinyl- N-octyl-D-cycloserine 3) Hydrochloride salt of N-octyl-D-cycloserine We have not yet tested these compounds in Ddl assays or against Mtb. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: A poster entitled "NMR metabolic analysis of Mycobacterium smegmatis D-cycloserine Resistant Mutants" covering the results of this research was presented at the 2008 University of Nebraska Redox Biology Center Annual Retreat. PROJECT MODIFICATIONS: Year 2 objectives: (1) Overproduce, purify and characterize the Mtb Alr as first priority. Develop reagents and assays for MurF. (2) Construct a M. smegmatis recombinant strain expressing the antisense message for the ddl gene under the control of a regulated promoter. Perform metabolomic analysis as describes above for the Alr to determine whether Ddl is the lethal target of DCS. We had to radically alter our plans since a publication from a competitor group constructed M.smegmatis alr deletion mutants in which approximately 99% of the alr coding sequences were deleted (Milligan et al., J. Bacteriol 189:8381-6, 2007). These mutants displayed an absolute dependency on D-alanine for growth. Milligan et al. asserted that our insertional mutants could have some residual Alr activity as a result of the joining of the two protein fragments potentially encoded for by the insertionally inactivated alr gene. However, Milligan et al. used Luria-Bertani broth with Tween (LBT) and 7H11 to test the dependency on the D-alanine for growth. In contrast, in our original study, we used Middlebrook 7H9 media. Two alternatives can be offered to resolve this issue: either insertion mutants were able to grow due to background Alr activity or the auxotrophy was dependent on the medium. To test these hypotheses, we constructed new independent alr mutants with the same deletion as those of Milligan et al. and compared their growth properties with our original insertion mutants. In this study, we showed that both insertion and deletion mutants have the same growth characteristics: they both grew in Middlebrook, but failed to grow in Luria-Bertani medium in the absence of D-alanine. Thus, the alr mutation confers auxotrophy for D-alanine only in suboptimal culture conditions, indicating that M. smegmatis may possess a novel pathway of D-alanine biosynthesis. These findings suggest that Alr is not a main target of antibacterial action in M. smegmatis, as we have described in our original studies. In addition, we compared the metabolomic profiles of wildtype and L-alanine dehydrogenase mutant strains. We also standardized a metabolomic flux analysis using a novel technology for 2-D NMR analysis. To this end, cells were labeled with 13C-D-alanine, allowing for analysis of both 1H and 13C NMR signals.

Impacts
These studies are significant in the context of discovering new drugs to treat mycobacterioses in animals and human beings that can overcome mechanisms of drug resistance.

Publications

• Steven Halouska presented a poster at the 2008 University of Nebraska Redox Biology Center Annual Retreat - NMR metabolic analysis of Mycobacterium smegmatis D-cycloserine Resistant Mutants by Steven Halouska, Ofelia Chacon, Zhengyu Feng, Nancy E. Caceres, Denise Zinniel, Robert Fenton, Raul Barletta and Robert Powers Department of Chemistry and Department of Veterinary and Biomedical Science, University of Nebraska 2008
• HarshDeep Dogra has submitted his thesis - Biochemical and genetic Characterization of D-alanine Ligase and D-alanine Racemase of Mycobacteria in order to obtain a Master of Science Degree in the Department of Veterinary & Biomedical Sciences 2008

Progress 01/20/07 to 09/30/07

Outputs
The objectives of this study were to (1) overexpress and characterize peptidoglycan biosynthetic enzymes of Mycobacterium tuberculosis, (2) to conduct functional and (3) structural NMR studies, (4) apply combinatorial chemistry and (5) identify lead compounds. (Objective 1) These studies were focused on the D-alanine ligase. We determined that D-alanyl-D-alanine was produced in equivalent molar amounts to the release of inorganic phosphate (Pi). A continuous Pi assay was developed to determine enzyme inhibition. Inhibition assays were carried out in a reaction mixture containing 0.1 mM enzyme, 1.0 mM D-alanine and 6.0 mM ATP. Under these conditions, we found that D-cycloserine and (S)-(+)-(1-Amino ethyl) phosphonic acid inhibited the enzyme with IC50 values of 0.3 and 0.5 mM, respectively. (Objectives 2 and 3): Principal component analyis of metabolmic data indicated signgicant differences between the D-alanine racemase mutant and wild type cells treated with D-cycloserine. (Objectives 4 and 5) N-octyl-D-cyloserine and 3-chloro-2,2-dimethyl-N-[4(trifluoromethyl)phenyl]propanamide were prepared by chemical synthesis. Compounds were crystallized from ethyl acetate-hexanes. For N-octyl-D-cycloserine, the residue was purified by flash column chromatography on silica with MeOH-CHCl3 (1:49) to give the desired amine (27 mg, 84% yield). All compounds synthesized had a melting point and chemical shifts for 1H and 13C NMR consistent with the structure. Dissemination: three posters were presented at the 2007 University of Nebraska Research Fair: (1) Principle Component Analysis to Determine the Lethal Target of D-cycloserine in Mycobacteria (Steven Halouska - Department of Chemistry), (2) Targeting the D-alanine-D-alanine Ligase of Mycobacterium tuberculosis for Drug Design (HarshDeep Dogra - Department of Veterinary and Biomedical Sciences) and (3) Approaches to the Design of Inhibitors for Targeting the D-alanyl-D-alanine Ligase of Mycobacterium tuberculosis (Judy Miska - Department of Chemistry). HarshDeep Dogra was awarded a prize in the Biological Sciences and Agricultural Sciences area. A manuscript was published in the Journal of Proteome Research (see below).

Impacts
The continuous inorganic phosphate (Pi) assay was established as the method of choice to screen for inhibitors of the D-alanine ligase. This knowledge will allow screening of a large number of compounds for inhibition. The IC50 values of D-cycloserine (0.3 mM) and phosphonic acid (0.5 mM) indicate that these compounds may target the ligase. However, the actual values indicate that they are relatively weak inhibitors. This knowledge will guide us in the synthesis of derivatives with greater binding affinity and inhibitory action. The metabolomics data indicate that D-cycloserine does not target D-alanine racemase as the main lethal target. This knowledge is useful to direct our search for new antimicrobial compounds to inhibitors of alternative targets such as D-alanine ligase. Two chemical compounds were made. Toxicity and inhibitory activity against mycobacterium tuberculosis D-alanine ligase and live bacilli will be determined. This knowledge will be used to determine whether these compounds could be developed into new agents agaisnt tuberculosis.

Publications

• Halouska Steven, Ofelia Chacon, Robert J. Fenton, Denise K. Zinniel, Raul G. Barletta and Robert Powers. 2007. Use of NMR Metabolomics to Analyze the Targets of D-cycloserine in Mycobacteria: Role of D-Alanine Racemase. Journal of Proteome Research. Published on web 11/03/2007.

Progress 07/01/06 to 01/19/07

Outputs
We have obtained large amounts of one of the enzymes in the murein (Mur) biosynthetic pathway. We have developed a high throughput assay and are synthesizing chemical inhibitors. We also developed a NMR metabolic profiling method to analyze the essential role of D-alanine racemase in the Mur pathway. We have prepared cell extracts of wild-type, susceptible and resistant Mycobacterium smegmatis strains, a model system for Mycobacterium tuberculosis, to conduct NMR profiling studies. The drug used for this study was the murein (peptidoglycan) synthesis inhibitor D-cycloserine (DCS). We analyzed the NMR data by the principal component analysis (PCA) methodology, a well established statistical technique that determines the direction of largest variations in the NMR data set. The results showed three distinct clusterings, indicating that the drug tested is active towards the system. The DCS susceptible strain TAM23 treated with and without DCS clustered in different locations, indicating that DCS is targeting a different protein in the system, which is a discovery of significance in the field. Since TAM23 is deficient in D-alanine racemase activity, this result indicates that this enzyme is not the lethal target of DCS. The wild-type (mcc155) and resistant GPM267 strains treated with DCS clustered with TAM23 treated with DCS indicating that DCS inhibits the same secondary protein present in TAM23. These results are consistent with one lethal target for DCS different from D-alanine racemase.

Impacts
Antimicrobial agents (antibiotics) continue to play an essential role in the fight against infectious diseases of human and veterinary importance. The widespread use of antibiotics has resulted in the emergence of drug resistance. Nonetheless, antibiotics still provide an essential means to accomplish these rapid intervention strategies. The expected outcome of this research effort will be the identification of candidate broad-spectrum antimicrobial agents that minimize the emergence of drug resistance.

Publications

• No publications reported this period