Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-06-26
2003-03-25
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S023000, C435S015000, C435S004000, C435S975000
Reexamination Certificate
active
06537770
ABSTRACT:
The present invention relates to a new assay for the detection of phospho-N-acetylmuramyl-pentapeptide translocase enzyme activity (hereinafter referred to as “translocase enzyme”).
Peptidoglycan is a major component of the bacterial cell wall that gives the wall its shape and strength. It is unique to bacteria and found in all bacteria, both gram-positive and gram-negative. Peptidoglycan is a polymer of glycan strands which are cross-linked through short peptide bridges. It consists of alternating &bgr;1-4 linked residues of N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). A pentapeptide chain is attached to MurNAc (MurNAc-pentapeptide) and the peptidoglycan polymers are crosslinked through these peptide chains.
Biosynthesis of peptidoglycan can be divided into three stages: firstly, synthesis of the precursors in the cytoplasm, secondly, transfer of the precursors to a lipid carrier molecule and, thirdly, insertion of the precursors into the cell wall and coupling to existing peptidoglycan.
Enzymes responsible for the biosynthesis of the peptidoglycan component of the bacterial cell wall are novel targets for the design of new antibiotics. Owing to the worldwide emergence of bacterial strains resistant to current antibiotics, it has become necessary to develop new antimicrobial agents. The translocase enzyme catalyses the first step in the membrane cycle of peptidoglycan biosynthesis, namely the transfer of phospho-N-acetylmuramyl-L-Ala-&ggr;-D-Glu-m-diaminopimellic acid-D-Ala-D-Ala from Uridine 5′-diphosphate phospho-N-acetylmuramyl-L-Ala-&ggr;-D-Glu-m-diaminopimellic acid-D-Ala-D-Ala (hereinafter referred to as “UDP-MurNAc-pentapeptide” or “UDP-MPP”) to a membrane-bound lipid carrier, undecaprenyl phosphate. The translocase enzyme is encoded by the mraY gene in
Escherichia coli.
the translocase enzyme is essential for bacterial viability (see D. Mengin-Lecreulx, L. Texier, M. Rousseaue and J. Van Heijernoot, J. Bacteriol., (1991), 173, 4625-4636).
No commercial antibiotics in current use are directed against the translocase enzyme. It therefore represents a target for novel antibacterial agents which has as yet been unexploited.
The translocase enzyme is usually assayed by radiolabelling the UDP-MurNAc-pentapeptide and monitoring the transfer of phospho-N-acetylmuramyl pentapeptide from the UDP-MurNAc-pentapeptide to undecaprenyl phosphate, resulting in the formation of a lipid intermediate, Lipid I. The radiolabelling is usually done either by using the enzyme Ligase to label the D-Alanine-D-Alanine end or by the in vivo incorporation on the membrane. Both these methods produce low yields and thus are not cost effective in developing high-throughput-screening (HTS) assays.
The translocase enzyme activity may alternatively be assayed using a fluorescent substrate such as dansyl chloride as described by Brandish et al., J. Biol. Chem., (1996), 271, 7609-7614. However, certain compounds may quench the fluorescence, thus resulting in picking up false inhibitors of the enzyme reaction.
It would be desirable to develop an assay for the translocase enzyme that is suitable for high-throughout screening.
In accordance with the present invention, there is therefore provided an assay for detecting phospho-N-acetylmuramyl-pentapeptide translocase enzyme activity, which comprises the steps of:
(1) incubating a reaction mixture comprising, in aqueous medium, N-succinimidyl [2,3-
3
H] propionate substituted UDP-MurNAc-pentapeptide, N-succinimidyl propionate substituted UDP-MurNAc-pentapeptide (non-radioactive), a source of divalent metal ions, a source of undecaprenyl phosphate, a source of translocase enzyme and a detergent, under conditions suitable for enzyme activity to occur;
(2) acidification of the reaction mixture with a suitable buffer comprising a quaternary ammonium salt at pH~4.2 to stop the enzyme reaction of step (1); and
(3) extraction of any undecaprenol-pyrophosphate-[2,3-
3
H]propionate-N-acetylmuramylpentapeptide product formed and measuring radioactivity using a scintillation counter.
In step (1), the UDP-MPP used may be any of those normally present in naturally occurring peptidoglycans. It is conveniently purified from bacteria or made enzymatically with precursors from bacteria, for example by methods similar to that described by Blaauwen et al.; J. Bacteriol. (1990), 172, 63-70. Alternatively, it may be isolated from cells of
B.subtilits
W23 by the methodology described by Lugtenberg et al.; J. Bacteriol. (1972), 109, 326-335. The preferred UDP-MPP to use is UDP-MurNAc-L-Alanine-&ggr;-D-glutamic acid-m-diaminopimellic acid-D-alanine-D-alanine from
Bacillus cereus.
The UDP-MPP thus obtained is reacted with N-succinimidyl [2,3-
3
H]propionate (commercially available from Amersham Ltd.) to obtain N-succinimidyl [2,3-
3
H]propionate substituted UDP-MurNAc-pentapeptide (hereinafter referred to as “
3
H-propionated UDP-MPP”).
The concentration of
3
H-propionated UDP-MPP used in the assay will typically be in the range from 2 to 50 &mgr;M, preferably from 2 to 40 &mgr;M and more preferably from 2 to 25 &mgr;M.
The concentration of the unlabelled, non-radioactive N-succinimidyl propionate substituted UDP-MPP (hereinafter referred to as “propionated non-radioactive UDP-MPP”) also used in the reaction may be in the range from 5 to 70 &mgr;M, preferably from 5 to 50 &mgr;M and especially from 8 to 30 &mgr;M.
Divalent metal ions used in the reaction are preferably magnesium ions. A suitable source of magnesium ions is magnesium chloride. The concentration of divalent metal ions used may be in the range from 20 mM to 100 mM, preferably from 20 mM to 80 mM, more preferably from 20 mM to 50 mM, e.g. 25 mM.
In addition, potassium chloride at a concentration in the range from 50 mM to 100 mM may be added to the reaction mixture.
The membranes of
Escherichia coli
bacteria may conveniently be used and indeed are preferred as a source of undecaprenyl phosphate and translocase enzyme. The quantity of membranes used will typically be in the range from 5 to 200 &mgr;g, preferably 50 &mgr;g, per 50 &mgr;l of the reaction mixture. The membranes may be prepared by methods known in the art.
The aqueous medium used in step (1) is preferably a buffer solution, e.g. of Tris [hydroxymethyl] aminomethane hydrochloride (“Tris-HCl”), having a pH of about 7.5. Tris-HCl is commercially available from the Sigma Aldrich Co. Ltd.
The reaction mixture may additionally contain 0.01 unit of alkaline phosphatase.
The detergent used may, for example, be Triton X-100 in a concentration of 0.1% w/v. The detergent may be effective in solubilising the bacterial membranes if these are used.
If the assay is intended to be used as a screen for identifying anti-bacterial compounds that are antagonists of the translocase enzyme, the reaction mixture in step (1) may further comprise one or more test compounds in varying concentrations. Since translocase is the enzyme required in the first step of peptidoglycan synthesis, it represents a suitable target for the development anti-bacterial drugs.
The reaction mixture of step (1) is maintained at a temperature in the range from 20° C. to 37° C., preferably 25° C., for a short period of time, e.g. up to 10 minutes, specifically 6-8 minutes.
The enzyme reaction is stopped by the addition of, for example, 6M pyridinium acetate and n-butanol (pH~4.2) in a 2:1 mixture. This constitutes step (2).
In step (3), the product is extracted using, for example, n-butanol. It is then quantified in a scintillation counter.
REFERENCES:
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Cook, Neil D. Scintillation proximity assay: a versatile high-throughput screening technology. Drug Discovery Today 1: 287-294, 1996.
Brandish, P. E. et al.Modes of Action of Tunicamycin, Liposidomycin B, and Mureidomycin A: Inhibition of Phospho-N-Acetylmuramyl-Pentapeptide Translocase from Escher
Das Kaveri
Ramachandran Janakiraman
AstraZeneca AB
Leary Louise N.
White & Case LLP
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