Use of the crystal structure of Staphylococcus aureus...

Details Use of the crystal structure of Staphylococcus aureus... Use of the crystal structure of Staphylococcus aureus...

2000-07-31

2003-10-07

Brusca, John S. (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Biological or biochemical

C702S027000, C435S004000, C435S193000, C530S350000, C536S023100

Reexamination Certificate

active

06631329

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the crystalline structure of isoleucyl-tRNA synthetase and the cognate tRNA
ile
and to methods of producing such crystals. The invention also relates to the atomic coordinates of isoleucyl-tRNA synthetase and the cognate tRNA
ile
, obtained by x-ray diffraction at high resolution. The present invention also relates to methods for identifying and designing new classes of ligands which target the isoleucyl-tRNA synthetases of specific organisms. The methods and compositions of the present invention find wide applicability in the design and production of antibiotics, insecticides, miticides and herbicides.
BACKGROUND
Mupirocin
The most important invention in medicine in this century is perhaps the discovery of penicillin by Alexander Fleming in 1928, a naturally occurring antibiotic that inhibits cell-wall synthesis in many pathogenic bacteria. In 1940, E. B. Chain and H. W. Florey were able to produce stable commercial formulations of this antibiotic. For this invention, Fleming, Chain, and Florey shared the Nobel Prize in medicine or physiology in 1945.
In the past half century, from penicillin to methicilin to vancomycin, over 130 related antibiotics have been discovered that inhibit cell-wall synthesis (Neu, 1991). The art of the discovery is relatively simple; it requires simply a combination of microbiology and organic chemistry. Any organic chemical that inhibits bacterial cell growth by acting on cell-wall synthesis are good antibiotics, since only bacteria, not human cells, have cell wall. In comparison, the same approach that has worked for the discovery of antibiotics that inhibit cell-wall synthesis has not worked well for the discovery of antibiotics that inhibit protein synthesis.
The antibiotic for inhibition of protein synthesis, pseudomonic acid, remains in its original form since it was first discovered about three decades ago by E. B. Chain and his colleagues (Fuller et al., 1971). However, it has been since renamed as mupirocin. Mupirocin is the active ingredient of Bactroban™, a trademark of SmithKline Beecham. All attempts so far have failed to modify this antibiotic with either improved stability against unknown human hydrolase(s) for in vivo use or improved selectivity for its pathogenic target enzyme over human enzyme, simply because no organic chemists know how to modify the antibiotic to achieve the above goals.
Staphylococcus aureus
(
SA
), present in about two-thirds of healthy individuals in the entire population, has a long association with nosocomial infection and is a virulent pathogen that is currently the most common cause of infections in hospitalized patients (Archer, 1998, Gould and Chamberlaine, 1995). In 1941, virtually all strains of
S. aureus
worldwide were susceptible to penicillin G, the first antibiotic used in clinics, but by 1944
, S. aureus
began to become resistant to the antibiotic, and by late 1980s, more than 95% of
S. aureus
worldwide were resistant to penicillin, amplicillin, and the antipseudomonas penicillins (Lyon and Skurray, 1987). In response, the pharmaceutical industry produced a second generation antibiotic, methicillin, a semisynthetic penicillin. However, methicillin-resistant
S. aureus
(MR
SA
) became a severe problem in the 1980s (Vandenbrouche-Grauls, 1994, Mulligan et al., 1993), and is resistant to all &bgr;-lactams because it produces a new penicillin binding protein to remove all related antibiotic, pencillins, cephalosporins, carbapenems, and penems (Lyon and Skurray, 1987; Ubukata et al., 1985; Murakami and Tomasz, 1989; Tesch et al., 1988; Chambers and Sachdeva, 1990). The emergence of MR
SA
as a major problem worldwide has resulted in an increased use of vanomycin, the only effective antibiotic and often reserved for use in patients who are gravely ill. Its increased use has created vancomycin-resistant pathogens including
S. aureus
(Flores and Gordon, 1997, Perl, 1999, Paterson, 1999, Neu, 1992).
Mupirocin, a derivative of pseudomonic acid from
Pseudomona fluorescens
(Fuller et al., 1971), is highly effective against MR
SA
(Bertino, 1997, Dacre et al., 1986). Differing from cell wall-inhibiting antibiotic, it binds isoleucyl-tRNA synthetase (IRS) as a competitive inhibitor for isoleucine and inhibits protein biosynthesis (Hughes and Mellows, 1978ab; Hughes and Mellows, 1980; Yanagisawa et al., 1994; Pope et al., 1998ab). Topical use of mupirocin has very successfully eradicated the nasal carriage of MR
SA
(Harbarth et al., 1999; Redhead et al., 1991; Casewell and Hill, 1989; Caderna et al., 1990). This is extremely important because the anterior opening to the nasal cavities (i.e., the naris or nares), are the major site where MR
SA
and susceptible staphylococci persist. Topical use also eradicated MR
SA
in skin and virginal infections after the failure of intervenous vancomycin therapy (Denning and Haiduven-Griffiths, 1988, Cool-Foley et al., 1991). Despite these success, a person could still die in a hospital in any major city with a resistant bacterial infection. Although mupirocin resistant
S. aureus
(MUR
SA
) is rare, it exists (Anthony et al., 1999; Schmitz et al., 1998; Gilbart et al., 1993; Farmer et al., 1992; Capobianco et al., 1989; Eltringham, 1997; Woodford et al., 1998).
Mupirocin is not very effective against bacteremia caused by MR
SA
because of its short half-life metabolic conversion in vivo from pseudomonic acid to inactive monic acid, which is rapidly cleared in the urine (Mellows, 1989). The pharmaceutical industry has been unsuccessful in slowing or halting the enzymatic hydrolysis by modifying the structure and function of the C1-C3 fragment, although the modified antibiotic retains good in vitro activity (Rogers 1980, Rogers and Coulton, 1882, Banks et al., 1989). This fragment has also been replaced by an unsaturated 5-membered heterocycle ring, but it must retain low-energy unoccupied molecular orbital for its inhibitory activity (Brown et al., 1997).
Selectivity of Isoleucyl-tRNA Synthetase
The high fidelity of genetic information transfer in translation is essential for the survival of organisms. Translation accuracy depends on the ability of amino acid tRNA synthethases to discriminate among tRNAs and among amino acids in amino acylation. Discrimination of L-isoleucine over L-valine by isoleucyl-tRNA synthetase is one of most difficult recognitions to achieve, because L-isoleucine and L-valine differ by only one methylene group in their aliphatic side chains. Additionally, this enzyme is the target of mupirocin, the only effective antibiotic that inhibits protein synthesis. This enzyme has therefore been extensively studied in over a half century, leading to the present invention (Silvian et al., 1999).
Isoleucyl-tRNA synthetase (IRS) selectively adds isoleucine to isoleucyl-tRNA, while rejecting all other amino acids and all other noncognate tRNAs. This enzymatic selectivity of isoleucine over valine is over 3000-fold (Loftfield, 1963; Loftfiled and Vanderjagt, 1972). If IRS were an inorganic catalyst, a free energy difference of one single methylene group between the two amino acids would provide only about 5-fold difference in selectivity (Pauling, 1958) based on an adsorption theory, which has successfully explained catalytic mechanisms for nearly all inorganic catalysts. According to the theory, inorganic catalysts (such as transition metal ions) accelerate rates of chemical reactions by increasing the collision frequency through adsorbing two reactants on catalysts' surface. The rate of enhancement is a function of adsorption of free energy, and the selectivity of given reactions is a function of free energy differences in the adsorption. A large discrepancy in selectivity of the synthetase led Baldwin and Berg (1966) to discover the hydrolytic activity of the enzyme and led Dingwall and Fersht (1979a,b) to propose a “double-sieve” hypothesis. This hypothesis predicted that there are two distinct enzymatically active sites, one hydrolytic and one synthetic. Amino acids that are larger tha

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