Recombinant Staphylococcus thioredoxin reductase and...

Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales

Reexamination Certificate

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C424S094100, C424S139100, C424S165100, C424S185100, C424S237100, C424S243100, C435S007330, C435S007700, C435S036000, C435S091100, C435S091500, C435S091510

Reexamination Certificate

active

06767536

ABSTRACT:

TECHNICAL FIELD
This invention relates generally to microbial metabolism and antimicrobial therapeutic agents. In particular, the invention relates to the bacterial enzyme thioredoxin reductase, to compounds that inhibit this enzyme, and to the use of these compounds as antimicrobial agents, particularly for the therapy of infections caused by Staphylococcus spp.
BACKGROUND OF THE INVENTION
The thioredoxin system is composed of NADPH, thioredoxin (Trx) and the flavoenzyme thioredoxin reductase (TrxB). Trx reduction by TrxB involves two half-reactions. In the first half-reaction, the FAD prosthetic group of TrxB is reduced by NADPH and electrons are transferred to cysteines present in the active site of TrxB. In the second half-reaction, oxidized Trx is reduced by TrxB. The thioredoxin system serves to transfer reducing equivalents for reductive enzymes such as ribonucleotide reductase, methionine sulfoxide reductase and vitamin K epoxide reductase. It also mediates protein folding and exerts specific redox control of some transcription factors to modulate their binding to DNA.
The thioredoxin system is of particular importance for redox metabolism in some Gram-positive bacteria. In this regard, certain Gram-positive bacteria, such as staphylococci, lack detectable glutathione (GSH) and glutathione reductase (GSR) which together play a key role in maintaining intracellular thiol-disulfide balance. GSH is the predominant thiol produced by aerobic eukaryotes and some Gram-positive bacteria, is believed to protect aerobic organisms from oxygen toxicity, and participates in a multitude of functions. For example, GSH plays a pivotal role in management of oxidative stress and maintenance and regulation of the redox balance. It acts as a cofactor for peroxide and ribonucleotide reductions, and serves in the conjugation and detoxification of foreign substances. Most organisms contain millimolar intracellular concentrations of GSH which, in concert with GSR and glutathione peroxidase, governs the redox status of the cellular environment. Thus, in microorganisms lacking the glutathione system, such as
Staphylococcus aureus
, the thioredoxin system, which is able to substitute for some of the glutathione-dependent processes, is of utmost importance.
The TrxB component of the thioredoxin system is a FAD-containing enzyme and belongs to a family of pyridine nucleotide-disulfide oxidoreductases. The bacterial enzyme obtained from
Streptomyces clavuligerus
, is a homodimer of 35 kDa subunits and has a native molecular weight of approximately 70 kDa. Aharonowitz et al. (1993)
J. Bacteriol
. 175:623-629. Each subunit of TrxB contains NADPH- and FAD-binding domains and includes an oxidoreductase active dithiol in the conserved sequence -CAT/VC-. Since the cysteine residues of TrxB are relatively inaccessible to the substrate thioredoxin, the enzyme appears to undergo a large conformational change during catalysis.
TrxBs from different mammalian species including calf (Holmgren, A. (1997)
J. Biol. Chem
. 252:4600-4606), rat (Luthman et al. (1982)
Biochem
. 21:66628-6633), and human (Arscott, et al. (1997)
Proc. Natl. Acad. Sci. USA
94:3621-3626), have been purified and biochemically characterized. The rat liver TrxB has been isolated as a 116 kDa homodimer of 58 kDa subunits, and the mass of human placental TrxB estimated to be 160 kDa by gel-filtration chromatography and 130 kDa (two 65 kDa subunits) by sucrose density gradient centrifugation. The size difference between human TrxB and the smaller bacterial TrxBs is primarily due to differences in the dimer-interface domain. The redox-active cysteines of human TrxB are located in the FAD domain with a 4-amino acid bridge linking the two cysteines. The active-site disulfide of bacterial TrxBs, on the other hand, is located within the NADPH domain and a 2-amino acid bridge links the two cysteines. The 3D structure of the human TrxB is likely to be more similar to GSR than to the bacterial TrxB. Thus, primary amino acid sequence alignment of human TrxB with bacterial TrxB sequences reveals just 23-31% identity, whereas alignment of the human TrxB with GSRs of different sources shows 35-44% identity. Arscott, et al. (1997)
Proc. Natl. Acad. Sci. USA
94:3621-3626.
Based on the significant differences that exist between the bacterial and the mammalian trxB genes, and the enzymes encoded thereby, the bacterial TrxB provides a potential target for the development of novel antibacterial drugs with a high degree of selectivity.
Antimicrobial agents commonly used to combat bacterial infections generally interfere with one or more critical steps in the metabolism of the bacterium, resulting in growth inhibition or death of the microbe. However, pathogenic microorganisms, including staphylococci, are developing resistance, and in many cases multiple resistances, to existing antimicrobial agents. In this regard,
S. aureus
is an opportunistic pathogen of increasing medical concern. It can be aggressively invasive, spreading rapidly through soft tissues, directly invading bones and even entering the bloodstream to produce septic shock and disseminated intravascular coagulation. Infections caused by staphylococci generally fall within one of two categories: those related to toxins produced by the bacterium exclusively, including gastroenteritis, toxic shock syndrome, scalded skin syndrome, and the like; and those related to direct invasion and systemic spread of the organism, including dermal infections, bone and joint infections, staphylococcal pneumonia and empyema, meningitis, cerebritis, endocarditis, bacteremia, septic shock, and the like.
These staphylococcal infections have traditionally been treated with &bgr;-lactam antibiotics. However, strains of &bgr;-lactam antibiotic-resistant staphylococci (BLARS), such as methicillin-resistant
S. aureus
(MRSA), have developed and become a widespread cause of fatal nosocomial infection. Infections caused by such resistant staphylococci are treated predominantly with “last resort” antibiotics such as vancomycin. Since resistance to these antibiotics would essentially exhaust the current therapeutic arsenal, it is essential that new antibacterial agents be identified.
SUMMARY OF THE INVENTION
The inventors herein have identified a bacterial thioredoxin reductase (TrxB) from Staphylococcus spp. that catalyzes, in two half-reactions, the specific NADPH-dependent reduction of thioredoxin (Trx), with the concomitant oxidation of NADPH to NADP
+
. In the first half-reaction, the FAD prosthetic group of TrxB is reduced by NADPH and electrons are transferred to cysteines present in the active site of TrxB. In the second half-reaction, oxidized Trx is reduced by TrxB.
The thioredoxin system provides a significant metabolic function in staphylococci and other Gram-positive bacteria that do not produce glutathione (GSH). The thioredoxin system catalyzes a broad range of protein thiol-disulfide exchange reactions, donates hydrogen for ribonucleotide reductase which is an essential enzyme in DNA synthesis, and is involved in redox regulation of numerous enzyme activities.
Staphylococcal TrxB differs significantly in its function from that of the mammalian enzyme in two important ways. First, it operates in an intracellular environment lacking GSH and GSH-dependent reductases. Second, its subunits are appreciably smaller than the mammalian enzyme and the dimeric enzyme possesses a substrate specificity distinct from its mammalian counterpart. Inhibition of Staphylococcal TrxB activity may cause depletion of reduced low molecular weight thiols, increase protein thiol oxidation, and interfere with DNA synthesis and radical scavenging. Such compromised cells are more likely to succumb to environmental challenges, such as those posed by the host immune system.
Consequently, bacterial TrxB provides an excellent target for the development of novel antibacterial drugs with a high degree of selectivity.
Such antibacterials act by inhibiting TrxB, thereby incapacitating the target bacterium, with few or no side-effects

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