Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-01-28
2003-10-07
Smith, Lynette R. F. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C514S012200, C514S013800, C514S014800, C530S300000, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000, C530S350000
Reexamination Certificate
active
06630583
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of novel antibiotic peptides, including naturally occurring peptides. The nucleic acid sequence encoding the peptide and the corresponding amino acid sequence are included, together with methods of using the same.
BACKGROUND OF THE INVENTION
Bacterial infections remain among the most common and deadly causes of human disease. Unfortunately, the overuse of antibiotics has led to antibiotic resistant pathogenic strains of bacteria. Indeed, bacterial resistance to the new chemical analogs of these drugs appears to be out-pacing the development of such analogs. For example, life-threatening strains of three species of bacteria (
Enterococcus faecalis, Mycobacterium tuberculosis
, and
Pseudomonas aeruginosa
) have evolved to be resistant against all known antibiotics. [Stuart B. Levy, “The Challenge of Antibiotic Resistance”, in
Scientific American
, pgs. 46-53 (March 1998)]
Classical penicillin-type antibiotics bind to cell wall synthetic enzymes and thereby deregulate the activity of a single class of proteins known as autolysins which leads to bacterial lysis and bacterial cell death. The development of new drugs which affect an alternative bacterial target protein would be desirable. Pneumococcus is a particularly relevant organism for such study because 1) it has only one predominant autolysin (LytA rather than the multiple autolysins of other bacteria), 2) the autolysin has been cloned and sequenced and can therefore be easily manipulated genetically, and 3) pneumococcus has only one growth zone so that is possible to study activation of the enzyme in a fairly defined region of the cell.
Most bacteria are stabilized by a cell wall consisting of a glycopeptide polymeric murein (peptidoglycan) that completely encloses the cell [Weidel & Pelzer et al.,
Enzymol
., 26:193-232 (1964)]. Expansion of the cell wall during bacterial growth and splitting of the septum for cell separation requires enzymes that can cleave this covalently closed network. In addition to acting as spacemaker enzymes for cell wall growth [Tomasz et al.,
Walter de Gruyter
, 155-172 (1983)], certain murein hydrolases also act as autolysins, putative suicide enzymes. The life and death dichotomy of autolysin function demonstrates the need for efficient and strict regulation of murein hydrolase activity, a paradigm conceptually similar to that for caspases in the process of eukaryotic apoptosis. Not surprisingly, the regulation of the autolysins is a highly sophisticated physiological task. For example, the enzymes must be controlled at their extracytoplasmic location. In addition, most bacteria possess multiple hydrolases which must be controlled in concert. Antibiotics such as penicillin induce bacteriolysis by interfering with the control of the endogenous autolytic enzymes, indicating the significant chemotherapeutic relevance of these enzymes.
Although the binding of antibiotics to cell wall synthetic enzymes has been very well characterized, it is unknown how this event leads to deregulation of autolytic enzymes. During normal cell growth, autolysin activity is believed to be subject to strong, prolonged downregulation. Thus, the expression of most hydrolases is constitutive throughout the cell cycle but the enzymes are only physiologically active during stationary phase lysis [Hakenbeck and Messer,
Eur. J. Biochem
, 129:1239-1244 (1977); Ronda et al.,
Eur. J. Biochem
., 164:621-624 (1987)]. Autolysin activity during exponential phase also remains curtailed even when the gene is constitutively expressed from a plasmid [Ronda et al.,
Eur. J. Biochem
., 164:621-624 (1987)]. This indicates regulation of autolysin activity is independent of transcription of the autolysin itself. In addition, cell wall hydrolases are continuously present on the cell surface and, since triggering of wall hydrolysis does not require the synthesis of new enzyme [Kitano and Tomasz,
Antimicrob. Agents Chemother
., 16:838-848 (1979)], these enzymes must be prevented from potential hydrolytic activity.
The most striking example of physiological down regulation of autolysis is the stringent response that occurs during deprivation of an essential nutrient [Cashel et al., The Stringent Response, In
Escherichia coli
and Salmonella: Cellular Molecular Biology, Neidhardt et al., Eds., (Washington, D.C.: ASM Press, (1996)]. Starved bacteria bind antibiotic normally, but do not lyse or die. Upon starvation, bacteria rapidly accumulate guanosine 3′,5′-bispyrophosphate (ppGpp), which is synthesized by ppGpp synthetase I encoded by the relA gene [Metzger et al.,
J. Biol. Chem
., 263:15699-15704 (1988); Schreiber et al.,
J. Biol. Chem
., 266:3760-3767 (1991); and Svitil et al.,
J. Biol. Chem
., 268:2307-2311 (1993)]. ppGpp in turn coordinately shuts down the synthesis of macromolecules such as DNA, phospholipids [Sokawa et al.,
Biochem. Biophys. Res. Commun
., 33:108-112 (1968)] and cell wall peptidoglycan [Ishiguro and Ramey,
J. Bacteriol
., 127:1119-1126 (1976)]. In this setting, antibiotic-induced lysis is blocked by an as yet uncharacterized defect in autolysin activation. This protection from death, termed phenotypic tolerance, is a property of all non-growing bacteria and forms the basis of antibiotic selection for auxotrophs [Hobby et al.,
Proc. Soc. Exp. Biol
., 50:281-285 (1942) and Tuomanen,
Revs. Infect. Dis
., 8
Suppl
. 3:279-291 (1986)]. Phenotypic tolerance during an infection is an important source of residual bacteria that survive antibiotic therapy in vivo, and can thereby promote the subsequent acquisition of antibiotic resistance and the concomitant failure of the antibiotic therapy [Handwerger and Tomasz,
Revs. Infect. Dis
., 7:368-386 (1985); Novak et al.,
Nature
, 399:590-593 (1999); Tuomanen et al.,
Antimicrob. Agents Chemother
., 30:521-527 (1986); Tuomanen et al.,
J. Bacteriol
, 170:1373-1376 (1988); and Tuomanen et al.,
J. Infect. Dis
., 158:36-43 (1988)].
Antibiotic tolerance, a phenomenon distinct from antibiotic resistance, was first described in 1970 in pneumococci and provided a significant clue to the mechanism of action of penicillin [Tomasz et al.,
Nature
, 227:138-140 (1970)]. Tolerant strains stop growing in the presence of conventional concentrations of antibiotic, but do not subsequently die. Tolerance arises when the bacterial autolytic enzymes, i.e., autolysins, fail to be triggered as the antibiotic inhibits the cell wall synthetic machinery. This explicitly implies that penicillin kills bacteria by activating a set of endogenous hydrolytic enzymes and that bacteria exhibit strategies to stop this activation resulting in survival of antibiotic therapy.
Tolerance is of clinical significance since it has been shown that the inability to eradicate tolerant bacteria leads to failure of antibiotic therapy in clinical infections [Handwerger and Tomasz,
Rev. Infect. Dis
., 7:368-386 (1985); Tuomanen E.,
Rev. Infect. Dis
., 3:S279-S291 (1986); and Tuomanen et al.,
J. Infect. Dis
., 158:36-43 (1988)]. Furthermore, tolerance is thought to be a prerequisite to the development of antibiotic resistance since it creates survivors of antibiotic therapy. These survivors can then acquire new genetic elements of resistance which allow growth in the presence of antibiotics. Virtually all resistant strains also have been shown to be tolerant [Liu and Tomasz,
J. Infect. Dis
., 152:365-372 (1985)]. Therefore, the identification of novel antibiotics which can lyse these “antibiotic-tolerant” bacteria is necessary.
Mechanistically speaking, tolerance arises in two settings: 1) all bacteria become phenotypically tolerant as growth rate decreases [Tuomanen E.,
Revs. Infect. Dis
., 3:S279-S291 (1986)] and 2) some bacteria are genotypically tolerant by virtue of acquisition of mutations. In both cases, the basic phenomenon is the down regulation of autolysin triggering. This down
Novak Rodger
Tuomanen Elaine I.
Butler, Snow O'Mara, Stevens & Cannada, PLLC
Smith Lynette R. F.
St. Jude Children's Research Hospital
Zeman Robert A.
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