Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-08-23
2004-05-11
Weddington, Kevin E. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S009100, C514S011400, C514S012200, C514S408000
Reexamination Certificate
active
06734165
ABSTRACT:
Throughout this application, various references are identified by citations or a number in parenthesis, in which case their full citations appear on the pages following the Detailed Description immediately preceding the claims. Disclosure of these references in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
FIELD OF THE INVENTION
The present invention relates to a method for re-sensitizing vancomycin resistant Gram-positive bacteria in which resistance results from the conversion of an amide bond to an ester bond in the cell wall peptide precursors of the bacteria which comprises using an antibacterial amount of vancomycin or a homolog of vancomycin and an amount of an agent effective to selectively cleave the ester bond so as to thereby re-sensitize vancomycin resistant bacteria.
BACKGROUND OF THE INVENTION
The introduction of the first antimicrobial agents allowed physicians and patients to manage infectious diseases effectively. Unfortunately, even before penicillin was introduced commercially, researchers had identified the first resistant
Staphylococcus aureus
(1). Since then, many new antimicrobials have been developed to combat the emergence of resistance. These advances increased the confidence that infectious diseases are non-fatal and still manageable. Nevertheless, with the medical progresses that allow people to live longer and the boost of various debilitating immune conditions (AIDS, cancer, organ transplants), a new human population emerged as being at great risk of infectious diseases caused in majority by two common nosocomial pathogens, Staphylococcus and Enterococcus species (2). The incidence of staphylococcal strains resistant to virtually all the antimicrobial agents except vancomycin has increased drastically during the last decade. Additionally, enterococcal infections are becoming increasingly resistant even to vancomycin, raising the alarming possibility that resistance genes will eventually be transmitted to staphylococci (3-5). One of the greatest concerns with vancomycin resistant enterococci (VRE) is that the resistance to vancomycin will be picked up by
Staphylococcus aureus
through genetic recombination. Many forms of
S. aureus
have already become resistant to methicillin and can now only be treated with vancomycin. There is significant concern that if
S. aureus
also becomes resistant to vancomycin, the health care profession will be left without treatment for these types of infections. Increasing resistance among several types of Gram-positive bacteria associated with common and potentially life-threatening infections complicate the treatment of serious infections and has been linked to extended hospitalizations, higher medical costs and high mortality rates.
Like the family of &bgr;-lactam antibiotics, vancomycin acts on peptidoglycan metabolism. The peptidoglycan is essential for bacterial survival because of its function as the exoskeleton that prevents cell rupture due to internal pressure. By binding to the D—Ala—D—Ala moiety of the bacterial cell wall precursors, vancomycin interferes with the growth of the peptidoglycan (6). In the resistant strains with vanA or vanB phenotype however, some of the D—Ala—D—Ala moiety of the cell wall precursors is substituted by analogous D—Ala—D—Lac ones (7-9). Only a small percentage of the enterococcus peptidoglycan layer is needed to be structurally altered from D—Ala—D—Ala to D—Ala—D—Lac to cause an increase in the vancomycin MIC (10% of the altered peptidoglycan increases the MIC of vancomycin from 2 to 32 ug/ml).
Resistant bacteria carry a transportable element encoding nine genes that contribute to the resistance phenotype (10). These gene products include VanS, a transmembrane protein that senses directly or indirectly the presence of vancomycin. Once autophosphorylated, VanS transmits a signal to a response regulatory protein VanR that activates transcription of the other resistance genes (11). VanA is involved in the synthesis of the depsipeptide D—Ala—D—Lac while VanH converts pyruvate into D-lactate. This pathway is essential for the resistance phenotype (12,13). VanX is a Zn
2+
dependent pepsidase that selectively cleaves D—Ala—D—Ala leading to an accumulation of the depsipeptide, and thus, of precursors with altered D—Ala—D—Lac termini (14,15). VanY is a membrane bound D—D-carboxypeptidase that hydrolyses the normal cell wall precursor lipid-intermediates, further increasing the pool of precursors with altered termini (16). However, the formation of D—Ala—D—Ala continues in the cell due to the activity of the native enterococcal D—Ala—D—Ala ligase. Because vancomycin binds to D—Ala—D—Ala substrates, a mechanism is required to prevent D—Ala—D—Ala from being incorporated into the cell wall. VanX and vanY perform this function. As a result of the incorporation of D—Ala—D—Lac by vancomycin resistant enterococcus (VRE), the affinity of vancomycin for the peptidoglycan layer diminishes over 1000-fold, leading to antibiotic resistance. VanA strain is the most common phenotype of VRE and is described by inducible, high-level resistance that is associated with the van genes that lead to D—Ala—D—Lac altered termini.
In order to bypass resistance, vancomycin has been modified to enhance its binding to D—Ala—D—Lac and inhibitors of the D—Ala—D—Lac biosynthetic pathway have been sought (17-19). Here we propose another approach—the selective and catalytic cleavage of the D—Ala—D—Lac depsipeptide by small molecules. By reducing the concentration of precursors with altered termini one would expect to re-sensitize the bacteria to vancomycin. A small molecule that performs such task could be used in concert with vancomycin (or vancomycin derivatives with higher affinity) in the treatment of vanA resistant strains.
SUMMARY OF THE INVENTION
One of the most challenging situations for the immuno-compromised patients is the development of vancomycin-resistant enterococci (VRE), an increasingly frequent cause of hospital-acquired infections in the United States. These organisms are resistant to virtually all currently available antibiotics including vancomycin, considered the agent of last resort for Gram-positive infections. VanA strain is the most common phenotype of VRE and is described by inducible, high-level resistance that is associated with the van genes that lead to D—Ala—D—Lac cell wall altered termini. Here we describe the development of small molecules that catalytically and selectively cleave the altered termini of the bacteria cell wall so as to disable the antibiotic-resistance mechanism in these pathogens. The molecules re-sensitize bacteria to the drug and could be used in concert with vancomycin in the treatment of VRE.
The invention provides a method of treating a subject afflicted with an infection caused by glycopeptide antibiotic resistant Gram-positive bacteria, such as vancomycin resistant Gram-positive bacteria, in which resistance results from the conversion of an amide bond to an ester bond in the cell wall peptide precursors of the bacteria which comprises administering to the subject an antibacterial amount of glycopeptide antibiotic, such as vancomycin or a homolog of vancomycin, and an amount of an agent effective to selectively cleave the ester bond so as to thereby treat the subject.
The invention also provides a method of killing glycopeptide antibiotic resistant Gram-positive bacteria, such as vancomycin resistant Van A, Van B, Van D, or Van G Gram-positive bacteria which comprises contacting the bacteria with an agent that selectively cleaves D—Ala—D—Lac cell wall depsipeptides in the bacteria in an amount effective to cleave such depsipeptides and an antibacterial amount of glycopeptide antibiotic, such as vancomycin or a homolog of vancomycin, so as to thereby kill the bacteria.
The invention further provides a method for determining whether a test compound selectively cleaves an ester bond present between two amino acid-like moieties in a depsipeptide whic
Boneca Ivo G.
Chiosis Gabriela
Still W. Clark
Cooper & Dunham LLP
The Trustees of Columbia University in the City of New York
Weddington Kevin E.
White John P.
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