Infectious complications of trauma injuries often require treatment of multi-drug resistant bacterial pathogens, and their secreted toxins. Bacterial toxins play a significant role in pathogenesis, virulence, tissue damage and inflammation. War wound infections have long posed a major challenge for military medicine and, infectious complications will remain a major cause of morbidity and mortality.
US casualties from Iraq and Afghanistan often have wounds that are colonized or infected with multidrug-resistant strains of Staphylococcus aureus. Other frequently identified resistant strains of bacteria are Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Overuse of broad-spectrum antibiotics may be the primary cause in emergence of these resistant strains [1]. In addition, these pathogens secrete toxins which enhance virulence and tissue damage. Such factors likely lead to hard to heal wounds.
When toxin producing pathogens invade and multiply in tissues, it is likely that during infection, sensitive cells will be exposed to their products, such as toxins that provoke active cellular responses. Synthesis and secretion of inflammatory cytokines is, of course, one of the countless possible reactions that will follow as a consequence of infections. S. aureus expresses numerous virulence factors including α-toxin/hemolysin that are aimed at weakening the immune system by direct inactivation of key innate response molecules. As a pore forming factor, α-toxin plays a major role in the pathogenesis of S. aureus infections and, therefore, is an important target for therapeutic intervention [8, 11]. Although early intervention with antibiotics can reduce the bacterial burden, inhibitors of secreted toxins could offer a therapeutic advantage in the later stages of infection involving toxemia. Currently, no such inhibitors are available to neutralize α-toxin mediated cytolytic and cytotoxic effects. Therefore, the present invention was undertaken to identify DNA based inhibitors/aptamers to detoxify α-toxin induced toxicity in S. aureus infections.
Aptamers represent a class of macromolecules with attractive drug properties which could be useful in the treatment of a variety of ailments. Aptamers are nucleic acids having specific binding affinity to multiple types of molecules through interactions other than classic Watson-Crick base pairing. Aptamers, like peptides generated by phage display or monoclonal antibodies (“MAbs”), are capable of specifically binding to selected targets, and modulating the target's activity. For example, binding of aptamers to a target may block the target's ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size, consists of 30-45 nucleotides, binds its target with sub-nanomolar affinity, and discriminates against closely related targets. Aptamers will typically not bind other proteins from the same gene family. A series of structural studies have shown that aptamers are capable of using the same types of binding interactions, such as hydrogen bonding, electrostatic complementarity, hydrophobic contacts and steric exclusion that drive affinity and specificity in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologics, for example speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial leads, including therapeutic leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled, and allows the generation of leads, including leads against both toxic and non-immunogenic targets.
Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments.
Aptamers (protein-binding oligonucleotides) have great potential as a new class of targeted therapeutics. These molecules are artificial, single-stranded nucleic acid ligands (DNA and RNA) generated against amino acids, proteins, drugs, viruses and whole cells [2]. They are discovered by an in vitro directed evolution process called SELEX but are ultimately chemically synthesized like small molecule therapeutics using solid-phase techniques that are easily scaled up and allow for modification and conjugation strategies [3, 4, and 5]. Aptamers emerged as a new class of molecules that rival antibodies in both therapeutic and diagnostic applications. One of the advantages of DNA-based aptamer drugs over currently available low molecular weight pharmaceuticals or antibodies is their selective recognition of molecular targets which imparts tremendous specificity of action with binding affinities in the low nano- to picomolar range. Because of their unique three dimensional structures, aptamers are able to bind to functional domains of the target, thereby modulating the biological function of the molecule. Their small size enables them to access epitopes that might otherwise be blocked or hidden. Aptamer selection and identification is much faster than monoclonal antibodies and allows optimization of binding affinity by successive rounds of selection and screening. Antibodies generally elicit strong immune responses due to their size, whereas aptamers show little or no immunogenicity. Further, aptamers are thermally stable and preserve their structure over a wide range of temperatures, exhibit low toxicity, and can be generated to recognize a wide range of targets, including small molecules and ions.
In spite of the infancy of aptamer therapeutics, these molecules have produced striking results in both preclinical and clinical studies. Several aptamers are currently in clinical trials, or being evaluated, for different diseases [25, 26]. To date, there are a few aptamers identified against bacterial and plant toxins that have the potential to inhibit the biological functions of the respective toxin both in vitro and in vivo [27-29]. Recent work by Liang et al. further reinforces the therapeutic potential of aptamers targeting rabies virus and demonstrated the aptamer mediated inhibition of rabies viral multiplication both in vitro and in vivo [30]. Most notably, the only DNA aptamer identified against one of the S. aureus exoproteins was against enterotoxin B (SEB) and is designated as APTSEB. This aptamer was developed not as a therapeutic, but to be used in the diagnostic platform to detect SEB [31].
The present invention provides novel small drug molecules or aptamers and method of using them to neutralize the lethal effects of Staphylococcus aureus alpha toxin for treatment of S. aureus infections.