We developed a range of small molecules to provide a generic toolbox for the modulation of microbiota. Such compounds are needed in diagnostics to suppress background flora in enrichment media but also in therapies to change population compositions where disturbed microbiota are a cause of disease. We synthesized a number of compounds containing three functional elements: an enzyme labile group to facilitate species selectivity and a biocide that is released upon cleavage by species selective enzyme activity and additional elements and to enhance chemical stability.
The enzyme-responsive inhibitors are non-inhibitory in their original state and exert their growth inhibition only after the enzymatic release of the biocide that acts through its metal chelating activity. Addition of metal ions is used to modulate the activity and prevent effects on the non-target populations.
Over millions of years of co-evolution, microbial communities (microbiota) have developed that live in close association with their hosts (e.g., animals or plants). It is now generally recognized that microbiota colonizing humans and animals play a key role in the host’s health and disease state. The human gut, for example, harbors one of the most complex microbiota, and disturbance of the normal gut microbiology have been implicated in many health and disease issues.
These findings have spurred intensive research to find ways of influencing microbiota to result in human health benefits. Early attempts to alter microbiota composition primarily involved the use of probiotic bacteria (e.g., Lactobacilli or Bifidobacteria) or prebiotic food components (e.g., nondigestable oligosaccharides) as food or feed additives. However, these approaches are limited in that they target undefined clinical indications by unknown mechanisms of action and often do not evoke the desired positive health effect. In case of probiotics, there is also a potential risk for transfer of drug-resistant genes or harmful infection and the problem of inconsistent product quality, leading to differing results.
Another approach is based on a medical procedure known as fecal microbiota transplantation (FMT). This procedure involves the infusion of a fecal suspension from a healthy donor into the gastrointestinal (GI) tract of a patient to restore the intestinal microbiota. It was found that FMT can have some therapeutic effects on gastrointestinal disorders, e.g. infections caused by Clostridium difficile. However, the efficacy of FMT remains in question. In addition, FMT is cumbersome, unpleasant and carries the risk of spreading infectious diseases.
A more recent strategy to modulate the microbiota composition and function is based on the use of antimicrobial peptides (AMPs). AMPs are promising alternatives to conventional antibiotics because of their natural, broad-spectrum antimicrobial properties and low propensity for development of induced resistance. AMPs, for example, have been demonstrated to positively modulate the intestinal microbiota and to alter the oral microbiota for caries prevention. However, AMPs have disadvantages that limit their use, including hemolytic activity toward human cells, rapid turnover in the human body, reduced activity due to their fragile chemical nature and/or sensitivity to the environment, limited scope of application and/or high cost of production.
Enzyme-activated inhibitors open the way
for new therapies and healthcare products
Intensive efforts have been made to develop small-molecule antimicrobials as alternatives to existing antimicrobial compounds. Scientists were compelled to look for new options for identifying novel antimicrobials. Among other things, they explored strategies for exploiting beneficial and commensal bacteria in microbiota, including the development of generic systems based on the principle of microbial auto-inhibition (i.e. the self-inhibition of a given microbial species by an inhibitory substance generated by the species itself).
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At Biosynth we designed the beta-galactosidase-responsive inhibitor GalactoZide™II and tested it on beta-galactosidase negative Salmonella enteritidis and beta-galactosidase positive Escherichia coli. The growth inhibition was determined by measuring optical density in broth and colony size and number on agar.
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GalactoZide™ - Z-4002_P00
In food and drinking water control, hygiene monitoring and clinical diagnostics predominant bacteria such as E.coli overgrow the target bacteria. As a result the detection limit for the target bacteria is diminished.
Enzyme-activated inhibitors have the potential
to revolutionize microbiological assays
As described, by adding GalactoZide™ to solid growth media inhibits the growth of unwanted E. coli background in a standard microbiological growth test. As a result, colonies of the bacteria of interest on agar plates are detected more easily and the detection limits are improved. Adding enzyme-activated inhibitors to growth media can also lower the incubation time of a plate assay considerably in some cases since distinct colonies are visible much earlier.
At Biosynth we designed more enzyme-activated inhibitors to target more bacteria that are typically seen as background flora in microbiological tests. These enzyme-triggered inhibitors can be applied to control background problems with high accuracy. In contrast to antibiotics, enzyme-activated inhibitors discriminate bacterial strains by their well characterized enzyme activity.
In addition to GalactoZide™, we designed the L-alanine aminopeptidase responsive inhibitor PeptiZide™ that targets specifically gram negative bacteria. Another example, GlucuroniZide™ uses E. coli's specific glucuronidase activity and allows to inhibit the growth of this bacteria. The following list summarizes new Biosynth products and examples of possible targets:
|GalactoZide™||E.coli (EHEC) / coliforms|
|PhosphoZide™||S. aureus (MRSA)|
|PeptiZide™ L-Pyr||Citrobacter spp.|
In summary, our product line of enzyme-responsive inhibitors GalactoZide™, PeptiZide™ and GlucuroniZide™ do inhibit specifically sub-populations of microbiota, while the non-targeted population is not affected. Such enzyme-responsive inhibitors are useful tools to control unwanted background for increased sensitivity of in vitro applications in food control, environmental safety, hygiene and clinical diagnostics.
With GalactoZide™ and other enzyme-triggered inhibitors a class of antimicrobial compounds becomes available that is capable of changing microbiota composition and function by selectively inhibiting distinct microbial species.
This provides a tool for use in therapy, in particular for use in the treatment of:
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For any further questions regarding our microbiology products please contact our team.
The GalactoZide™ Principle
We provide more information about the mode of actions of enzyme-activated inhibitors in our brief handout.