Technologies
Abstract
DNA
topoisomerases (Topos) are enzymes responsible for the relaxation of the
positive and negative supercoiled DNA as well as the resolution of DNA knots
and catenanes during vital biological processes. DNA Topos create transient DNA
break which results in changes in DNA topology, a DNA property that affects
genomic stability and the susceptibility to cancers or certain hereditary
diseases. Therefore, DNA Topos are an important target for certain antibiotics
and/or anticancer drugs.Commonly
used assays to test DNA Topos activities include agarose gel electrophoresis
and fluorescently labeled DNA molecules. However, the agarose gel
electrophoresis method is labor-intensive, time-consuming, and does not apply
to high throughput screening (HTS) assays. On the other hand, using
fluorescently labeled DNA molecules is expensive and the method could interfere
with the final detection signal. Hence, there is a need for methods and assays to
identify Topo inhibitors as potential initiators for drug development. FIU
scientists have developed assays and to screen compound libraries to identify
potential inhibitors targeting different DNA topoisomerases, such as bacterial
DNA topoisomerases I and II, and DNA gyrases. The method is based on using a
circular plasmid DNA, with at least one hairpin structure, and T5 exonuclease
(T5E) ability to digest the circular plasmid DNA in a specific configuration.Benefit
Applicable for high throughput screening assaysCost-effective assaysSensitive assaysMarket Application
Drug development
Abstract
FIU inventors have developed novel reagents for the study of DNA topology
and topoisomerases. These tools are also useful for screening Anti-Cancer Drugs
and Antibiotic Targeting DNA topoisomerases. FIU inventors have created a
method to mass-produce a type of fluorescently labeled circular DNA molecules
with high yields to study DNA topology and topoisomerases by fluorescence
resonance energy transfer (FRET), and to screen anti-cancer drugs and
antibiotics targeting DNA topoisomerases.In a typical assay to study DNA supercoiling and
topoisomerases, agarose gel electrophoresis is usually used to resolve DNA
topoisomers produced in the reaction mixtures. Since gel electrophoresis is
time-consuming it is desirable to develop other assays, such as
fluorescence-based assays, to study DNA topology and DNA topoisomerases. The
novel fluorescently labeled circular DNA molecules provide a quick and low-cost
method to study DNA topology and topoisomerases through FRET.Benefit
Provides tools to study DNA topology and topoisomerases in real time by FRETFacilitates the development of kits and reagents for high throughput screening (HTS) against DNA topoisomerases for anticancer drug and antibiotics discovery using FRETAllows for the production of fluorescence-labeled DNA molecules at milligram rangeProvides high quality of relaxed and supercoiled DNA moleculesMarket Application
The immediate and future applications of this technology are to provide a type of novel reagents to study DNA topology and topoisomerases by FRET and a new method for high throughput drug screening against DNA topoisomerases for anticancer drug discovery and antibiotics discovery
Abstract
Prokaryotic DNA
gyrase is a type II topoisomerase that can introduce (-) supercoils to the DNA
substrates with the hydrolysis of ATP. Because DNA gyrase only exists in
bacterial cells and is an essential enzyme to bacteria, it is possible to
identify inhibitors targeting DNA gyrase without affecting host human enzymes.
Additionally, DNA gyrase can form covalent enzyme-DNA complex intermediates.
This property makes gyrase an excellent bactericidal target for developing
antibiotics. Fluoroquinolones are among the most successful
antibiotics targeted to DNA gyrase; however, since fluoroquinolones have been
explored extensively in terms of improving spectrum and potency, and overcoming
resistance, the limits of what these compounds can provide might have been reached.
Therefore, it is necessary to identify new types of compounds targeting
DNA gyrase. One challenge is to develop screening assays to identify inhibitors
in small molecule libraries that may target DNA gyrase. Additionally, there is
not cell-based method for identifying inhibitors targeting DNA gyrase. Since
compounds identified by the in vitro biochemical methods may not enter into
cells and function as antimicrobial agents, cell-based methods are preferable.FIU scientists
have developed materials and methods for
transcription regulation via divergently coupled promoters and their use in the
preparation of cells, polynucleotides, and assays for identifying gyrase
inhibitors. In this invention, a first promotor is linked to a first gene, and
a second promoter (linked to a second gene) is divergently coupled to the first
promoter. Transcription of the linked second gene under the control of the
second promoter is inhibited by negative supercoiling of the second promoter. A
compound is identified as a gyrase inhibitor based on differential expression
of genes under the control of divergently coupled promoters in the presence of
the compound. A compound that is not a gyrase inhibitor cannot inhibit the expression
of the first gene and cannot prevent negative supercoiling of the second
promoter. The materials include plasmids and E. coli strains to
identify inhibitors targeting bacterial DNA gyrase.Benefit
Provides cell-based assays for identifying inhibitors targeting DNA gyrase in small molecule librariesMarket Application
Identification/screening of antibiotics for drug repurposing and/or development
Abstract
Bacteria are rapidly evolving to be resistant to
currently available antibiotics. In our fight against disease-causing
pathogenic bacteria, it is crucial to synthesize compounds that can selectively
work against bacteria while avoiding antibiotic resistance mechanisms. Enzyme
inhibitors that selectively target bacterial enzymes, such as DNA topoisomerases (Topos),
over their human equivalent offer a unique benefit in that regard. DNA Topos are enzymes responsible for the relaxation of (+) and (-) supercoiled (sc) DNA and the resolution of DNA knots and catenanes during essential biological processes, such as DNA replication, transcription, recombination, and maintenance of chromosome structure. DNA Topos that control DNA topology inside cells are, thus, important targets for certain antibiotics and anticancer drugs.FIU scientists have synthesized Hoechst 33258 based bisbenzimidazoles containing a
terminal hydrophobic group.
These bisbenzimidazoles display topoisomerase I inhibition that is much better
than Hoechst 33342 or Hoechst 33258, with IC50 values in the range
of 2.47-6.63 μM. These compounds also display selective inhibition of E. coli topoisomerase I over DNA gyrase, human topoisomerases I and II, and effectively
inhibit bacterial growth.Benefit
Effective and selective inhibition of E. coli topoisomerase I over DNA gyrase, human topoisomerase I and IIAvoids antibacterial resistance mechanismsMarket Application
The compounds can be used as antibacterial agents while avoiding problems associated with drug resistance in bacteriaPublications
Ranjan et al. Med. Chem. Commun.,
2014, 5, 816-825
