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Greenville, SC, United States

Kumar S.,Clemson University | Kumar S.,Yale University | Spano M.N.,Nubad, Llc | Arya D.P.,Clemson University
Bioorganic and Medicinal Chemistry | Year: 2015

Abstract DNA-protein recognition has shown us the importance of DNA shapes in the recognition process. Specific high-affinity targeting of DNA shapes by small molecules is desirable for many biological applications that involve regulation of DNA based processes. Here, the effect of linker length and rigidity on the affinity of a conjugated neomycin dimer for a specific DNA shape (B∗ form) AT-rich DNA was explored. Binding constants approximating 108 M-1 for optimal linker lengths of 18-19 atoms are reported herein. © 2015 Elsevier Ltd. Source


Small molecule fluorescent probes for established drug targets such as nucleic acids including DNA and RNA has been developed and disclosed herein. These nucleic acid probes bind to multiple DNA and RNA structures, and to sites crucial for nucleic acid function, such as DNA and RNA major grooves. Displacement of the probes by other binders such as small molecule compounds and/or proteins illicits a fluorescence change in the probe that once detected and analyzed provide binding information of these other binders of interest. Similarly, changes in fluorescence upon binding of the probes to nucleic acid have been applied to screen nucleic acid of different sequence and conformation. The nucleic acid probes and method of uses disclosed herein are advantageously suitable for high-through put screening of libraries of small molecule compounds, proteins, and nucleic acids.


Small molecule fluorescent probes for established drug targets such as nucleic acids including DNA and RNA has been developed and disclosed herein. These nucleic acid probes bind to multiple DNA and RNA structures, and to sites crucial for nucleic acid function, such as DNA and RNA major grooves. Displacement of the probes by other binders such as small molecule compounds and/or proteins illicits a fluorescence change in the probe that once detected and analyzed provide binding information of these other binders of interest. Similarly, changes in fluorescence upon binding of the probes to nucleic acid have been applied to screen nucleic acid of different sequence and conformation. The nucleic acid probes and method of uses disclosed herein are advantageously suitable for high-through put screening of libraries of small molecule compounds, proteins, and nucleic acids.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 464.38K | Year: 2015

DESCRIPTION provided by applicant The world is rapidly heading towards a pre andapos s scenario when it comes to fighting infectious disease Antimicrobial resistance is a growing problem on a global scale greatly hampering our abilities to quell worldwide epidemics such as tuberculosis and malaria as well as the simple staphylococcus infection The proposed project is significant and has huge potential for impact on public health because unless innovative strategies are developed to produce robust and effective new classes of antibiotics health care costs will continue to climb and we will completely lose our ability to combat even the most common infection Current antibiotic treatments originated predominantly from natural products produced by fungi and bacteria that were able to inhibit the growth of other organisms usually by inhibiting cell wall synthesis or maintenance or by inhibiting protein synthesis Since penicilln was first isolated by Fleming in most of the subsequent generations of antibiotics remain very similar to the original natural products with functional groups modified to increase their activity across a broader range of pathogens and decrease their side effect profiles Oxazolidones glycopeptides lactams and quinolones show some promise for the future but gram negative bacterial infections still remain problematic Nucleic acids are promising avenues for drug design both as therapeutics and as targets However specificity is often a problem with small molecule nucleic acid binders such as intercalators groove binders and even aminoglycosides Here we propose an innovative plan for identification of and both functional and mechanistic assaying of a novel class of aminoglycoside nucleic acid conjugate ligands that are specific for an aminoglycoside targeting riboswitch and render it inactive in vivo This riboswitch is a key switch in the mechanism responsible for conferring antibiotic resistance in dozens of pathogenic bacterial strains and has never before been targeted for possible therapeutic development to our knowledge The designed ligands which are aminoglycoside conjugates have the potential to be both specific for this riboswitch target and useful against a broad spectrum of infectious bacteria including gram negative strains First as outlined in Specific Aim we will obtain a model riboswitch aptamer domain that has been synthesized commercially with FRET donor and acceptor dyes in different regions of the construct We will perform a fluorescence assay to rapidly screen approximately novel aminoglycoside nucleic acid conjugates developed at NUBAD LLC for binding to the riboswitch target and identify promising ligands with high specificity and affinity for the target riboswitch as outlined in Specific Aim In vivo assays will be used Specific Aim to identify lead compounds that are uptaken by aminoglycoside resistant cells and render them susceptible aminoglycosides once again In order to verify that the compounds indeed inhibit the riboswitchandapos s mechanism of action mechanistic assays will be performed Specific Aim The riboswitch will be positioned within a reporter plasmid so that it is under control of an IPTG inducible tac promoter Ptac that will be positioned upstream of the gal reporter gene Function of the riboswitch will be assessed by agar diffusion analysis in the presence of aminoglycosides and selected identified conjugate ligand binders As a result of this study several lead compounds will be identified that are taken up by pathogenic bacteria restore aminoglycoside susceptibility to resistant bacteria and specifically target the aminoglycoside binding riboswitch as their primary mechanism of action Future phases of this project will focus on developing these lead compounds for development as therapeutics NUBAD LLC is a drug discovery company devoted to identifying therapeutic agents that target nucleic acids We develop novel probes assays and small molecule therapeutics targeting RNA and DNA structures identified as targets in human disease and this project is extremely well suited to NUBADandapos s aims and its employeesandapos specific skill sets PUBLIC HEALTH RELEVANCE Antimicrobial resistance occurs when microorganisms often infectious bacteria viruses and certain parasites are no longer sensitive to drugs that were previously used to treat them this is of global concern because it hampers our ability to control infectious disease and increases the costs of health care In order to combat this world wide problem innovative strategies for antibiotic drug design must be implemented The proposed research describes the in vitro identification and in vivo functional characterization of an extremely promising new class of molecules that can specifically target a region of bacterial RNA that is known to confer antibiotic resistance in many pathogenic bacterial strains


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 285.50K | Year: 2011

DESCRIPTION (provided by applicant): NUBAD, LLC is a drug discovery company devoted to identifying therapeutic agents that target nucleic acids. This project is designed to synthesize and characterize a novel class of fluorescently-labeled aminoglycosidesthat exhibit a dramatic change in fluorescence upon RNA binding. Aminoglycosides are a potent class of antibiotics that block bacterial protein synthesis by binding to a conserved site on ribosomal RNA. It is the purpose of the project to develop an innovative fluorescence-based screen for A-site targeted drugs for rapid screening of RNA binding antibacterials. PUBLIC HEALTH RELEVANCE: The aminoglycoside class of antibiotics block bacterial protein synthesis by binding a conserved site on ribosomal RNA. These antibiotics are clinically important agents that combat a broad spectrum of bacterial infections but can cause serious side effects such as kidney damage and hearing loss. The emergence of antibiotic resistance and high level of toxicity associated with the current arsenal of drugs demands the development of efficient methods of screening for novel antibiotics with the desirable properties of high potency and low toxicity. NUBAD, LLC, Pendleton, SC in collaboration with PI's lab, Clemson University, SC, has recently identified a fluorescent neomycin conjugate that is being used to probe drug binding to RNA targets. This proposal describes a phase I project that will determine the technical and scientific feasibility of using a fluorescent-neomycinprobe in an innovative screen suitable for a high-throughput screen for new aminoglycoside based antibiotics. This phase I application will develop the probe for high throughput applications and then use the targeted screen to check for assay robustness. Selected compounds will then be checked for antibacterial activity and mechanism of action.

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