Nubad, Llc

Greenville, SC, United States

Nubad, Llc

Greenville, SC, United States

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Watkins D.,Nubad, Llc | Kumar S.,Clemson University | Kumar S.,Yale University | Green K.D.,University of Kentucky | And 3 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2015

The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


Watkins D.,Nubad, Llc | Norris F.A.,Nubad, Llc | Norris F.A.,Clemson University | Kumar S.,Clemson University | Arya D.P.,Clemson University
Analytical Biochemistry | Year: 2013

The development of new antibacterial agents has become necessary to treat the large number of emerging bacterial strains resistant to current antibiotics. Despite the different methods of resistance developed by these new strains, the A-site of the bacterial ribosome remains an attractive target for new antibiotics. To develop new drugs that target the ribosomal A-site, a high-throughput screen is necessary to identify compounds that bind to the target with high affinity. To this end, we present an assay that uses a novel fluorescein-conjugated neomycin (F-neo) molecule as a binding probe to determine the relative binding affinity of a drug library. We show here that the binding of F-neo to a model Escherichia coli ribosomal A-site results in a large decrease in the fluorescence of the molecule. Furthermore, we have determined that the change in fluorescence is due to the relative change in the pK a of the probe resulting from the change in the electrostatic environment that occurs when the probe is taken from the solvent and localized into the negative potential of the A-site major groove. Finally, we demonstrate that F-neo can be used in a robust, highly reproducible assay, determined by a Z′-factor greater than 0.80 for 3 consecutive days. The assay is capable of rapidly determining the relative binding affinity of a compound library in a 96-well plate format using a single channel electronic pipette. The current assay format will be easily adaptable to a high-throughput format with the use of a liquid handling robot for large drug libraries currently available and under development. © 2012 Elsevier Inc. All rights reserved.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 294.58K | Year: 2012

DESCRIPTION (provided by applicant): One of the challenges of research in infectious diseases is to find ways to use the increasing knowledge of the mechanisms underlying disease transformation and progression to develop novel therapeutic strategies for AIDS. Targeting specific RNA- protein interactions, such as Tat-TAR or Rev-RRE, which are involved in proliferation and survival of HIV-1 is a promising approach. Our preliminary results show the ability of novel ligands to stabilize TAR RNA, inhibit Tat-TARinteraction at nanomolar concentrations and inhibit HIV-1 in MT-2 cells. These preliminary results will now be built upon to develop a library of conjugates to target Tat-TAR interaction that bind with high affinity and specificity to TAR. Proposed studies will further help establish the efficacy of this approach. The work proposed here, a multidisciplinary effort encompassing organic synthesis, biophysical chemistry and HIV pathogenesis describes the development of small molecule mediated inhibition of Tat-TAR inhibitors as HIV-1 therapeutics. The success of the proposed work would be a significant addition to currently available protein- specific approaches in AIDS therapy and RNA targeting. We propose using a 31 nt TAR target sequences to design conjugates that can be employed to inhibit Tat-TAR interaction; opening possibilities for developing small molecule RNA targeted HIV-1 therapeutics. PUBLIC HEALTH RELEVANCE: Several decades of research on the RNA structure has shown it to be an establisheddrug target, well known as a receptor for small molecule antibiotics. Though the bacterial ribosome has been a well known receptor for antibiotics blocking protein synthesis since the discovery of streptomycin in the 1940s, new antibacterial and antiviralapproaches are urgently needed to combat drug resistance, which severely limits the effectiveness of current antibiotics. To investigate the advantage of small molecule-based specificity coupled with charge/shape complementarity, we have initiated a program in the development of a approaches using multimeric ligands (consisting of ligands with independent binding sites) that can be used to target a specific RNA. This proposal focuses on the development of small molecule aminosugars (neamine) conjugates asan example of this approach. A comprehensive approach to identifying essential drug targets in multiple pathogens can be combined with our complementary approach of developing small molecules that bind with high affinity in a specific fashion to previously known as well as rapidly identified, new RNA targets. The inhibition of the Tat/TAR interaction, which facilitates HIV RNA transcription subsequently arrests HIV replication. The central hypothesis of this application is that conjugation of two ligandswith an independent binding sites can be conjugated with an appropriate linker to provide a high affinity TAR specific ligand, capable of inhibiting the Tat/TAR interaction at nanomolar concentrations. Furthermore, the assay is applicable to RNA based drugdiscovery where two pharmacophores with independent binding sites can be combined to select a high affinity ligand. Ultimately, the discovery of a TAR binding ligand with improved affinity and specificity over currently available molecules will provide abetter understanding for the potential use of a novel target for implementation in the fight against HIV. NUBAD is well equipped to synthesize the molecules and carry out the biophysical assays for inhibition. Select compounds identified from the assay that inhibit tat-TAR interaction at nanomolar Kd will be tested for inhibition of HIV.


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.


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: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 300.00K | Year: 2016

PROJECT SUMMARY Aminoglycosides are one of the cheapest and well known antibiotics in clinical use for over years but one of the major limitations in their use is their ototoxicity We are developing fast and low cost methods to develop aminoglycosides with anti ribosomal activities and reduced toxicity In this project we will identify novel aminoglycoside antibacterials that show reduced ototoxicity Complexes between ribosomal components will be exploited as targets for small molecule drug libraries that inactivate the ribosome stopping bacterial protein synthesis and causing bacterial death while reducing toxicity This work addresses an important health issue antibiotic ototoxicity and presents creative steps towards a novel solution to this problem The work proposed here a multidisciplinary effort encompassing antibacterial screening and ototoxicity studies using zebrafish and guinea pig models describes the development of novel aminosugar rRNA binders as non toxic antibacterial therapeutics The success of the proposed work would be a significant addition to currently available approaches in antibacterial therapy We propose using a zebrafish assay to identify conjugates that show reduced toxicities opening possibilities for developing reduced toxicity RNA targeted aminosugar therapeutics PROJECT NARRATIVE The proposed project presents a strategy for developing novel aminoglycoside therapeutics with reduced ototoxicities with evasion of resistance 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 a strategy for improving the therapeutic index of aminoglycosides by designing novel structures that lower their ototoxicity and evade common resistance pathways


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 325.00K | Year: 2016

DESCRIPTION provided by applicant Nucleic acids are avenues for drug design both as therapeutics and as targets Here we propose to establish new methods for identifying antibiotic ribosome targets and lead compounds Targeting specific RNA such as rRNA which are involved in proliferation and survival of bacteria is a promising approach We are developing fast and low cost methods to screen sequence specific small molecules for novel anti ribosomal activities We will construct sequence specific ribosomal targeting oligomers as antibacterials that can be effectively delivered inside the cell Complexes between ribosomal components will be exploited as targets for small molecule drug libraries that inactivate the ribosome stopping bacterial protein synthesis and causing bacterial death NUBADs unique experimental approaches and technologies will allow us to target ribosomal regions not previously explored for susceptibility against anti bacterial agents This work addresses an important world health issue antimicrobial resistance and presents creative steps towards a novel solution to this problem The work proposed here a multidisciplinary effort encompassing solid phase organic synthesis oligonucleotide delivery RNA targeted screening and antibacterial studies describes the development of sequence specific cell permeable binders of rRNA as antibacterial therapeutics The success of the proposed work would be a significant addition to currently available ribosome specific approaches in antibacterial therapy We propose using a small rRNA target sequences to design conjugates that can be employed to inhibit bacterial growth opening possibilities for developing sequence specific RNA targeted therapeutics PUBLIC HEALTH RELEVANCE The work proposed here a multidisciplinary effort encompassing organic synthesis oligonucleotide delivery RNA targeted screening and antibacterial studies describes the development of sequence specific cell permeable binders of rRNA as antibacterial therapeutics The success of the proposed work would be a significant addition to currently available ribosome specific approaches in broad spectrum antibacterial antibiotic development in particular for targeting gram negative pathogens We propose using a small rRNA target sequences to design conjugates that can be employed to inhibit bacterial growth opening possibilities for developing sequence specific RNA targeted therapeutics


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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