MADISON, WI, United States
MADISON, WI, United States
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Patent
BellBrook Labs | Date: 2014-06-11

A cell culture device for the study of barrier function and differentiation are provided by this invention.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 353.33K | Year: 2016

Project Summary The broad goal of this project is the pharmacological validation of GIV a nonreceptor guanine nucleotide exchange factor GEF for liver fibrosis GIV sits at the hub of profibrogenic signaling in liver fibrosis and is required for the key physiological events that lead to this condition the migration proliferation and extracellular matrix deposition of hepatic stellate cells HSC GIV is an ideal drug target as it is strongly ov erexpressed in the disease state and functions independently of the extracellular signaling receptors used to instigate fibrosis The aims of the proposal are to develop a biochemical primary screening assay and a multiparametric phenotypic secondary assay in HSCs The primary assay is based on the steady state GDP levels associated with GEF and its cognate G protein G i The GEF activity assay will be used to screen an orthogonally pooled compound library Hits will be confirmed and shown to be GEF specific Following hit expansion functional activity will be verified using the phenotypic assays In phase II these compounds will be profiled for selectivity and tested in animal models Project Narrative This proposal will help to find new drugs for liver fibrosis a potentially deadly condition that can develop into cirrhosis and or liver cancer It is prevalent in patients with alcoholism and is more likely to affects patients who are obese or have met abolic syndrome Exciting new laboratory findings indicate a new approach is warranted and this proposal seeks to realize the potential to better understand and potentially treat this disease


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 234.94K | Year: 2016

Project Summary Abstract High throughput screening HTS is a powerful method for the discovery of new drug leads for target enzymes In HTS assays the activity of the target enzyme is often evaluated by quantifying a small molecule or cofactor that is produced or consumed by the enzyme While antibodies are a mainstay of small molecule detection as says they do have severe limitations which are largely tied the challenge of functionalizing small molecule targets without masking key functional groups As a result conjugation of the target molecule to a carrier pro tein for antibody generation is laborious and often decreases the specificity of the antibodies generated Addi tionally HTS assays that use small molecule binding antibodies require that a labeled version of the target be produced to act as a competitor in the assay We propose that DNA structure switching SS biosensors can overcome these limitations as these sensors provide a direct fluorescence readout upon target binding and do not require that the target be covalently labeled Additionally these biosensors utilize nucleic acid ap tamers which can be generated using in vitro selection methods that do not necessarily require that the target be modified or immobilized While SS biosensors hold tremendous potential for use in HTS and other small molecule detection assays the currently available protocols for generating these biosensors are time consuming and at times unreliable Thus we propose an improved method for the in vitro selection of SS bio sensors that is anticipated to provide more efficient enrichment of functional sequences This will both reduce the time required and increase the success rate for generating biosensors to new small molecule targets of interest In Aim we will develop and implement this selection method to generate a SS biosensor for Coen zyme A CoA In Aim we will utilize this biosensor to produce a fluorescence polarization HTS assay for CoA the product of histone acetyltransferases HATs The immediate impact of this research will be an im proved HTS assay for screening HATs which will address a significant unmet need in drug discovery for a number of epigenetic diseases including neurological disorders cancers and cardiovascular disease From a broader perspective this research will provide a rapid and reliable method for generating SS biosensors for small molecule targets which will accelerate development of HTS assays for diverse enzyme drug targets Project Narrative High throughput screening HTS has proven to be a highly effective method for drug discovery The proposed research will develop new methods for HTS against protein targets that play key roles in multi ple diseases with an initial focus on diseases with an epigenetic basis


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 225.00K | Year: 2015

DESCRIPTION provided by applicant Neurodegenerative diseases that affect motor neurons such as PD ALS and HD represent an enormous unmet medical need that is growing with the aging population Existing treatments have little or no effect on the course of disease and patients have to cope with the loss of brain and body function for the rest of their lives Defects in the transport of organelles and biomolecules through axons is a hallmark of early stage disease and there is strong evidence that it contributes to the andquot dying backandquot pathology seen in most motor neuron diseases which is characterized by early degeneration of the synapse and the axon followed by damage to the soma and eventual neuronal loss Therefore discovery of drugs that prevent or rescue axonal transport defects is a compelling strategy for early intervention in neurodegeneration However the difficulty in tracking cargo movement through axons has prevented the scaling of assays for high throughput screening HTS and discouraged the use of physiologically relevant models incorporating glial cells and three dimensional D tissue architecture We will overcome these technical hurdles by using BellBrookandapos s proprietary iuvo R microchannel plate technology to enable automated high content analysis HCA of axonal transport in D cocultures of neurons and astrocytes derived from induced pluripotent stem cells iPSC Flow based collagen patterning in microchannels will be used to align both cell types with the longitudinal axis of the channel Alignment of neurons combined with the height restrictions imposed by the microchannel will make it vastly simpler to track the movement of axonal cargoes allowing the use of streamlined image acquisition that is scalable for high throughput screening HTS Moreover a uniform cell polarity along collagen andquot tracksandquot will resemble in vivo tissue architecture much more closely than monolayers of randomly oriented neurons This is a collaborative effort leveraging the microfluidics and expertise of Dr David Beebe John D MacArthur Professor in the Department of Biomedical Engineering at UW Madison and Dr Robert Loweryandapos s long track record in developing enabling HTS assay products at BellBrook Labs We will optimize D coculture and neural cell alignment iuvo R Microchannel plate at BellBrook Aim while Dr Beebeandapos s methods in the existing group develops a new prototype microchannel slide channels with modifications to optimize optics and axon patterning for image based Aligned D neural coculture for HTS imaging of axonal transport Microchannel plates analysis of axonal transport Aim Lastly we will have arrays of precisely patterned channels in a standard SLAS footprint shown is iuvo R use the new device to streamline methods for Microchannel with channels Longitudinal alignment of astrocytes gray and neurons along collagen fibrils combined with optimization of channel design will enable image based tracking of axonal cargo transport rapid scalable imaging of axonal transport The microchannel plates are compatible establishing feasibility for scaling the device and with standard liquid dispensing and automated microscopy equipment enabling robust high throughput assays for axonal transport in a D tissue like microenvironment methods to HTS in Phase II To our knowledge the proposed microchannel platform for aligned D cocultures will be the first in vitro model for probing axonal transport ina D tissue like microenvironment and the first HTS scalable assay An HTS compatible phenotypic assay platform for axonal transport could enable the discovery of drugs that prevent or slow neuronal loss early in the disease process and thereby accelerate the development of more effective treatments for devastating neurodegenerative diseases that affect a growing fraction of our aging population PUBLIC HEALTH RELEVANCE Transport of organelles and biomolecules between cell bodies and nerve endings known as axonal transport is critical to the function or neurons and this process often becomes defective early in the course of neurodegenerative diseases like Parkinsonandapos s disease and Multiple Sclerosis This proposal seeks to develop in vitro assays to identify new drugs that will prevent or rescue axonal transport defects so that neurodegeneration can be slowed or prevented before irreversible damage occurs


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2015

DESCRIPTION provided by applicant Inflammation has both beneficial and harmful effects in chronic neurodegenerative diseases such as Alzheimerandapos s disease AD and Parkinsonandapos s disease PD and paracrine signaling is central to the inflammatory response Rapid advances in in vitro models that incorporate different neural cell types in the context of the brainandapos s uniqu extracellular matrix ECM are helping to define the paracrine signaling pathways that contribute to neuroinflammatory disease pathologies However to be useful for drug discovery these models must be amenable to analysis on automated high throughput screening HTS platforms Unfortunately dynamic non invasive detection of soluble molecules in ECM is not feasible with the current immunodetection methods used for automated high content analysis HCA which typically involve multiple components and wash steps To overcome this technical gap we propose to develop fluorescent andquot single reagentandquot paracrine biosensors based on aptamers that transduce signals intramolecularly to quantum dots Unlike antibodies aptamers can be readily engineered to incorporate ligand dependent structural switches that can be harnessed for Forster resonance energy transfer FRET signals And quantum dots have distinct advantages over organic fluors including greater brightness resistance to photobleaching and multiplexing capability As a first step we are proposing to develop a fluorescent biosensor using an existing aptamer to TNF a key inflammatory signal molecule Proof of concept for the TNF andapos AptaFluorandapos will be demonstrated in BellBrookandapos s iuvo r Microchannel Array Plates an alternative to multiwell plates which enable dynamic imaging of ECM biology on automated HCA platforms Phase I studies will lay the technical foundation for development of AptaFluor biosensors for several inflammatory cytokines and their incorporation into mutlitplex assays in Phase II AptaFluors in combination with BellBrookandapos s iuvo microchannel plates would be an extremely powerful platform for probing cell cell signaling in ECM as it would allow non invasive spatially resolved detection of soluble signaling molecules in real time using existing high content analysis platforms PUBLIC HEALTH RELEVANCE Harmful brain inflammation is a hallmark of neurodegenerative diseases such as Alzehimerandapos s and Parkinsons diseases and inflammation is controlled largely by signals sent from one cell type to another To accelerate discovery of more effective therapies for these disabling diseases we are proposing to develop biosensors for detecting the inflammatory signals in the context of the gelatinous matrix that supports cells in the brain


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2014

Not Available


Patent
BellBrook Labs | Date: 2012-03-23

This invention provides methods to determine the activity of methyltransferase enzymes which employ S-adenosylmethionine (SAM) as a substrate and transfer a methyl group to convert this substrate to S-adenosylhomocysteine (SAH), by measuring SAH conversion to AMP.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.93K | Year: 2014

DESCRIPTION (provided by applicant): Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer, diabetes and inflammation, and high throughput screening for histone methyltransferase (HMT) inhibitorsis an area of intense drug discovery effort. However, there are significant shortcomings with existing HMT enzyme assay methods, and these are slowing exploration of the therapeutic potential of these emerging targets. Detection of specific methylation events can be quite complicated, and detection of S-adenosylhomocysteine (SAH), the invariant product of all HMT reactions, would be preferred in most cases. However, HMTs are very poor catalysts and many have very low SAM requirements - a combination of factors that creates very stringent sensitivity requirements for SAH-based assay methods. Moreover, direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S- adenosylm


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 733.59K | Year: 2012

DESCRIPTION (provided by applicant): Approaches to drug screening typically involve dramatic compromises in order to achieve high throughput and hold down costs. Examples include the use of immortalized, heterologous cell lines, and genetic engineering toover-express the target protein and/or incorporate a fluorescent surrogate reporter to display the result. At least partly because such systems often produce responses that are not relevant to the actual human disease state, high throughput screening has not historically delivered a strong return on investment. More representative primary cell models are often available for tissues and diseases, but these models are underutilized in screening because of the lack of technological solutions and high costs. For similar reasons, more relevant assay technologies such as endogenous immunocytochemistry and electrophysiology are not commonly employed in HTS. The goal of this project is to bring the gold standard, organotypic cell model for airway epithelia into truehigh throughput screening, and enable the use of more informative high content assays including endogenous CFTR trafficking and airway surface liquid height. In addition to applications for cystic fibrosis and COPD, the resultant device will also be valuable for skin models and drug transport studies. The device consists of an array of 96 microchambers in standard microplate format. The microchamber design is small enough to be compatible with 384-well plate densities, so 384 microchambers per plate is readily achievable. The plate includes specialized features for compatibility and ease-of-use with standard liquid handling robotics, and high resolution microscopy. Importantly, the device is simple enough to be produced at a cost consistent with the cost constraints of HTS labs, and confers dramatic savings in primary cell and media consumption. The feasibility study on a small-scale device was successfully completed, indicating that the highly miniaturized approach is compatible with organotypic epithelialairway culture at air-liquid interface. Although not an aim of this proposal this device is designed to be integrated into an electrophysiology instrument designed specifically for this culture system. PUBLIC HEALTH RELEVANCE: The project seeks todevelop an automatable device that uses the best possible cell models for airways disease, and the best possible tests or assays for the critical functions of human airways. The goal is to help improve the success ratios of bringing a candidate drug to the market for airways diseases like cystic fibrosis and chronic obstructive pulmonary disorder. The device will also be useful for finding drugs for other diseases like skin cancer and certain kidney diseases.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 470.46K | Year: 2016

DESCRIPTION provided by applicant Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer diabetes and inflammation and high throughput screening for histone methyltransferase HMT inhibitors is an area of intense drug discovery effort However there are significant shortcomings with existing HMT enzyme assay methods and these are slowing exploration of the therapeutic potential of these emerging targets Detection of specific methylation events can be quite complicated and detection of S adenosylhomocysteine SAH the invariant product of all HMT reactions would be preferred in most cases However HMTs are very poor catalysts and many have very low SAM requirements a combination of factors that creates very stringent sensitivity requirements for SAH based assay methods Moreover direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S adenosylmethionine SAM which differ by a single methyl group The available SAH assays rely largely on enzymatic conversion of SAH to a detectable product and are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases The lack of suitable assay reagents is delaying and in some cases preventing the screening of potential therapeutic targets To overcome this technical gap we are using microbial SAH sensing RNA aptamers or andquot riboswitchesandquot that bind SAH with nanomolar affinity and exquisite selectivity In Phase I we established the critical technical feasibility for this approach by showing that SAH binding to a riboswitch can be transduced into fluorescence polarization FP and time resolved F rster resonance energy transfer TR FRET signals without disrupting affinity or selectivity To achieve this we split the riboswitch into two halves such that SAH binding induces assembly of a trimeric complex this modification vastly improved the sensitivity selectivity and stability of the signaling We used the split aptamer assays called AptaFluor SAH to detect SAH produced by several HMTs at levels several fold below the sensitivity limit for current assays In Phase II we will leverage recent advances in aptamer and nanoparticle technologies to make the novel FP and TR FRET based assays suitable for industrial HTS validate them extensively for inhibitor screening and profiling with HMTs and establish stability and manufacturing aspects required for commercialization In addition we will develop an ultrasensitive ELISA like assay for detecting HMT activity in biological samples using an innovative split aptamer proximity ligation method By enabling direct highly sensitive detection of SAH in homogenous the FP and TR FRET AptaFluor SAH assay will provide a universal HMT assay platform for inhibitor discovery and lead optimization and allow pursuit of otherwise intractable targets The solid phase AptaFluor SAH assay will enable discovery of biomarkers and development of companion diagnostic assays for clinical development of HMT targeted therapies Taken together these developments will accelerate screening of new HMT targets and development of small molecule drugs for cancer diabetes and other diseases with an epigenetic basis PUBLIC HEALTH RELEVANCE The regulation of gene expression by chemical modification called epigenetics is a promising new area for discovering improved drugs for cancer and other debilitating diseases We are developing new screening assays for important epigenetic drug targets based on naturally occurring microbial chemical sensing molecules called riboswitches

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