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MADISON, WI, United States

Staeben M.,BellBrook Labs
Assay and drug development technologies | Year: 2010

Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.

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

BellBrook Labs | Date: 2014-06-11

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

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

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

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