| Methods for cyclic nucleotide determination -> Monitor Keywords |
|
|
 
Methods for cyclic nucleotide determinationRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving Avidin-biotin BindingMethods for cyclic nucleotide determination description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070172896, Methods for cyclic nucleotide determination. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims priority to U.S. Provisional Application No. 60/742,922 filed Dec. 6, 2005, which is incorporated herein in its entirety. FIELD OF THE INVENTION [0002] The present invention relates in general to cellular analysis tools and more particularly to methods for detecting or determining cyclic nucleotide concentrations in samples. BACKGROUND OF THE INVENTION [0003] The second messengers, adenosine 3', 5'cyclic monophosphate (cAMP) and guanosine 3', 5'cyclic monophosphate (cGMP), are important intracellular mediators of a variety of cellular functions including cell growth, differentiation, apoptosis, and cell death. Production of cAMP is controlled through the adenylyl cyclase family of enzymes, which convert adenosine triphosphate (ATP) to cAMP and inorganic pyrophosphate (PPi). The adenylyl cyclases are activated or inhibited via direct interaction with membrane bound G-protein coupled receptor (GPCR) .alpha.-subunits. When an .alpha.-subunit of a stimulatory GPCR is activated, designated G.sub..alpha.s, adenylyl cyclase converts ATP to cAMP and PPi. Conversely, when an .alpha.-subunit of an inhibitory GPCR is activated, designated G.sub..alpha.i, an inhibitory effect on adenylyl cylase is exerted and the conversion of ATP to cAMP and PPi is not realized. G-protein coupled receptors play a prominent role in a wide variety of biological processes such as neurotransmission, cardiac output, and pain modulation. Their importance in developing new medically useful compounds is well understood; as such they are highly targeted in drug discovery research. [0004] The intracellular concentration of cAMP is also affected by another group of enzymes, cyclic nucleotide phosphodiesterases (PDE), which catalyze the hydrolysis of cAMP to AMP and cyclic cGMP to GMP. Phosphodiesterases function in conjunction with adenylyl cyclases and guanylate cyclases to regulate the amplitude and duration of cell signaling mechanisms that are mediated by cAMP and cGMP. Phosphodiesterases therefore regulate a wide range of important biological responses to first messengers such as hormones, light, and neurotransmitters. There are two classes of PDEs; Class I are found in the cytoplasm or bound to intracellular organelles or membranes of all eukaryotic cells, whereas Class II PDEs are not well characterized and have only been found in lower eukayotes. Cellular responses controlled by Class I phosphodiesterases, through control of cAMP and cGMP conversion, include neuronal responses, aldosterone production, regulation of platelet aggregation, insulin regulation, emesis, regulation of smooth muscle tension, visual phototransduction, and modulation of T-cell responsiveness. Numerous clinically important compounds are known to inhibit phosphodiesterases including; rolipram, theophylline, and sildenafil. Therefore, inhibitors of phosphosdiesterases are also important targets in drug discovery. [0005] The second messenger cAMP is known to activate cAMP dependent protein kinase (PKA). Mammalian holo-PKA is a tetramer, made up of two regulatory and two catalytic subunits. cAMP binds to the regulatory subunits, thereby dissociating holo-PKA into its catalytic and regulatory subunits. Once released, the free catalytic subunits are capable of phosphorylating a multitude of cellular proteins, thereby causing changes in cellular functions such as muscle contraction, activation of cell cycle, activation of transcriptional activity, and DNA processing. [0006] Because the activation or inhibition of GPCR and subsequent activation or inhibition of adenylyl cyclase results in an increase or decrease in intracellular cAMP, agents that affect their activity are important targets for drug discovery. Drugs that target GPCR account for many of the medicines sold worldwide due to the tremendous variety of biological processes relating to G-protein coupled receptors. Examples of drugs that influence GPCR include Claritin.RTM. and Alavert.RTM. (loratadine) which are used for relieving allergy symptoms, Paxil.RTM. (paroxetine HCl) for relief of depression, and Vasotec.RTM. (enalapril maleate) for relief of hypertension. Because of their importance, various GPCR assays have been developed to determine the effect of agonists and antagonists on these system components, mainly by assaying for the increase or decrease in cAMP levels. Limitations of these methods include non-homogeneous assays that require multiple dispensing steps, long incubation times, and the need for expensive equipment. [0007] Therefore, what are needed are assays that require less manipulation than currently available technologies (e.g. two steps or less), assays that provide shorter incubation times (e.g., less than 1 hour), and assays that utilize low cost equipment while maintaining high throughput system (HTS) capabilities (e.g., luminescent based equipment). Such streamlining and cost effectiveness will allow for faster and easier evaluation of targets for drug discovery. Furthermore, luminescent based assays are not prone to interference from fluorescence; that is useful in screening large libraries of chemicals to discover the next potential drug. SUMMARY OF THE INVENTION [0008] The present invention relates in general to cellular analysis tools and more particularly to methods for detecting or determining cyclic nucleotide concentrations in samples. [0009] Cyclic nucleotides, such as cAMP and cGMP, increase or decrease in response to a variety of substances that interact with cellular proteins. The methods described herein provide for the detection of such changes. In one embodiment, the methods described herein permit cyclic nucleotides to be detected and correlated with the effect of a stimulus on cellular proteins. [0010] In one embodiment, methods as described herein monitor the binding of cyclic nucleotides to an enzyme that is dependent upon cyclic nucleotide binding in order to activate the enzyme (e.g. cAMP dependent protein kinase, or PKA). For example, once cAMP binds to PKA, PKA transfers a phosphate from adenoside triphosphate (ATP) to a suitable PKA substrate (e.g. Kemptide). The phosphorylation event is detected by various known methods, and the output of each detection method is correlated to the amount of cyclic nucleotide present in a sample. Suitable detection methods include, but are not limited to, methods based on luminescence, radioactivity, and fluorescence. [0011] In one embodiment, a method to determine adenylyl cyclase activity in a sample is provided. Said method utilizes the activation of PKA to provide an activity that can be detected, measured and subsequently correlated to adenylyl cyclase activity. For example, if adenylyl cyclase is stimulated, cAMP is produced which activates PKA, whose activity is detected and correlated to adenylyl cyclase activity. [0012] In another embodiment, a method to determine phosphodiesterase activity in a sample is provided. Said method utilizes the activation of PKA to provide an activity that is detected, measured, and subsequently correlated to phosphodiesterase activity. For example, if a phosphodiesterase is inhibited, cAMP is not converted to AMP or cGMP is not converted to cGMP, therefore cAMP and cGMP can activate PKA, whose activity is detected and correlated to phosphodiesterase activity. [0013] In further embodiments, methods for monitoring the activation of a G-protein coupled receptor (GPCR) by an agonist, or its inhibition by an antagonist, are provided. For example, the level of cAMP found upon addition of agonist or antagonist to a sample comprising a GPCR is detected and measured through the activation of PKA. Such activity (or lack thereof) is detected by a measurable output that is correlated to cAMP levels or amounts. [0014] In one embodiment, samples used in practicing the methods as described herein comprise a lysate. In some embodiments, the sample lysate is derived from prokaryotes or eukaryotes such as bacteria, yeast or mammalian cells. In some embodiments, said sample comprises plasma membranes, cellular membranes, and/or organellar membranes. Membrane preparations as described herein have furnished unexpected results, such that the membrane preparations maintain the integrity and functionality of processes, proteins and receptors (Examples 9-11) associated with the membranes. This allows for targeted membrane functional assays to be performed using the methods as described herein, without accompanying cell lysate components found in a normal cell lysate. [0015] Measurable output may be in the form of bioluminescence, chemiluminescence, radioactivity, or differential output based on different fluorescence technologies (e.g. fluorescence polarization, fluorescence resonance energy transfer, and immunoassay). In one embodiment, the measurable output is in the form of bioluminescence. For example, the coleopteran (firefly) luciferase enzyme utilizes ATP and other factors to convert beetle luciferin to oxyluciferin, a byproduct of the reaction being light. Once PKA is activated, the amount of PKA activation is dependent on the amount of cAMP present, PKA utilizes a phosphate from ATP to phosphorylate a receptive substrate, thereby causing the concentration of ATP to decrease in a sample, thereby causing a decrease in luminescence, or light output. As such, as cAMP concentration in a sample increases a reciprocal decrease in luminescence is seen which is correlated to the amount of cAMP, adenylyl cyclase, and/or GPCR activity present in the initial sample. [0016] In one embodiment, the present invention provides a method for determining the amount of cyclic nucleotides in a sample comprising a sample with may contain a cyclic nucleotide, adding to said sample an inactive enzyme capable of being activated by said cyclic nucleotide, adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and determining the amount of cyclic nucleotide present in said sample based on said signal. In some embodiments, said sample comprises a lysate. In some embodiments, the sample lysate is derived from bacteria, yeast or mammalian cells. In some embodiments, said sample comprises plasma membranes, cellular membranes, and/or organellar membranes. In some embodiments, said cyclic nucleotide is cAMP or cGMP. In some embodiments, said inactive enzyme is a cAMP dependent protein kinase or a cGMP dependent protein kinase. In some embodiments, said detection system comprises a substrate capable of being phosphorylated by PKA or PKG. In some embodiments, said substrate comprises SEQ ID NO: 1. In some embodiments, said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal wherein said enzyme is luciferase. In some embodiments, said substrate comprises a radioactively labeled biotinylated substrate further comprising SEQ ID NO: 1. In some embodiments, said detection system further comprises a streptavidin coated binding surface. In some embodiments, said substrate comprises a fluorescently labeled substrate further comprising SEQ ID NO: 1, wherein said fluorescent label is preferentially rhodamine. In some embodiments, the method of the present invention further comprises the addition of one or more inhibitors of phosphodiesterases, and/or the addition of an agonist or antagonist capable of affecting cyclic nucleotide amounts in said sample. In some embodiments, said agonist or antagonist modulates adenylyl cyclase activity and/or GPCR activity and/or PDE activity. [0017] In one embodiment, the present invention provides a method for determining adenylyl cyclase activity in a sample comprising a sample that may contain adenylyl cyclase, adding to said sample an inactive enzyme capable of being activated by cAMP, adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and determining adenylyl cyclase activity present in said sample based on said signal. In some embodiments, said sample comprises a lysate. In some embodiments, the sample lysate is derived from bacteria, yeast or mammalian cells. In some embodiments, said sample comprises plasma membranes, cellular membranes, and/or organellar membranes. In some embodiments, said inactive enzyme is a cAMP dependent protein kinase. In some embodiments, said detection system comprises a substrate capable of being phosphorylated by PKA. In some embodiments, said substrate comprises SEQ ID NO: 1. In some embodiments, said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal wherein said enzyme is luciferase. In some embodiments, said substrate comprises a radioactively labeled biotinylated substrate further comprising SEQ ID NO: 1. In some embodiments, said detection system further comprises a streptavidin coated binding surface. In some embodiments, said substrate comprises a fluorescently labeled substrate further comprising SEQ ID NO: 1, wherein said fluorescent label is preferentially rhodamine. In some embodiments, the method of the present invention further comprises the addition of one or more inhibitors of phosphodiesterases, and/or the addition of an agonist or antagonist capable of affecting adenylyl cyclase activity. [0018] In one embodiment, the present invention provides a method for determining phosphodiesterase activity in a sample comprising a sample that may contain a phosphodiesterase, adding to said sample an inactive enzyme capable of being activated by cAMP, adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and determining phosphodiesterase activity present in said sample based on said signal. In some embodiments, said sample comprises a lysate. In some embodiments, the sample lysate is derived from bacteria, yeast or mammalian cells. In some embodiments, said sample comprises plasma membranes, cellular membranes, and/or organellar membranes. In some embodiments, said phosphodiesterase is a cyclic nucleotide phosphodiesterase. In some embodiments, said cyclic nucleotide is cAMP or cGMP. In some embodiments, said inactive enzyme is a cAMP dependent protein kinase or a cGMP dependent protein kinase. In some embodiments, said detection system comprises a substrate capable of being phosphorylated by PKA or PKG. In some embodiments, said substrate comprises SEQ ID NO: 1. In some embodiments, said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal wherein said enzyme is luciferase. In some embodiments, said substrate comprises a radioactively labeled biotinylated substrate further comprising SEQ ID NO: 1. In some embodiments, said detection system further comprises a streptavidin coated binding surface. In some embodiments, said substrate comprises a fluorescently labeled substrate further comprising SEQ ID NO: 1, wherein said fluorescent label is preferentially rhodamine. In some embodiments, the method of the present invention further comprises the addition of one or more inhibitors of phosphodiesterase activity. [0019] In one embodiment, the present invention provides a method for determining G-protein coupled receptor activity in a sample comprising a sample that may contain a GPCR, adding to said sample an inactive enzyme capable of being activated by cAMP, adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and determining GPCR activity present in said sample based on said signal. In some embodiments, said sample comprises a lysate, more preferably a lysate derived from mammalian cells. In some embodiments, said sample comprises plasma membranes. In some embodiments, said inactive enzyme is a cAMP dependent protein kinase. In some embodiments, said detection system comprises a substrate capable of being phosphorylated by PKA. In some embodiments, said substrate comprises SEQ ID NO: 1. In some embodiments, said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal wherein said enzyme is luciferase. In some embodiments, said substrate comprises a radioactively labeled biotinylated substrate further comprising SEQ ID NO: 1. In some embodiments, said detection system further comprises a streptavidin coated binding surface. In some embodiments, said substrate comprises a fluorescently labeled substrate further comprising SEQ ID NO: 1, wherein said fluorescent label is preferentially rhodamine. In some embodiments, the method of the present invention further comprises the addition of one or more inhibitors of phosphodiesterase activity and/or addition of an agonist or antagonist of GPCRs. [0020] In one embodiment, the present invention provides a kit for determining the concentration of cyclic nucleotides in a sample comprising a cyclic nucleotide, a protein kinase, ATP, a protein kinase substrate, and instructions for using said kit in determining said concentration of said protein kinase substrate. In some embodiments, said kit further comprises a luminescent detection system. In some embodiments, said kit further comprises a fluorescent detection system. In some embodiments, said kit further comprises a radioactive detection system. [0021] In one embodiment, the present invention provides a kit for determining the cyclic nucleotide phosphodiesterase activity in a sample comprising substrates fror cAMP and cGMP, a protein kinase, a protein kinase substrate, and instructions for using said kit in determining said activity of said cyclic nucleotide phosphodiesterase. In some embodiments, said kit further comprises a luminescent detection system. In some embodiments, said kit further comprises a fluorescent detection system. In some embodiments, said kit further comprises a radioactive detection system. Continue reading about Methods for cyclic nucleotide determination... Full patent description for Methods for cyclic nucleotide determination Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for cyclic nucleotide determination patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Methods for cyclic nucleotide determination or other areas of interest. ### Previous Patent Application: Method for quantitative detection of biological toxins Next Patent Application: Methods and compositions for diagnosing ankylosing spondylitis using biomarkers Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Methods for cyclic nucleotide determination patent info. IP-related news and info Results in 0.32557 seconds Other interesting Feshpatents.com categories: Daimler Chrysler , DirecTV , Exxonmobil Chemical Company , Goodyear , Intel , Kyocera Wireless , 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
