RNA Aptamer of ACE

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Contents

Overview

We are proposing the selection for a novel aptamer that binds to Angiotensin Converting Enzyme (ACE) in order affect its ability to convert angiotensin I into angiotensin II. Angiotensin II is a protein that causes the muscles surrounding blood vessels to contract and narrow blood vessels. This narrowing of blood vessels can have negative effects such as increased blood pressure and hypertension. ACE inhibitors exist that are shown to decrease blood pressure but have negative side affects that result from the degradation of the drug. RNA aptamer drugs have the potential to have the same inhibitor effects with less side affects.

Project Detail and Methods

We will use SELEX to select the aptamer that binds to ACE with the highest affinity and specificity. We immobolize ACE to a nickel agarose resin by a His tag. Then we will wash the aptamer library over the the resin to select for the aptamers that bind. We will also use negative selection to remove the aptamer that binds to resin simply.

After purification of the aptamers, we will select for the aptamers with the greatest negtive impact on ACE activity. We will perform an assay with angiotensin I and ACE and measure the amounts of angiotensin I and angiotensin II after incubation. Ideally, our aptamer will inhibit ACE activity, and thus the sample with the highest angiotensin I will have the lowest activity.


Motivation

  • Untreated hypertension tends to shorten life expectancy by approximately 5 years (Franco 2005)
  • Angiotensin II is involved in the vasoconstriction of arteries, decreased levels of angiotensin II decrease blood pressure
  • Inhibition of the rennin angiotensin system (RAS) has consistently been found to reduce progressive deterioration of kidney function (Van der Meer 2010)
    1. occurs through reduction of blood pressure and proteniuria (abnormal amount of protein in urine)
    2. two different methods of inhibition of RAS: angiotensin converting enzyme (ACE) inhibitors (specifically called kininase II and bradykinin dehydrogenase) and angiotensin II receptor type I blockers (ARBs), ARBs typically much more expensive than ACE inhibitors
    3. ACE inhibitors have similar effect to ARBs, can be used in combination to increase the inhibition of RAS more than with a single RAS inhibitor (Catapano 2009)

Background

  • Renin-Angiotensin System (RAS)
    1. Angiotensinogen, produced in the liver, is cleaved by renin in the kidneys to form angiotensin I which is then activated by ACE (kininase II and bradykinin dehydrogenase) in lungs to form angiotensin II
    2. Angiotensin II binds to AT receptors vascular smooth muscle cells and cause vasoconstriction
  • ACE inhibitors (rimipril, lisinopril)
    1. ACE inhibitors after-load reducers meaning they dilate the arteries after the heart, makes it easier for the heart to pump
    2. Cause chronic dry cough in 12% of patient: it is not well understood but it is thought that ACEs metabolize bradykinins, when ACE is inhibited bradykinins collect and are thought to be connected to the formation of NO in the body, thus the cough to expel
  • Anginotension II Type I Receptor Blockers (ARBs)
    1. Relatively expensive drugs that block the binding between angiotensin II and the receptor protein on the smooth vascular cells
    2. They then cannot constrict to increase blood pressure
    3. In a study of 129 patients with a history of ACE inhibitor-induced, a randomized double blind study on coughing also found that the occurrence of cough after 3 and 6 weeks of therapy was significantly less with the ARB valsartan (19.5%) and hydrochlorothiazide (19%) than with lisinopril (68.9%) (Benz 2005)
    4. Other study says that there is decreased angioedema while using an ARB as opposed to ACE inhibitor (Makani 2010)
  • Most patients must take more than one blood pressure medication to achieve target blood pressure
  • Tetrahydroimidazopyridines (PDs) have been discovered that block the AT2 receptors, our aptamer will give evidence to the importance of the specific sites on the receptor that activate bradykinin
    1. NO is also produced by AT2 receptor, if we can activate NO but inhibit bradykinin, we can decrease cough while keeping vasodilation ability

Experimental Overview

  • Express the type-1 Angiotensin II (A2)receptor
    1. Modify the intracellular domain to contain a binding attachment for immobilization (See P. Ghanouni 2001)
  • Immobilize protein on bead or micro array (see Yan 2009 and Neumann 2002 - M1 antibody immobilization)
    1. The transmembrane protein is first solubilized in a micelle by a detergent
    2. BSA-biotin is deposited onto gold or glass which it spontaneous binds
      • the BSA-biotin is resitant to high and low salt washes and to washes with detergent (dodecyl-beta-D-maltoside [DDM]) that holds the transmembrane protein
    3. Avidin is then coated over the BSA-biotin
    4. the TM protein can be stuck to the avidin directly by a cystiene residue on the TMP bound to biotin or by a monoclonal antibody bound to biotin.
  • Create an RNA aptamer library that has a polyethelyne glycol (PEG) molecule attached to the 5' end
    1. PEG increases the half life of the aptamer for in vivo studies by decreasing rate of renal degradation (Bailon 2009)
  • Use SELEX (with a 40 nt variable region as recommended by Daniels et al. for receptor G-proteins) to select an aptamer that binds the A2 recetpor
    1. Run a negative selection on the immobilization structure without the receptor protein
    2. Run SELEX on the immobilized receptor
    3. Isolate enriched aptamers with high affinity and specificity for A2 receptor.
  • Assay Kd of Angiotensin II binding A2 receptor
    1. Angiotesin II and receptor/micelle complex
    2. Angiotensin II and each aptamer separately and A2 receptor/micelle complex
    3. Angiotensin II and known A2 receptor blockers and A2 receptor/micelle complex

References

  1. Van der Meer IM, Cravedi P, Remuzzi G. The role of renin angiotensin system inhibition in kidney repair. Fibrogenesis and Tissue Repair 2010; 3:7
  2. Catapano F, Chiodini P, De Nicola L, Minutolo R, Zamboli P, Gallo C, Conte G. Antiproteinuric response to dual blockade of the rennin-angiotensin system in primary glomerulonephritis: meta-analysis and metaregression. Am J Kidney Dis 2008; 52:475-485
  3. Bailon P, Won CY. 2009. PEG-modified biopharmaceuticals. Expert Opin. Drug Deliv. 6:1–16
  4. Benz J, Oshrain C, Henry D, et al. Valsartan, a new angiotensin II receptor antagonist: a double-blind study comparing the incidence of cough with lisinopril and hydrochlorothiazide. J Clin Pharmacol 1997; 37:101-107.
  5. Daniels et al. Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin, NTS-1. Analytical biochemistry (2002)
  6. Franco OH, Peeters A, Bonneux L, de Laet C. Blood pressure in adulthood and life expectancy with cardiovascular disease in men and women. Hypertension 2005; 46:280-6.
  7. Makani et al. DO ANGIOTENSIN RECEPTOR BLOCKERS INCREASE THE RISK OF ANGIOEDEMA?. J Am Coll Cardiol 2010
  8. Background: http://en.wikipedia.org/wiki/ACE_inhibitor
  9. ACE with drug inhibitor:http://molvis.sdsc.edu/fgij/fg.htm?mol=3L3N
  10. Aptamer Binding Protein:http://www.ncbi.nlm.nih.gov/pubmed/19734942
  11. Aptamber Binding Angiotensin I:http://www.ncbi.nlm.nih.gov/sites/entrez
  12. DNA and protein sequence of type-1 angiotensin II receptor [Homo sapiens] :http://www.ncbi.nlm.nih.gov/protein/4501997?report=genpept
  13. Immobilize G protein receptors:http://www.nature.com/nbt/journal/v17/n11/pdf/nbt1199_1105.pdf; http://www.stanford.edu/group/Zarelab/pub%20links/680.pdf
  14. Structure of type-1 angiotensin II receptor: http://www.jbc.org/content/285/4/2284.full.pdf+html
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