RNA Aptamer of ACE

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

• 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

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)
• Create an RNA aptamer library
• Use SELEX 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

• Websites

1. Background

• http://en.wikipedia.org/wiki/ACE_inhibitor

1. ACE with drug inhibitor

• http://molvis.sdsc.edu/fgij/fg.htm?mol=3L3N

1. Aptamer Binding Protein

• http://www.ncbi.nlm.nih.gov/pubmed/19734942

1. Aptamber Binding Angiotensin I

• http://www.ncbi.nlm.nih.gov/sites/entrez

1. DNA and protein sequence of type-1 angiotensin II receptor [Homo sapiens]

• http://www.ncbi.nlm.nih.gov/protein/4501997?report=genpept

1. 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

1. Structure of type-1 angiotensin II receptor

• http://www.jbc.org/content/285/4/2284.full.pdf+html