Daniel Albrecht
9/11/14
Dr. Chambers Lab
499Y proposal

Synthesis of a Click Chemistry Small Molecule Tracking Agent for GluA2 Unedited Receptors in ALS

1. The currently incurable neurodegenerative disease ALS causes excitotoxic cell death in motor neurons, leading to difficulty moving, speaking, swallowing, and breathing. Patients typically live just 3-5 years after the initial presentation of symptoms, with little recourse.1 The only available medication, Riluzole, affords only a fraction of the patients three additional months of life. Research into the mechanisms of ALS have revealed calcium mediated excitotoxic cell death as the primary culprit of motor neuron loss. Excitotoxicity is thought to be caused by an abundance of glutamate binding on overexpressed calcium permeable AMPA receptors (CP-AMPARs).2 Overexpression of CP-AMPARs on motor neurons of diseased patients leads to calcium intake levels that are fatal for the cell.3 Calcium impermeability of AMPA receptors is dependent upon the inclusion of an mRNA-editing event of a subunit called GluA2. Adenosine deaminase acting on RNA 2 (ADAR2) mediates the editing of GluA2 subunits in CP-AMPARs.4 ADAR2 has been shown to be significantly down-regulated in patients with ALS, leading to an overabundance of GluA2 lacking CP-AMPARs.5 Increasing cystolic Ca2+ levels causes a build-up of Ca2+ in the mitochondria that are slow to level off.6 Chronically high Ca2+ levels have been reported in ALS nerve cells7 and may lead to apoptosis.8 Additionally, fragmentation of the golgi apparatus and the endoplasmic reticulum have been detected in ALS patient motor neurons.9
The rate of cycling and prevalence of these receptors is still a topic of debate. The purpose of this proposal is to perform a three step synthesis under the guidance of Dr. Chambers to develop a covalently binding marker for CP-AMPARs with a click-chemistry moiety that can be used to bind a fluorophore or other tracking agent. With these markers, the behavior of the receptors can be studied throughout their lifetime.
2. Many readings are given as references at the end of the proposal. Other readings include:
Stromgaard K, Brierley MJ, Andersen K, Slok FA, Mellor IR, et al. (1999) Analogues of neuroactive polyamine wasp toxins that lack inner basic sites exhibit enhanced antagonism toward a muscle-type mammalian nicotinic acetylcholine receptor. Journal of Medicinal Chemistry 42: 5224-5234.

Kumar R, Ramachandran U, Raichur S, Chakrabarti R, Jain R (2007) Synthesis and evaluation of N-acetyl-L-tyrosine based compounds as PPARalpha selective activators. Eur J Med Chem 42: 503-510.

Agarwal A, Srivastava K, Puri SK, Sinha S, Chauhan PMS (2005) Solid support synthesis of 6-aryl-2-substituted pyrimidin-4-yl phenols as anti-infective agents. Bioorganic & Medicinal Chemistry Letters 15: 4923-4926.

Pisani L, Muncipinto G, Miscioscia TF, Nicolotti O, Leonetti F, et al. (2009) Discovery of a Novel Class of Potent Coumarin Monoamine Oxidase B Inhibitors: Development and Biopharmacological Profiling of 7-[(3-Chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one Methanesulfonate (NW-1772) as a Highly Potent, Selective, Reversible, and Orally Active Monoamine Oxidase B Inhibitor. Journal of Medicinal Chemistry 52: 6685-6706.

3. EHS requires trainings in Lab Safety, Hazardous Waste Management and Right to Know policies. These trainings have previously been completed and licensure for all trainings expires 1/6/2015. The trainings will need to be repeated prior to beginning the 499T term, but remain applicable during 499Y.
4. Before the end of the withdrawal period, I will prepare a draft of the thesis that focuses on background information and references while the experimental data is still being gathered. This draft will be expanded upon as the semester continues. The work in the first semester will primarily involve the synthesis of the