开题报告内容：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）
Opening report
Cocaine is a highly abusive and addicting drug. Cocaine acts by blocking the reuptake of the neurotransmitters serotonin (5-HT), dopamine (DA), and norepinephrine (NE). This results in higher concentrations of these three neurotransmitters in the human brain. It can easily cross the blood–brain barrier and acts very fast.[8] [4] It is important that we should avoid it from happening and find treatments for cocaine abuse and addiction.
The dopamine transporters (DAT), serotonin transporters (SERT), and norepinephrine transporters (NET), which are called as monoamine transporters (MATs) together, have a huge impact on regulating the neuronal response to the neurotransmitters. MATs terminate the action of the neurotransmitters by transporting them from the synaptic cleft back into the presynaptic neurons where they are either stored in vesicles or degraded. These three neurotransmitters are responsible for controlling a number of physiological, emotional, and behavioral functions.[1] MATs are major drug targets for the treatment of many psychiatric diseases such as anxiety, depression, obesity, drug abuse, obsessive compulsive disorder, attention deficit hyperactive disorder (ADHD), and schizophrenia. When blocking these proteins, the concentration of the neurotransmitter within the synapse is increased hereby relieving the symptoms of many mental illnesses.[5] They are also the target of psycho-stimulants such as amphetamine, cocaine, and ecstasy.[6]
Since drug addiction is an enormous burden to the society and human health, it is extremely important to understand the molecular mechanism of how these compounds interact with the MATs. Drugs of abuse include inhibitors like cocaine and a class of compounds such as amphetamine, which are able to reverse the direction of transport in MATs by a mechanism that is still not fully understood.
Typical DAT blockers fully block the DA re-uptake, and also block the binding of another blocker. Behaviorally, typical DAT inhibitors are also known to stimulate locomotor behavior, and reinforcing behavior. As a result, they often suffer from abuse, just like cocaine. In contrast, atypical DAT ligands deviate from those expected effects, both in vitro and in vivo. Atypical DAT inhibitors have potential as treatments for stimulant abuse, and they may also provide important basic information about DAT function. Examples of atypical DAT inhibitors are benztropone, modafinil, sydnocarb and GBR 12909.[5]
The dopamine transporter (DAT) is the major site of action for psycho-stimulants like amphetamine and cocaine and as a consequence of great clinical interest. We have recently discovered a novel allosteric site (named as the A2 site) in DAT and we are studying if targeting the A2 site has clinical potential in reducing the psychostimulant effects. A class of atypical DAT inhibitors has been demonstrated to display interesting behavioral profiles and we hypothesize that this is caused by the interaction of these compounds with A2. In this project, we will use various biochemical methods to test if the atypical DAT inhibitors interact with A2 and how they modulate dopamine transporter function.
Luckily, in May 2015 a library of new crystal structures of dDAT with various ligands bound was published. With the help of these crystal structures, we were able to directly visualize the binding of various DAT inhibitors within the DAT binding site and.[6] Through these structures, we learnt that cocaine, and other cocaine-like typical DAT inhibitors block the DA transport by directly binding to the DA-binding site, i.e. the orthosteric site S1. This means that cocaine and other cocaine-like compounds are competitive inhibitors of DA-transport.[5] In further studies, scientists have also compared the binding of drugs to dDAT and LeuBAT (bacterial transporter which transports leucine) and find it is possible to reveal the binding of drugs to the human MATs through computational modeling and experimental validation.[6]
To successfully achieve the crystallization of the dDAT protein bound to various DAT inhibitors, two different types of dDAT chimeras were used: dDATCrystal and the other is dDATmfc. dDATCrystal was a thermo-stabilized variant of wild-type dDAT and lacks neurotransmitter transport activity, while the dDATmfc has fewer thermo-stabilizing mutations and possesses dopamine transport activity.[4] For my project, I am using the DNAs of these two variants of the dDAT to do biochemical assessment with atypical DAT inhibitors. I want to test the hypothesis of whether the atypical DAT inhibitors bind to the A2 site and how the binding of these inhibitors affect the interaction of typical DAT blockers like cocaine. I will use the cDNA of dDATcryst and dDATmfc to express these proteins in COS-7 cells and run pharmacological assays to study the effects of atypical DAT inhibitors such as modafinil, sydnocarb and benztropine on the DA-transport. To achieve this, I need high concentrations of the cDNA of dDATcryst and dDATmfc. To make more cDNA, I will use the method of bacterial transformation for making larger amounts of the cDNA. In this method, the cDNA is introduced into the E.coli bacteria. Transformation is a key step in DNA cloning. The bacteria is given a heat shock, which causes DNA of drosophila to insert in some of them. The basic answer is that a heat shock makes the bacterial membrane more permeable to DNA molecules, such as plasmids. It appears that the heat shock causes the formation of pores in the bacterial membrane, through which the DNA molecules can pass. After transformation, bacteria are plated on agar plates containing an antibiotic. The antibiotic, such as carbenicillin, is added to avoid the growth of native bacteria. Bacteria with a plasmid are antibiotic-resistant, and each one will form a colony. Plates are put in 37 degrees Celsius incubator overnight and let colonies grow.
Then QiagenTM midi-prep protocol will be followed to further grow the transformed bacteria. The bacteria will be then lysed to release the cDNA. cDNA will be filtered so they can be separated from impurity, washed and dried. Finally, DNA is dissolved in liquid. Then the concentration of DNA can be measured and stored in freezers. The concentration of DNA is good if it is larger than 1mu;g/mu;L.[7] The plasmid is then ready for use in cell culture experiments.
