Cathinones are psychostimulant compounds heavily implicated as drugs of abuse. They exert their physiological actions at the monoamine transporters, which are responsible for maintaining synaptic neurotransmitter homeostasis. Monoamine transporters produce currents during transport and have been shown to depolarize cell membranes and activate voltage-gated calcium channels in mammalian expression systems. This phenomenon is harnessed in an assay which measures these induced calcium transients, allowing for quantification of pharmacodynamic effects of compounds at monoamine transporters. It is unknown if this electrical coupling occurs in neurons, but the implications if it does are significant. In the current work, fluorescent resonance energy transfer studies of HEK cells expressing hDAT suggest that a subpopulation of monoamine transporters and calcium channels may be interacting directly. Additionally, this work presents calcium assay data comparing several novel methcathinone analogs. Of the compounds tested, a single α-methyl substituent at the α-carbon yields the greatest potency at hDAT. The implications of these results shed light on future psychostimulant studies and further define the physiological relationship of the components of a system used to study these compounds.

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This dissertation work investigated the effect of acute and repeated in vivo administration of lobeline on dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) function. The effects of lobeline were then compared to the effects of acute and repeated in vivo administration of methylphenidate and amphetamine to determine if lobeline produced similar effects compared to these Attention Deficit Hyperactivity Disorder (ADHD) medications. These medications are considered the first line of pharmacotherapy for ADHD, although there is a growing concern associated with their potential for abuse and other side effects. This merits the need for novel ADHD treatments that have a safer side effect profile. If lobeline alters DAT and VMAT2 function in the same way as methylphenidate or amphetamine, further investigation may be necessary to evaluate lobeline as a potential treatment for ADHD. Kinetic analysis of [3H]dopamine (DA) was utilized to determine the effect on DAT and VMAT2 function in rat striatum. Results from the DAT experiments, revealed that lobeline as well as amphetamine had no effect on DAT function. However, methylphenidate increased DAT function after acute and 7-day treatment. None of the drug treatment regimens altered Km. To determine if the methylphenidateinduced increase in DAT function was due to DAT trafficking, biotinylation and Western blot analyses were performed. Acute administration of methylphenidate did not alter surface DAT, however repeated administration of methylphenidate for 7 days decreased intracellular DAT, suggesting that methylphenidate redistributes DAT in a time-dependent manner. Similar results were found in the VMAT2 experiments. Lobeline and amphetamine had no effect on VMAT2 function after acute or repeated administration. Amphetamine decreased the Km after repeated administration for 7 days. Methylphenidate increased VMAT2 function after acute and repeated administration for 7 days. The overall results of these experiments suggest that methylphenidate interacts with DAT and VMAT2 in a different manner than amphetamine and lobeline. In addition, since lobeline and amphetamine had no effect on DAT and VMAT2 function, further investigation is warranted to elucidate the underlying mechanisms of the therapeutic actions of these agents. This additional information will aid in the development of novel treatments for ADHD.

Abstract FUNCTIONAL AND BIOCHEMICAL CONSEQUENCES OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE HUMAN VESICULAR MONOAMINE TRANSPORTER 1 GENE (SLC18A1) By Sally Gamal Shukry, B.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Advisor: Jennifer K. Stewart Associate Professor and Graduate Director, Department of Biology Single nucleotide polymorphisms (SNP) in the human VMAT1 gene (SLC18A1) have been associated with schizophrenia in three different populations: Han Chinese, Western European and Japanese. Effects of these mutations on transport function of the hVMAT1 protein have not been reported. The goal of this study was to investigate functional and biochemical differences in human VMAT1 proteins with a threonine or proline at amino acid position 4 (Thr4Pro) and a serine or threonine at position 98 (Ser98Thr). COS1 cells were transfected with variant SNPs coding for 4Thr/98Ser, 4Pro/98Ser, or 4Thr98Thr. Western blotting demonstrated robust over expression of the genes and no differences in electrophoretic mobility of the proteins. Maximal transport of serotonin by the VMAT1 protein with 4Pro/98Ser was less than that of the 4Thr/98Ser or the 4Thr/98Thr. Response of the 4Pro/Ser98 to the VMAT inhibitor reserpine was lower than that of the 4Thr/98Thr. These findings suggest mechanisms for human VMAT1 links to schizophrenia.

Depression is a major psychological disorder that affects a person's mental and physical abilities. The National Institute of Mental Health (NIMH) classified it as a serious medical illness. It causes huge economic, as well as financial impact on the people, and it is also becoming a major public health issue. Antidepressant drugs are prescribed to mitigate the suffering caused by this disorder. Different generations of antidepressants have been developed with dissimilar mechanisms of action. According to the Center for Disease Control, the usage of antidepressants has skyrocketed by 400 percent increase over 2005- 2008 survey period. This dramatic rise in usage indicates that these are the most prescribed drugs in the US. Even with the FDA mandated "black box" warning of increased suicidal thoughts upon use of selected antidepressants, these drugs are still being used at a higher rate. <br>All classes of antidepressants are plagued by side effects with mainly sexual dysfunction common among them. To avoid the adverse effects, an emphasis is to discover novel structural drug scaffolds that can be further developed as a new generation of antidepressants. The importance of this research is to discover structurally novel antidepressants by performing in silico virtual screening (VS) of chemical databases using the serotonin transporter (SERT). In the absence of a SERT crystal structure, a homology model was developed. The homology model was utilized to develop the first structure-based pharmacophore for the extracellular facing secondary ligand binding pocket. The pharmacophore captured the necessary drug-SERT interaction pattern for SERT inhibitory action. This pharmacophore was employed as one of the filters for VS of candidate ligands. The ten compounds identified were purchased and tested pharmacologically. Out of the ten hits, three structurally novel ligands were identified as lead compounds. Two of these compounds exhibited selectivity towards SERT; the remaining lead compound was selective towards the dopamine transporter and displayed cocaine inhibition. The two SERT selective compounds will provide new opportunities in the development of novel therapeutics to treat depression. <br>For dopamine transporter (DAT), the study was based on recently developed structurally diverse photo probes. In an effort to better understand the binding profile similarities among these different scaffolds, the photo probes were docked into DAT. The finger print analysis of the interaction pattern of docked poses was performed to identify the inhibitor-binding sites. <br>For mitogen activated protein kinase 5 (MEK5), given the lack of structural information, a homology model of MEK5 was developed to guide the rational design of inhibitors. Docking of known MEK5 inhibitors into the homology model was performed to understand the inhibitory interaction profile. Several series of analogues were designed utilizing the generated interaction profile. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

Abstract Patients with idiopathic Parkinson's disease (PD) may suffer from autonomic nervous system dysfunction even in the early phase of the disease. We assessed the autonomic cardiovascular and sudomotor regulation in de novo PD patients with and without medication. We also measured the dopamine (DAT) and serotonin transporter (SERT) uptake in the PD patients using 2β-carboxymethoxy-3β-(4-iodophenyl)tropane (β-CIT) SPECT and studied the clinical correlates of the uptake. Sixty PD patients were included in the study and randomised to receive levodopa, bromocriptine or selegiline (n=20 in each) as their treatment. Thirty patients were examined with β-CIT SPECT. The results of the patients were compared with those of healthy controls and within the subgroups at different time points. Cardiovascular autonomic regulation was assessed using standard cardiovascular reflex tests at baseline, after six months' medication and following a 6-week washout period. The heart rate (HR) and blood pressure (BP) regulation was impaired in PD patients at baseline, and PD medications modified the responses further. Bromocriptine and selegiline, in contrast to levodopa, increased the orthostatic BP fall and suppressed the BP response to isometric exercise. The long-term cardiovascular autonomic function was evaluated from ambulatory ECG recordings by analysis of traditional spectral and non-spectral components of HR fluctuation together with two-dimensional vector analysis and power-law relationship analysis of the HR dynamics. All spectral measures and the slope of the power-law relationship demonstrated impaired tonic cardiovascular regulation in the PD patients. Sympathetic sudomotor activity was evaluated using the sympathetic skin response (SSR). The major finding was suppression of the SSR amplitudes with an inverse correlation to clinical disability, whereas PD medication seemed to have only minor effects. The changes in amplitude and repetitiveness of the SSRs with normal adaptation suggest deficits at several levels of the SSR reflex arc. DAT uptake, assessed by β-CIT SPECT, was diminished in the striatum and especially the putamen of the PD patients, and correlated with the results of the cardiovascular reflex tests and ambulatory ECG recordings. Simultaneous measurement of SERT binding demonstrated decreased SERT availability in the thalamic and frontal areas. The results demonstrate disturbances of the reflectory and tonic cardiovascular autonomic regulation caused by PD itself. PD medications further modify the reflectory responses. The degenerative process in PD also involves the sympathetic sudomotor pathway. β-CIT SPECT provides a useful method for simultaneous assessment of DAT and SERT binding, demonstrating the deficit of serotonin metabolism in PD.

Substance use disorders are serious health concerns in the United States. Furthermore, the National Survey on Drug Use and Health reports a continuous increase in substance use disorders in the United States during the last 10 years. However, there are not many effective pharmacotherapeutics available for substance use disorders. The current dissertation is focused on research aimed at discovering pharmacotherapeutics for substance use disorders. First part of dissertation focused on discovering methamphetamine (METH) use disorder therapeutics targeting specific mechanism of METH action on dopaminergic neurons. The second part of dissertation focused on opioids and cocaine use disorder therapeutics targeting rewarding pathway commonly activated by opioids and cocaine. With respect to METH, it induces release of dopamine (DA) in neuronal terminals by interacting with the vesicular monoamine transporter-2 (VMAT2) and DA transporter (DAT). VMAT2 inhibitors have been found by our research group to decrease METH-evoked DA release, METH-induced hyperlocomotion, and METH self-administration in rats. However, these VMAT2 inhibitors lacked selectivity and tolerance developed to these pharmacologic effects after repeated administration, thereby limiting their potential as pharmacotherapeutics for METH use disorders. In the current study, analogs from a novel scaffold were found to selectively inhibit VMAT2 and were evaluated using neurochemical and behavioral pharmacological approaches. R- and S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11610 and GZ-11608, respectively) exhibited 94- to 3450-fold selectivity for VMAT2 over human-ether-a-go-go (hERG) channel, DAT, serotonin transporter, and nicotinic acetylcholine receptors. GZ-11608 competitively and concentration-dependently inhibited METH-evoked DA release via VMAT2. Also, GZ-11610 (56-300 mg/kg, oral) and GZ-11608 (300 mg/kg, oral; 10-30 mg/kg, s.c.) reduced METH-induced hyperlocomotor activity in METH-sensitized rats. Furthermore, GZ-11608 (1-30 mg/kg, s.c.) inhibited METH self-administration, cue- and METH-induced reinstatement in a dose-dependent manner, and 30 mg/kg (s.c.), 10 mg/kg (s.c.), and 17 mg/kg (s.c.) produced significant effect, respectively. Importantly, the GZ-11608-induced decrease in METH self-administration was not surmounted by increasing the amount of METH available. GZ-11608 did not substitute for METH and did not serve as a reinforcer in rats self-administering METH and drug naïve rats, respectively. Thus, these VMAT2 inhibitors incorporating a new scaffold are novel leads for new pharmacotherapeutics to treat METH use disorders. Substances with high abuse potential including opioids and cocaine elevate extracellular DA concentration in the nucleus accumbens, and this mechanism has long been considered to underly substance-induced reward. DA in the nucleus accumbens originates from DA neuron cell bodies located in the ventral tegmental area in the midbrain. Interestingly, M5 muscarinic acetylcholine receptors (mAChRs) are proteins that are highly expressed on ventral tegmental area DA neurons. Also, studies investigating M5 mAChRs knockout mice showed reduced responding for cocaine in cocaine self-administration and decreased time spent in cocaine-paired and morphine-paired place preference studies. Pharmacological inhibition of M5 mAChRs function via microinfusing mAChR antagonists exhibiting no selectivity among M1-M5 mAChRs subtypes into the ventral tegmental area where expression of M5 mAChRs are dominant, reduced morphine-induced hyperlocomotion and cocaine seeking behaviors in rats. These studies support therapeutic potential of M5 mAChRs selectivity antagonists in opioids and cocaine use disorders. Thus, in the current study, affinity of a series of pethidine and quinuclidinyl N-phenylcarbamate analogs for M5 mAChRs was evaluated using in vitro and ex vivo neuropharmacological assays. Among the pethidine analogs, compound 6a showed the highest binding affinity at M5 (Ki = 0.38 µM), but also high affinity at M1 and M3 mAChRs (0.67 and 0.37 µM, respectively). Among the quinuclidinyl N-phenylcarbamate analogs, compound 13c exhibited the highest affinity at M5 (Ki = 1.8 nM), but also high affinity at M1, M2, M3 and M4 mAChRs (Ki = 1.6, 13, 2.6, 2.2 nM, respectively). Also, 13c acted as an agonist of mAChRs on oxotremorine-induced DA release from rat striatal slices. In addition, compound 13b was found exhibiting the highest selectivity (17-fold) at M3 over M2 mAChRs, suggesting potential of 13b as a chronic obstructive pulmonary disease therapeutics. Taken together, these novel analogs serve as leads for further discovery of subtype-selective M5 mAChR antagonists that may have potential as therapeutics for substance use disorders, as well as for chronic obstructive pulmonary disease.