<p>The G protein-coupled receptors (GPCRs) are important targets in drug discovery. In several cases, the endogenous ligands that activate the GPCRs of pharmaceutical interest are peptides. Unfortunately, peptides are in general not suitable as drugs, since the peptide structure is associated with several disadvantages, such as low oral bioavailability, rapid degradation and low receptor subtype selectivity. Thus, there is a strong need for drug-like nonpeptide ligands to peptide-activated GPCRs. However, to discover nonpeptide ligands that mimic the effect of the endogenous peptide, i.e. peptidomimetics, is a tremendous challenge. In fact, morphine and the related opioids were the only known examples of peptidomimetics before 1995 and these ligands were known long before the native endogenous peptide ligands were discovered. </p><p>The main objective of the work described in this thesis was to design, synthesize and biologically evaluate selective nonpeptide agonists to the peptide-activated GPCR AT₂. The AT₂ receptor belongs to the renin–angiotensin system, where the octapeptide angiotensin II (Ang II) is the major effector peptide. Ang II mediates its effects through the two GPCRs AT₁ and AT₂. The AT₁ receptor is already an established target in the treatment of hypertension. The physiological role of the AT₂ receptor, which is up-regulated in certain pathological conditions, is not fully understood but it seems to include positive effects such as vasodilatation, tissue repair, tissue regeneration and neuronal differentiation. </p><p>In the current investigation we started from the nonpeptide and nonselective (AT₁/ AT₂) compound L-162,313. This ligand is a known AT₁ receptor agonist but its effect on the AT₂ receptor was unknown at the start of this project. We were able to show that it acts as an agonist also at the AT₂ receptor. Furthermore, stepwise synthetic modifications of L-162,313 led to the identification of the first selective nonpeptide AT₂ receptor agonist. Following the discovery of this compound several selective nonpeptide AT₂ receptor agonists were identified. It was also revealed that a minor structural alteration of one of these compounds interconverted the functional activity from agonism to antagonism. The structural requirement for agonism vs antagonism was therefore studied. The functionality switch was suggested, at least partly, to be due to the spatial relationship between the methyleneimidazole group and the isobutyl side chain of the compounds. To further investigate the bioactive conformation(s) of this series of compounds enantiomerically pure analogues with conformationally constrained isobutyl chains were prepared. This study revealed that the direction of the isobutyl side chain determine whether the compounds act as agonists or antagonists at the AT₂ receptor. Further investigations are required to fully elucidate the bioactive conformation(s) of these nonpeptide AT₂ receptor agonists.</p><p>We believe that the selective nonpeptide AT₂ receptor agonists and antagonists identified in this thesis will serve as important research tools in the continuing investigation of the physiological role of the AT₂ receptor. We also believe that these drug-like compounds might provide potential leads in drug discovery processes.</p>

The G protein-coupled receptors (GPCRs) are important targets in drug discovery. In several cases, the endogenous ligands that activate the GPCRs of pharmaceutical interest are peptides. Unfortunately, peptides are in general not suitable as drugs, since the peptide structure is associated with several disadvantages, such as low oral bioavailability, rapid degradation and low receptor subtype selectivity. Thus, there is a strong need for drug-like nonpeptide ligands to peptide-activated GPCRs. However, to discover nonpeptide ligands that mimic the effect of the endogenous peptide, i.e. peptidomimetics, is a tremendous challenge. In fact, morphine and the related opioids were the only known examples of peptidomimetics before 1995 and these ligands were known long before the native endogenous peptide ligands were discovered. The main objective of the work described in this thesis was to design, synthesize and biologically evaluate selective nonpeptide agonists to the peptide-activated GPCR AT2. The AT2 receptor belongs to the renin–angiotensin system, where the octapeptide angiotensin II (Ang II) is the major effector peptide. Ang II mediates its effects through the two GPCRs AT1 and AT2. The AT1 receptor is already an established target in the treatment of hypertension. The physiological role of the AT2 receptor, which is up-regulated in certain pathological conditions, is not fully understood but it seems to include positive effects such as vasodilatation, tissue repair, tissue regeneration and neuronal differentiation. In the current investigation we started from the nonpeptide and nonselective (AT1/ AT2) compound L-162,313. This ligand is a known AT1 receptor agonist but its effect on the AT2 receptor was unknown at the start of this project. We were able to show that it acts as an agonist also at the AT2 receptor. Furthermore, stepwise synthetic modifications of L-162,313 led to the identification of the first selective nonpeptide AT2 receptor agonist. Following the discovery of this compound several selective nonpeptide AT2 receptor agonists were identified. It was also revealed that a minor structural alteration of one of these compounds interconverted the functional activity from agonism to antagonism. The structural requirement for agonism vs antagonism was therefore studied. The functionality switch was suggested, at least partly, to be due to the spatial relationship between the methyleneimidazole group and the isobutyl side chain of the compounds. To further investigate the bioactive conformation(s) of this series of compounds enantiomerically pure analogues with conformationally constrained isobutyl chains were prepared. This study revealed that the direction of the isobutyl side chain determine whether the compounds act as agonists or antagonists at the AT2 receptor. Further investigations are required to fully elucidate the bioactive conformation(s) of these nonpeptide AT2 receptor agonists. We believe that the selective nonpeptide AT2 receptor agonists and antagonists identified in this thesis will serve as important research tools in the continuing investigation of the physiological role of the AT2 receptor. We also believe that these drug-like compounds might provide potential leads in drug discovery processes.

We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein γ2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the γ2 subunit (Gγ2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a δ-opioid receptor agonist. In the present study, we examined the involvement of Gγ2 in antinociception mediated by other (μ- or κ-opioid, cannabinoid, α2-adrenoreceptor) analgesic agents in a warm (55°C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein γ2 subunits in supraspinal antinociception.

abstract: Patients with schizophrenia have deficits in sensorimotor gating, the ability to gate out irrelevant stimuli in order to attend to relevant stimuli. Prepulse inhibition (PPI) of the startle response is a reliable and valid model of sensorimotor gating across species. Repeated D2-like agonist treatment alleviates prior PPI deficits in rats, termed a PPI recovery, and is observable 28 days after treatment. The aim of the current project is to illuminate the underlying mechanism for this persistent change of behavior and determine the clinical relevance of repeated D2-like agonist treatment. Our results revealed a significant increase in Delta FosB, a transcription factor, in the nucleus accumbens (NAc) 10 days after repeated D2-like agonist treatment. Additionally, we investigated if Delta FosB was necessary for long-lasting PPI recovery and discovered a bilateral infusion of dominant-negative Delta JunD prevented PPI recovery after repeated D2-like agonist treatment. To further develop the underlying mechanism of PPI recovery, we observed that dominant negative mutant cyclic adenosine monophosphate (cAMP) response biding element protein (CREB) prevented repeated D2-like agonist-induced Delta FosB expression in the NAc. We then compared our previous behavioral and intracellular findings to the results of repeated aripiprazole, a novel D2-like partial agonist antipsychotic, to determine if repeated D2-like receptor agonist action is a clinically relevant pharmacological approach. As compared to previous PPI recovery and Delta FosB expression after repeated D2-like agonist treatment, we found similar PPI recovery and Delta FosB expression after repeated aripiprazole treatment in rats. We can conclude that repeated D2-like agonist treatment produces persistent PPI recovery through CREB phosphorylation and Delta FosB, which is necessary for PPI recovery. Furthermore, this pharmacological approach produces behavioral and intracellular changes similar to an effective novel antipsychotic. These findings suggest the underlying intracellular mechanism for sustained PPI recovery is clinically relevant and may be a potential target of therapeutic intervention to alleviate sensorimotor gating deficits, which are associated with cognitive symptoms of schizophrenia. / Dissertation/Thesis / Ph.D. Psychology 2013

abstract: Stroke remains the leading cause of adult disability in developed countries. Most survivors live with residual motor impairments that severely diminish independence and quality of life. After stroke, the only accepted treatment for these patients is motor rehabilitation. However, the amount and kind of rehabilitation required to induce clinically significant improvements in motor function is rarely given due to the constraints of our current health care system. Research reported in this dissertation contributes towards developing adjuvant therapies that may augment the impact of motor rehabilitation and improve functional outcome. These studies have demonstrated reorganization of maps within motor cortex as a function of experience in both healthy and brain-injured animals by using intracortical microstimulation technique. Furthermore, synaptic plasticity has been identified as a key neural mechanism in directing motor map plasticity, evidenced by restoration of movement representations within the spared cortical tissue accompanied by increase in synapse number translating into motor improvement after stroke. There is increasing evidence that brain-derived neurotrophic factor (BDNF) modulates synaptic and morphological plasticity in the developing and mature nervous system. Unfortunately, BDNF itself is a poor candidate because of its short half-life, low penetration through the blood brain barrier, and activating multiple receptor units, p75 and TrkB on the neuronal membrane. In order to circumvent this problem efficacy of two recently developed novel TrkB agonists, LM22A-4 and 7,8-dihydroxyflavone, that actively penetrate the blood brain barrier and enhance functional recovery. Findings from these dissertation studies indicate that administration of these pharmacological compounds, accompanied by motor rehabilitation provide a powerful therapeutic tool for stroke recovery. / Dissertation/Thesis / Ph.D. Neuroscience 2013

Congenital myasthenic syndromes (CMS) are a rare group of heterogeneous disorders, characterised by compromised neuromuscular transmission and symptoms of fatiguable muscle weakness. CMS is caused by mutations in genes that affect the structure and function of the neuromuscular junction (NMJ). In about 20% of CMS cases, patients have mutations in the gene DOK7; the protein product, DOK7, is crucial for maintaining the dense aggregation of acetylcholine receptor (AChR) clusters at the NMJ. DOK7-CMS patients do not respond to treatment with acetylcholinesterase inhibitors which are the first line treatment for most forms of CMS. Instead, a dramatic response to beta-2 adrenergic receptor (ADRB2) agonists, such as salbutamol, is observed. The aim of this project was to investigate the molecular mechanisms that underlie the beneficial effects of ADRB2 agonists. Firstly, NMJ functioning was modelled in vitro by studying AChR clusters formed on cultured C2C12 mouse myotubes in the presence of WT DOK7. Overexpression of mutant DOK7 led to a significant reduction in the number of AChR clusters, explaining the pathogenic effect of the mutation. Importantly, incubation of myotubes with salbutamol increased the number of AChR clusters and their stability. The results provide the first evidence that ADRB2 agonists directly affect proteins located at the NMJ. However, this disease model suffers from limitations. The rest of the thesis focussed on developing alternative cell culture models to explore the AChR clustering pathway. The first model combined optogenetics and fluorescence lifetime microscopy to study the effects of ADRB2 activation on AChR cluster stability in single live cells. The second used CRISPR/Cas9 genome editing tools to directly introduce Dok7 mutations to the genome of C2C12 cells, thereby overcoming some of the drawbacks associated with DOK7 overexpression. Further manipulations of these novel model systems will be used in the future to examine in more detail the molecular events underlying the pathogenic effects of DOK7 mutations and the mechanisms of ADRB2 agonists.

Release of endomorphin (ENDO)-2 like substances from the dorsal horn of the isolated rat spinal cord was measured by the immobilized-antibody microprobe technique. Spinal cords were removed from anesthetized 4-6 week old rats and superfused with oxygenated Krebs solution at room temperature. Glass microprobes coated with ENDO-2 antibodies were inserted into the dorsal horn of the lumbar spinal cord 1.5 mm lateral to the midline to a depth 2.5 mm below the dorsal surface of the cord. Each probe remained in situ for 10 min periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same spinal segment. There was no detectable basal release of immunoreactive endomorphin-2 like substance (irENDO) from the dorsal horns during the pre-stimulation, nor following the stimulation period. A significant release of irENDO was measured during the electrical stimulation. These results provide the first evidence of a irEndo release that is correlated spatially with the dorsal horn laminae I and II where ENDO-2-immunoreactive fibers are concentrated in the dorsal horn in response to electrical activation of primary afferent fibers.

No / A sensitive and rapid high performance liquid chromatography method has been developed and used for the simultaneous determination of formoterol and budesonide in Symbicort Turbuhaler when assessing the aerodynamic characteristics of the emitted dose using Pharmacopoeial methods. This capability results in both time and cost saving. The mobile phase composition was acetonitrile-5 mM sodium dihydrogen orthophosphate, pH 3 (60: 40% v/v), and was passed at 1.5 ml min-1 through a C18 column with a UV detection (wavelength 214 nm). The method was shown to give good analytical performance in terms of linearity, precision (using phenylpropanolamine as an internal standard), sensitivity and solution stability. The intra-day precision for both formoterol and budesonide were 0.75% and 1.11%, respectively (n = 10). The limit of quantitation for formoterol was 10 ¿g L-1 and for budesonide was 120 ¿g L-1, and the limit of detection were 3 and 30 ¿g L-1, for both formoterol and budesonide, respectively. The method has been applied to determine the content of the emitted dose and the fine particle dose of Symbicort Turbuhaler.

Many peptide receptors are of pharmaceutical interest and there is thus a need for new ligands for such receptors. Unfortunately, peptides are not suitable as orally administrated drugs since they are associated with poor absorption, rapid metabolism and low sub-receptor selectivity. One approach that should allow identification of more drug-like ligands is to use the structural information of the endogenous ligand to develop peptidomimetic compounds. The main objective of the work described in this thesis was to convert angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) to small drug-like compounds with retained bioactivity at the AT2 receptor. The study was performed step-wise via incorporation of well-defined secondary structure mimetics and repeated truncation of the peptide. Five scaffolds, comprising a benzene ring as a central element, suitable as a γ-turn or dipeptide mimetics were designed and synthesized. In order to decorate the scaffolds, a method of microwave-assisted alkoxycarbonylation was developed. After incorporation of the scaffolds into Ang II-related peptides or peptide fragments, the affinities for both the AT1 and the AT2 receptor were determined. In the first series of ligands, two tyrosine-related scaffolds were introduced as γ-turn mimetics in Ang II. All five pseudopeptides exhibited good affinities for the AT2 receptor. One compound was chosen for functional studies and was shown to act as an AT2 receptor agonist. After truncation of Ang II it was shown that C-terminal pentapeptide analogs were AT2 receptor selective agonists. A series of pseudopeptides comprising tyrosine-related scaffolds, derived from the pentapeptides, displayed high AT2 receptor affinities. Two compounds had agonistic effect at the AT2 receptor. This study revealed that the N-terminal part was of less importance while a C-terminal Ile residue was a key element for enhanced AT2 receptor affinity. In the final set of compounds, the peptide was truncated to tripeptide C-terminal fragments. After replacing His-Pro by a histidine-related scaffold small drug-like peptidomimetic compounds with nanomolar affinity for the AT2 receptor were identified.

Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.