There is a dogma in molecular biology that biological functions of peptides are determined by their structure ("function" code), coded in their primary structure ("structure" code). This work describes a new approach that attempts to elucidate these relationships by peptide topology design based on intriguing conformational properties of pipecolic acid based amino acids--like 1,2,3,4 tetrahydroisoquinoline (Tic). Opioid peptides, owing to the heterogeneity of opioid receptors, display a wide variety of physiological actions. The mu opioid receptor selective octapeptide I (D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂) is a model compound for topographical modifications induced by sequential substitutions by Tic residue. Thus, the closely related peptides I and II (Gly-D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂, obtained by coupling Gly residue to I) have contrasting affinities for the mu opioid receptor (IC₅₀ = 1.2 and 278 nM, respectively). Conformational analysis of I and II by means of 1D and 2D ¹H NMR spectroscopy allowed to determine dramatic differences in the side chain orientation of D-Tic in both peptides and to propose features of the bioactive conformation. The extended conformation of I (due to g(-) side chain conformation of D-Tic) is well recognized by the mu receptor in contrast to the folded conformation of II (due to a g(+) side chain conformation of D-Tic¹, that places the aromatic ring on the opposite side of the molecule), which is not. Peptide III (D-Phe-Cys-Tic-D-Trp-Orn-Thr-Pen-Thr-NH₂), featuring replacement of Tyr³ by Tic³, binds very weakly to the mu opioid receptor, due to rotation of the Tic aromatic side chain to the opposite side of the molecule (Tic side chain is in a g(+) conformation again). As these substitutions conserve the conformation of the backbone, constrained cyclic amino acids (picolic acid derivatives) can modify the topography of the peptide in a predictable manner, and (in conjunction with biological data) disclose structural elements of bioactive conformations. The mechanisms of pipecolic acid side chain rotamer selection, will be discussed in the context of design principles.

The metabolism of ADP-ribose polymers (PAR) is involved in several cellular processes with a primary focus on maintaining genomic integrity. PAR metabolism following genotoxic stress is transient due to a close coordination between poly(ADP-ribose) polymerases (PARPs) which synthesize PAR and poly(ADP-ribose) glycohydrolase (PARG) which degrades PAR. PARP-1 inhibitors have emerged as promising anticancer therapeutics by increasing chemotherapy sensitivity and selectively target tumors harboring DNA repair defects. Several pharmaceutical companies have PARP-1 inhibitors in clinical trials for treatment of cancer. PARP-1 inhibitors are generally well tolerated, although they typically have poor selectivity among PARPs, and potentially other NAD binding enzymes. The promise of PARP-1 inhibitors as cancer therapeutics has led this dissertation research towards developing alternative tools and approaches to target PAR metabolism.One approach described is an evaluation of high-throughput PARP-1 screening assays as potential tools to discover new classes of PARP-1 inhibitors. These assays were compared to a widely used radiolabeling PARP-1 assay. They were found to offer several advantages that include simplicity, sensitivity, reproducibility, accuracy and eliminating the need for radioactive materials.The primary focus of this dissertation research was to develop PARG inhibitors as an alternative way of targeting PAR metabolism. Lack of viable genetically engineered animals, effective siRNA, and useful pharmacological 20 inhibitors has prevented PARG from being evaluated as a therapeutic target. This dissertation describes the first systematic approach, using Target related Affinity Profiling (TRAP) technology, for the discovery of PARG inhibitors. Identification of several hits led to the first detailed structural activity relationship (SAR) studies defining a pharmacophore for PARG inhibition. Interestingly, these molecules show varying degrees of PARP-1 inhibition, providing the first direct evidence for homology in the active sites of PARP-1 and PARG. Evaluation of a lead inhibitor has provided the first evidence for PARG inhibition in intact cells. Further optimization resulted in a cell permeable inhibitor with reduced toxicity and poor selectivity, providing evidence for a new class of inhibitors that disrupt PAR metabolism by inhibiting both enzymes. The use of dual PARG/PARP-1 inhibitors represents a new approach for therapeutic development of anticancer agents. Finally, directions aimed to overcome remaining challenges are discussed.

Millions of people in the US currently suffer from chronic pain but available therapeutics do not provide effective chronic pain treatment. Opiate therapy is still the gold standard for chronic pain management with detrimental side effects, such as tolerance, addiction, constipation, and respiratory depression that limit their therapeutic potential. Opiates exert their positive and negative effects by activating the μ opioid receptor (MOR). Conversely, the κ opioid receptor (KOR) has been shown to modulate the tolerance and addiction produced by MOR agonists and is also involved in mood modulation (anxiety and depression). Therefore, blocking KOR activation results in positive effects against opiate side effects and stress-related depression. Dynorphin A (Dyn A) is the endogenous opioid peptide for the KOR. Structure-activity relationship (SAR) studies were carried out to develop a KOR selective antagonist based on the Dyn A structure. A minimum Dyn A pharmacophore with improved stability, no cell toxicity, and antagonist activity was discovered. Peptidomimetic enkephalin analogues previously developed in our group as MOR and δ opioid receptor (DOR) agonists have shown multifunctional activity, with MOR/DOR agonist and KOR antagonist activities. To our knowledge, this finding is first of its class for the opioid receptors. Novel design and synthesis of KOR selective ligands based on our multifunctional enkephalin analogues was done. Successful peptide synthesis resulted in analogues with high stability in rat plasma and no cell toxicity.

Aerosols play important roles in atmospheric and environmental processes. Not only do they impact human health, they also affect visibility and climate. Despite recent advances made to under their sources and fate, there remains a limited understanding of the mechanisms that lead to the formation of aerosols and their ultimate fate in the atmosphere. These knowledge gaps provide the crux of the research reported herein, which has focused on identifying novel sources of atmospheric aerosol, characterizing its physical and optical properties, and rationalizing these properties using an in-depth knowledge of the molecular level mechanisms that led to its formation. Upon mowing, turfgrasses emit large amounts of green leaf volatiles which can then be oxidized by ozone to form SOA. Overall, the mowing of lawns has the potential to contribute nearly 50 µg SOA per square meter of lawn mowed. This SOA contribution is on the same order of magnitude as other predominant SOA sources (isoprene, monoterpenes, sesquiterpenes). Turfgrasses represent an interesting and potentially meaningful SOA source because they contribute to SOA and also because they cover large land areas in close proximity to oxidant sources. Another related SOA precursor is sugarcane, which is in the same family as turfgrass and is among the largest agricultural crops worldwide. Globally, the ozonolysis of sugarcane has the potential to contribute 16 Mg SOA to the atmosphere, compared to global estimates of SOA loading that range from 12-70 Tg SOA. In order to fully understand the role of atmospheric SOA on the radiative budget (and therefore climate), it is also important to understand its optical properties; its ability to absorb vs scatter light. Turfgrass and sugarcane produced SOA that was weakly absorbing while its scatter efficiency was wavelength and size-dependent. Interestingly, SOA formed under both dry (10% RH) and wet (70% RH) conditions had the same bulk chemical properties (O:C), yet significantly different optical properties, which was attributed to differences in molecular-level composition. The work presented herein represents a unique, inclusive study of SOA precursors. A complete understanding of the chemistry leading to SOA formation is used to understand its physical and optical properties and evaluate these large-scale effects of SOA from these precursors.

The overall aim of this work was to apply HINT, an empirical scoring function based on the understanding of hydrophobicity, to analyze and predict the binding affinities and biological activities of colchicine-site anticancer agents. The second, concurrent aim was to improve the scoring function by incorporating tautomerism within the modeling process. Our belief is that proper evaluation of tautomeric forms for small molecules will improve performance of virtual screening. The novel pyrrole-based compounds targeting the colchicine site were docked into the receptor using HINT as a rescoring function. Two distinct binding modes dictated by the size and shape of a subpocket were predicted to differentiate the highly active compounds from the weak ones. Of the residues predicted to participate in binding for the active binding mode, Cys241β was revealed to form a weak but critical hydrogen bond with the ligand. A larger collection of colchicine-site agents, biologically tested in the same laboratory including our pyrrole-based compounds were subject to 3D quantitative structure-activity relationship (QSAR) study. Using results on docking the pyrrole compounds as a guide, relative binding poses and QSAR models were built to facilitate ligand design and optimization. A new 3D modeling approach was introduced to visually highlight the unique features of highly active compounds and the commonality of all compounds in the dataset using HINT maps and successfully tested on the colchicine-site agents. These results will provide valuable guidance in the future design and development of new colchicine-site agents. To incorporate tautomerism within HINT, we proposed and developed two workflow approaches: a general search tool using a simple and intuitive algorithm analyzing hydrogen shift patterns to identify and enumerate tautomeric structures, and a database that contains commonly observed tautomeric structures. The first approach was designed for small-scale docking studies and the second approach was designed for large-scale virtual screening. The tautomer module in HINT will give more accurate modeling results when the compound encountered is able to tautomerize.

Methcathinone (MCAT) is the β-ketone analog of methamphetamine, and like its amphetamine analog, MCAT functions as a monoamine releaser that selectively promotes the release of dopamine (DA) and norepinephrine (NE) over serotonin (5-HT). MCAT produces amphetamine-like psychostimulant effects and is classified as a Schedule I drug of abuse by the United States Drug Enforcement Administration (DEA). Recently, synthetic MCAT analogs have emerged as designer drugs of abuse in Europe and the United States and have been marketed under deceptively benign names like “bath salts” in an attempt to evade legal restriction. These dangerous, recently emergent and novel drugs of abuse display varying selectivity to promote release of DA/NE vs. 5-HT, and selectivity for DA neurotransmission is believed to correlate with abuse liability. The goal of this dissertation was to conduct preclinical research to examine structural determinants of abuse-related behavioral and neurochemical effects produced by a series of synthetic MCAT analogs. Specifically, this project focused on one feature of the methcathinone scaffold: the para substituent of the benzene ring. A series of six novel MCAT analogs will be examined to evaluate how physicochemical parameters (steric, Es; electronic, σp; lipophilic, πp) of the para substituent influence in vitro monoamine transporter selectivity as well as in vivo neurochemical and behavioral effects. Results from this body of work implicate steric factors as being particularly important in determining a compound’s abuse-related neurochemical and behavioral effects. Thus, these data not only offer an improved understanding of the mechanism of abuse-related drug effects produced by synthetic MCAT analogs, but also help in the generation of homology models of the human DA and 5-HT transporters (DAT and SERT, respectively).

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22397号 / 薬科博第119号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Esticolisinas I e II, citolisinas purificadas da anêmona marinha Stichodactyla helianthus, agem lisando membranas biológicas e modelo. O mecanismo de ação proposto consiste na formação de um poro toroidal com o envolvimento do domínio N-terminal. Diferentes aspectos da interação entre peptídeos derivados do N-terminal das toxinas (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, e StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) com membranas modelo - micelas e bicamadas - foram estudados com o objetivo de contribuir para a elucidação do mecanismo de ação das toxinas em nível molecular. O emprego dos peptídeos teve como base a hipótese de que fragmentos proteicos podem ser capazes de mimetizar a estrutura e atividade das proteínas inteiras. O análogo contendo o aminoácido paramagnético TOAC (N-TOAC-StII11-30) também foi estudado. Estudos conformacionais foram realizados empregando-se as técnicas espectroscópicas de dicroísmo circular (CD), ressonância paramagnética eletrônica (EPR) e fluorescência. Foram ainda realizados estudos de predição de estrutura e modelagem molecular. Espectros de CD mostraram que os peptídeos adquirem conformação em α-hélice ao interagir com membranas modelo, de acordo com a conformação observada nessa região para as toxinas. Variando a composição lipídica das membranas modelo estudadas, foi possível investigar a contribuição de forças eletrostáticas de de interações hidrofóbicas para a ligação do peptídeo. Ensaios de supressão de fluorescência de lípidos contendo grupamentos fluorescentes em diferentes posições pelo resíduo paramagnético TOAC e espectros de ressonância paramagnética eletrônica (EPR) permitiram localizar o resíduo TOAC na interface membrana-água, corroborando o modelo proposto do poro toroidal. A análise dos espectros de CD e EPR também permitiu obter as constantes de ligação dos peptídeos com micelas e bicamadas. Os peptídeos também foram capazes de mimetizar as toxinas do ponto de vista funcional, como mostrado por testes de vazamento de carboxifluoresceína e atividade hemolítica. Peptídeos curtos, contendo partes da sequência de StII1-30, sintetizados com o objetivo de examinar uma eventual atividade antimicrobiana, demonstraram baixa atividade, bem como ausência de atividade hemolítica e de toxicidade para células humanas. / Sticholysins I and II, cytolysins purified from the sea anemone Stichodactyla helianthus, act by lysing biological and model membranes. The proposed mechanism of action consists in the formation of a toroidal pore with the involvement of the N-terminal domain [1]. Different aspects of the interaction between peptides from the toxins\' N-termini (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, and StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) and model membranes - micelles and bilayers - have been studied to contribute to the elucidation of the toxins mechanism of action at the molecular level. The use of peptides was based on the hypothesis that potein fragments can eventually mimic the structure and activity of the whole protein. An analogue containing the paramagnetic amino acid TOAC (N-TOAC-StII11-30) was also studied. Conformational studies were performed making use of the spectroscopic techniques circular dichroism (CD), electron paramagnetic resonance (EPR), and fluorescence. Studies of structure prediction and molecular modeling were also performed. CD spectra showed that the peptides acquired α-helical conformation upon interaction with model lipid membranes, in agreement with the conformation found for these segments in the whole proteins. Making use of membranes of variable lipid composition, it was possible to assess the contribution of electrostatic and hydrophobic interactions for peptide binding. Fluorescence quenching of labeled lipids by paramagnetic TOAC and EPR spectra allowed us to locate the TOAC residue at the membrane-water interface, corroborating the proposed model of the toroidal pore. The CD and EPR studies also allowed us to obtain the binding constants for the peptide-micelle and peptide-bilayer interaction. The peptides were also capable of mimicking the toxins function, as shown by assays of carboxyfluorescein leakage and hemolytic activity. Short peptides containing parts of StII1-30\'s sequence were synthesized with the aim of testing their antimicrobial activity. The peptides displayed low antimicrobial activity, as well as lack of hemolytic activity and toxicity against human cells.

by Paula A. Livingston. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web. / Two protein tyrosine phosphatases, dual specificity phosphatase PVP and low molecular weight phosphatase WZB were purified and characterized. PVP was expressed as inclusion bodies and a suitable purification and refolding method was devised. Enzyme kinetics revealed that p-nitrophenylphosphate and (Sb(B-naphthyl phosphate were substrates with KM of 4.0mM and 8.1mM respectively. PVP showed no reactivity towards phosphoserine. Kinetic characterization of WZB showed that only pnitrophenylphosphate was a substrate with no affinity for Ç-naphthyl phosphate and phosphoserine. Optimal conditions for activity with PNPP were found at a pH of 5 with a KM of 1.1mM, kcat of 35.4s-1 and kcat/KM of 32.2s-1mM-1. Inhibition studies showed that phosphate, fluoride, and molybdate were competitive inhibitors with Ki of 3.2mM, 71.7mM, and 50.4(So(BM respectively and hydrogen peroxide abolished activity. Active site mutants of WZB Cys9Ser and Asp115Asn showed no activity.

Esticolisinas I e II, citolisinas purificadas da anêmona marinha Stichodactyla helianthus, agem lisando membranas biológicas e modelo. O mecanismo de ação proposto consiste na formação de um poro toroidal com o envolvimento do domínio N-terminal. Diferentes aspectos da interação entre peptídeos derivados do N-terminal das toxinas (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, e StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) com membranas modelo - micelas e bicamadas - foram estudados com o objetivo de contribuir para a elucidação do mecanismo de ação das toxinas em nível molecular. O emprego dos peptídeos teve como base a hipótese de que fragmentos proteicos podem ser capazes de mimetizar a estrutura e atividade das proteínas inteiras. O análogo contendo o aminoácido paramagnético TOAC (N-TOAC-StII11-30) também foi estudado. Estudos conformacionais foram realizados empregando-se as técnicas espectroscópicas de dicroísmo circular (CD), ressonância paramagnética eletrônica (EPR) e fluorescência. Foram ainda realizados estudos de predição de estrutura e modelagem molecular. Espectros de CD mostraram que os peptídeos adquirem conformação em α-hélice ao interagir com membranas modelo, de acordo com a conformação observada nessa região para as toxinas. Variando a composição lipídica das membranas modelo estudadas, foi possível investigar a contribuição de forças eletrostáticas de de interações hidrofóbicas para a ligação do peptídeo. Ensaios de supressão de fluorescência de lípidos contendo grupamentos fluorescentes em diferentes posições pelo resíduo paramagnético TOAC e espectros de ressonância paramagnética eletrônica (EPR) permitiram localizar o resíduo TOAC na interface membrana-água, corroborando o modelo proposto do poro toroidal. A análise dos espectros de CD e EPR também permitiu obter as constantes de ligação dos peptídeos com micelas e bicamadas. Os peptídeos também foram capazes de mimetizar as toxinas do ponto de vista funcional, como mostrado por testes de vazamento de carboxifluoresceína e atividade hemolítica. Peptídeos curtos, contendo partes da sequência de StII1-30, sintetizados com o objetivo de examinar uma eventual atividade antimicrobiana, demonstraram baixa atividade, bem como ausência de atividade hemolítica e de toxicidade para células humanas. / Sticholysins I and II, cytolysins purified from the sea anemone Stichodactyla helianthus, act by lysing biological and model membranes. The proposed mechanism of action consists in the formation of a toroidal pore with the involvement of the N-terminal domain [1]. Different aspects of the interaction between peptides from the toxins\' N-termini (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, and StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) and model membranes - micelles and bilayers - have been studied to contribute to the elucidation of the toxins mechanism of action at the molecular level. The use of peptides was based on the hypothesis that potein fragments can eventually mimic the structure and activity of the whole protein. An analogue containing the paramagnetic amino acid TOAC (N-TOAC-StII11-30) was also studied. Conformational studies were performed making use of the spectroscopic techniques circular dichroism (CD), electron paramagnetic resonance (EPR), and fluorescence. Studies of structure prediction and molecular modeling were also performed. CD spectra showed that the peptides acquired α-helical conformation upon interaction with model lipid membranes, in agreement with the conformation found for these segments in the whole proteins. Making use of membranes of variable lipid composition, it was possible to assess the contribution of electrostatic and hydrophobic interactions for peptide binding. Fluorescence quenching of labeled lipids by paramagnetic TOAC and EPR spectra allowed us to locate the TOAC residue at the membrane-water interface, corroborating the proposed model of the toroidal pore. The CD and EPR studies also allowed us to obtain the binding constants for the peptide-micelle and peptide-bilayer interaction. The peptides were also capable of mimicking the toxins function, as shown by assays of carboxyfluorescein leakage and hemolytic activity. Short peptides containing parts of StII1-30\'s sequence were synthesized with the aim of testing their antimicrobial activity. The peptides displayed low antimicrobial activity, as well as lack of hemolytic activity and toxicity against human cells.