Calmodulin (CaM) is an essential protein found in all eukaryotes ranging from vertebrates to unicellular organisms such as Paramecia. CaM is a calcium sensor protein composed of two domains (N and C) responsible for the regulation of numerous calcium-mediated signaling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. Since the discovery of CaM, most studies have focused on the role of its calcium-saturated form. However, an increasing number of target proteins have been discovered that preferentially bind apo (calcium-depleted) CaM. My study focused on understanding how apo CaM recognizes drugs and protein sequences, and how those interactions differ from those of calcium-saturated CaM. I have used spectroscopic methods to explore CaM binding the drug Trifluoperazine (TFP) and the IQ-motif of the type 2 Voltage-Dependent Sodium Channel (Nav1.2IQp). These studies have shown that both TFP and Nav1.2IQp preferentially bind to the "semi-open" conformation of apo CaM. TFP was shown to be an unusual allosteric effector of calcium binding to CaM. Using 15N-HSQC NMR spectroscopy, I determined the stoichiometry of TFP binding to apo Cam to be 2:1 and to (Ca2+)4-CaM to be 4:1 TFP:CaM. That difference in stoichiometry determined whether TFP decreased or increased the affinity of CaM for calcium. Analysis of residue-specific chemical shift differences indicated that TFP binding to apo and (Ca2+)4-CaM perturbed the C-domain more than the N-domain, prompting high-resolution structural studies of the isolated C-domain of CaM. Crystallographic studies of TFP bound to a calcium-saturated C-domain fragment of CaM (CaM76-148) revealed that CaM adopted an "open" tertiary conformation. The unit cell contained two protein and 4 drug molecules. The orientation of TFP revealed that its trifluoromethyl group was found in two alternative positions (one in each protein in the unit cell), and that Met 144 acted as a gatekeeper to select the orientation of TFP. In contrast to TFP binding to the "open" conformation of calcium-saturated CaM76-148, my NMR studies showed that TFP bound the "semi-open" conformation of apo CaM76-148. TFP interacted with CaM residues near the perimeter of the hydrophobic pocket, but did not contact residues that are solvent-accessible only in the "open" form. Allosteric effects due to TFP binding were observed in the calcium-binding loops of apo CaM76-148. These properties suggest that TFP may antagonize interactions between apo CaM and target proteins such as ion channels that preferentially bind apo CaM. Nav1.2, is responsible for the passage of Na+ ion across cellular membranes. Apo binding of CaM to Nav1.2 poises it for action upon calcium release in the cell. My NMR studies of CaM binding to the Nav1.2 IQ-motif sequence (Nav1.2IQp) showed that the C-domain of apo CaM was necessary and sufficient for binding. My high-resolution structure of the isolated C-domain of CaM bound to Nav1.2IQp revealed that the domain adopted a "semi-open" conformation. At the interface between the IQ-motif and CaM, the highly conserved I and two Y residues of Nav1.2IQp interacted with hydrophobic residues of CaM, while the invariant Q residue interacted with residues in the loop between helices F and G of CaM. This is the first CaM-IQ complex to be determined by NMR; the only other available structure of apo CaM bound to an IQ-motif was determined crystallographically. To accomplish its regulatory roles in response to cellular Ca2+ fluxes, CaM has evolved multiple binding interfaces that are allosterically linked to its Ca2+-ligation state. My studies of CaM binding to TFP and NaV1.2 demonstrate the versatility of CaM functioning as a regulatory protein comprised of domains having separable functions.

Salvia miltiorrhiza (SM) preconditioning was reported to be helpful in the early recovery of gastrointestinal motility in the intestinal congestion of rats with hepatic ischemia reperfusion. The aim of this study was to determine whether SM stimulates contraction of isolated terminal ileum of Sprague-Dawley rat ex vitro and the mechanisms which regulates that. The roots of SM were extracted by ethanol. One of the indicative marker of SM, Tanshinone IIA, was identified and quantified with high performance liquid chromatography (HPLC), and the results showed that Tanshinone IIA was 1190 £gg/ml in SM extract. The effects of contractile activity of SM extract at various cumulative dosages on the rat isolated terminal ileum were studied in organ bath. The area under curve above the baseline of contractile graphy of SM extract on isolated terminal ileum was recorded. In order to explore the contractile mechanism of SM extract on isolated terminal ileum, the individual pretreatment or use of atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), Ca2+ free Kreb¡¦s solution with EGTA, or trifluoperazine (a calmodulin blocker) was given and then cumulative dosages (40 £gL, 100 £gL, 180 £gL, 280 £gL) of SM extract were added. In addition, we used Fura-2 pentakis acetoxymethyl ester to detect the change of intracellular calcium concentration of intestinal epithelial cell-6 (IEC-6) induced in 1/1000-time or 1/10000-time dilution of SM extract. The result indicated SM extract significantly simulated the contraction of isolated terminal ileum in a dose-dependent manner. The individual addition or use of atropine, tetrodotoxin, nifedipine, or Ca2+ free Kreb¡¦s solution with EGTA all could not down-regulate significantly the contraction of SM extract on isolated terminal ileum. Trifluoperazine significantly down-regulated the contraction of SM extract on isolated terminal ileum. In addiation, SM extract was able to increase cytosolic calcium concentration of IEC-6 cells. In conclusion, the mechanisms of contraction of SM extract on isolated terminal ileum of rat were involved in calmodulin/Ca2+ associated contraction pathway.

Thesis (M.S.)--Duquesne University, 2009. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 82-90) and index.

Orientador: Prof. Dr. Tiago Rodrigues / Among the main problem faced during the treatment of cancer patients is resistance to chemotherapy. Among the multiple drug resistance mechanisms (MDR), the best characterized involves the activity of P-glycoprotein (Pgp), the product of the MDR1 gene expression. The reversal of MDR due to Pgp inhibition has been observed by several drugs, including phenothiazines, which has currently been the focus of studies suggesting their use in antitumor therapy. Studies with copolymers of ethylene oxide and propylene oxide, termed poloxamers, demonstrated the ability to inhibit Pgp on MDR cells suggesting their use in antitumor formulations. Thus, this work aims to study cell death induced by phenothiazines inserted in nanostructured systems (liposomes, gold nanoparticles and polymeric micelles) in human myeloblastic leukemia cells K562 and Lucena 1, only the last one overexpressing the MDR1 gene. The results showed that the polymeric micellar systems consisting of poloxamers are more suitable for potentiation of the effect of phenothiazines. The characterization of micellar systems assays indicated that the structures are organized in a lamellar shape and the particle size is about 60 nm at 25°C and 25 nm at 37°C. The in vitro release assays showed that formulations were able to decrease the releasing by 80% when compared to phenothiazines in the free form. Formulations containing PL407/L81 (poloxamer 407 and Pluronic®L81) showed higher capacity to enhance the cytotoxic effect of phenothiazines. The formulation containing trifluoperazine (TFP) inserted in PL407/L81 system caused a significant decrease in the EC50 when compared to the free form only after 48 hours of incubation in K562 lines and Lucena 1. The formulation containing thioridazine (TR) significantly alter the EC50 for 24 hours in the cell line K562 and the both strains after 48 hours of incubation. For formulation containing chlorpromazine (CPZ) was significantly decreased upon incubation for 24 to 48 hours in both cell lines. The death induction profile was similar for all phenothiazines suggesting predominantly apoptosis and late apoptosis. The PL407 / L81 system was able to inhibit the activity of Pgp in resistant strain Lucena 1, this effect was also observed for TR and TFP which had the effect potentiated after insertion in the system containing PL407/L81. However, CPZ has not been capable of inhibiting Pgp in the absence of system PL407/L81 indicating that this system is essential for the enhancement of the cytotoxic effect of CPZ in the line Lucena 1. The tests on non-tumor cell line showed that the mononuclear PL407 / L81 system does not have cytotoxicity, and also is able to decrease the cytotoxic effects of phenothiazines. These data indicate that system containing PL407 and L81 has the greatest potential for application in anti-tumor therapy with the additional benefit of reduced cytotoxic effect on normal cells. / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2015. / Um dos principais problemas enfrentados no tratamento de pacientes com câncer é a resistência a quimioterápicos. Entre os mecanismos de resistência a múltiplas drogas (MDR), o melhor caracterizado envolve a atividade da glicoproteína P (Pgp), produto da expressão do gene MDR1. A reversão da MDR por inibição da Pgp foi observada por diversos fármacos, entre eles as fenotiazinas que atualmente tem sido alvo de estudos que sugerem sua utilização na terapia antitumoral. Estudos com co-polímeros constituídos por óxido de etileno e óxido de propileno, denominados poloxamers, demonstraram a capacidade de inibição da Pgp em células MDR sugerindo sua aplicação em formulações contendo antitumorais. Dessa forma, este trabalho tem como objetivo estudar a morte celular induzida por fenotiazinas inseridas em sistemas nanoestruturados (lipossomas, nanopartículas de ouro e micelas poliméricas) em células leucêmicas mieloblásticas humanas K562 e Lucena 1, sendo que apenas a última apresenta superexpressão do gene MDR1. Os resultados indicaram os sistemas micelares poliméricos constituídos de poloxamers são mais adequados para potencialização do efeito das fenotiazinas. Os ensaios de caracterização dos sistemas micelares indicaram que as estruturas se organizam de forma lamelar e possuem tamanho de partícula de aproximadamente 60 nm, a 25°C, e de 25 nm, a 37°C. Nos ensaios de liberação in vitro, as formulações diminuíram 80% da liberação quando comparadas a liberação das fenotiazinas na forma livre. As formulações contendo PL407/L81 (poloxamer 407 e Pluronic®L81) apresentaram maior capacidade de potencialização do efeito citotóxico das fenotiazinas. A formulação contendo trifluoperazina (TFP) inserida no sistema PL407/L81 promoveu diminuição significativa do EC50 em relação à forma livre apenas após 48 horas de incubação nas linhas K562 e Lucena 1. A formulação contendo tioridazina (TR) alterou significativamente o EC50 em 24 horas na linhagem K562 e em ambas a linhagens após 48 horas de incubação. Para a formulação contendo clorpromazina (CPZ) a diminuição foi significativa na incubação por 24 e 48 horas, em ambas as linhagens. O perfil de indução de morte foi semelhante para todas as fenotiazinas sugerindo, predominantemente, apoptose tardia e apoptose. O sistema PL407/L81 foi capaz de inibir a atividade da Pgp na linhagem resistente Lucena 1, este efeito também foi observado para TR e TFP que tiveram o efeito potencializado após a inserção no sistema contendo PL407/L81. Contudo, CPZ não foi capaz de inibir a Pgp na ausência do sistema PL407/L81 indicando que este sistema é essencial para a potencialização do efeito citotóxico da CPZ na linhagem Lucena 1. A avaliação na linhagem mononuclear normal mostrou que o sistema PL407/L81 não apresenta citotoxidade e, ainda, é capaz de diminuir o efeito citotóxico das fenotiazinas. Estes dados indicam que sistema contendo PL407 14,5% e L81 0,5%, possui maior potencial para aplicação na terapia antitumoral com o benefício adicional de diminuição do efeito citotóxico em células normais.

Calmodulin is a highly conserved calcium ion binding protein expressed in all eukaryotic species. The 149 amino acid residues in the primary structure are organized in seven α helices with the highly flexible central α helix connecting the two non-cooperative domains of calmodulin. Each domain contains two EF-hand motifs to which calcium ions bind in a cooperative manner, hence the binding of four calcium ions saturate one calmodulin molecule. In the cardiovascular area calmodulin is involved in the activation of cardiac muscle contraction, and mutations that arise in the genetic sequence of the protein often have severe consequences. One such consequential mutation that can arise brings about the replacement of the highly conserved aspartic acid with glycine at position 130 in the amino acid sequence. In this research, the thermal and chemical stability within the C domain of the D130G variant of human calmodulin was investigated using a new method only requiring circular dichroism spectroscopic measurements. Affinity studies within the C domain of the D130G variant of human calmodulin were performed using fluorescence spectroscopy, and the ligands chosen for this purpose were trifluoperazine and p- HTMI. All analytical experiments were performed with the C domain of wild type human calmodulin as a reference. From the new method, it was concluded that the C domain of the D130G variant of human calmodulin has a slightly decreased stability in terms of Tm and Cm values compared to the C domain of wild type human calmodulin. The affinity analyses indicated that neither trifluoperazine nor p-HTMI discriminates between the C domain of the D130G variant of human calmodulin and the C domain of wild type human calmodulin in terms of dissociation constants. The pivotal outcome from this research is that the new method is applicable for determination of the stability parameters Tm and Cm of heat stable proteins.

Each year countless lives are affected and about half a million people die from malaria, a disease caused by parasites originating from the Plasmodium family. The most virulent species of the parasite is Plasmodium falciparum (P. falciparum).   Calmodulin (CaM) is a small, 148 amino acid long, highly preserved and essential protein in all eukaryotic cells. Previous studies have determined that CaM is important for the reproduction and invasion of P. falciparum in host cells. The primary structure of human CaM (CaMhum) and CaM from P. falciparum (CaMpf) differ in merely 16 positions, making differences in their structures and ligand affinity interesting to study. Especially since possible inhibitors of CaMpf in favor of CaMhum, in extension, could give rise to new malaria treatments.   Some antagonists, functioning as inhibitors of CaM, have already been analysed in previous studies. However, there are also compounds that have not yet been studied in regards to being possible antagonists of CaM. This study regards three known antagonists; trifluoperazine (TFP), calmidazolium (CMZ) and artemisinin (ART) and also three recently created fentanyl derivatives; 3-OH-4-OMe-cyclopropylfentanyl (ligand 1), 4-OH-3OMe-4F-isobutyrylfentanyl (ligand 2) and 3-OH-4-OMe-isobutyrylfentanyl (ligand 3).   Bioinformatic methods, such as modelling and docking, were used to compare the structures of CaMhum and CaMpf as well as observe the interaction of the six ligands to CaM from both species. In addition to the differences in primary structure, distinguished with ClustalW, disparities in tertiary structure were observed. Structure analysis of CaMhum and CaMpf in PyMOL disclosed a more open conformation as well as a larger, more defined, hydrophobic cleft in CaMhum compared to CaMpf. Simulated binding of the six ligands to CaM from both species, using Autodock 4.2, indicated that TFP and ART bind with higher affinity to CaMhum which is expected. Ligand 2 and ligand 3 also bound with higher affinity and facilitated stronger binding to CaMhum, which is reasonable since their docking is based on how TFP binds to CaM. However, ligand 1 as well as CMZ both bound to CaMpf with higher affinity. Despite promising results for ligand 1 and CMZ, no decisive conclusion can be made solely based on bioinformatic studies.    To gain a better understanding on the protein-ligand interactions of the six ligands to CaMhum and CaMpf, further studies using e.g. circular dichroism and fluorescence would be advantageous. Based on the results from this study, future studies on the binding of CMZ and ligand 1 to CaM as well as ligands with similar characteristics would be especially valuable. This is because they, based on the results from this study, possibly are better inhibitors of CaMpf than CaMhum and thereby could function as possible antimalarial drugs.