An introduction, background and recent advances in the areas of microtubule-binding agents and heat shock protein 90 (HSP90) inhibitors as anticancer agents are briefly reviewed. The work in this dissertation is centered on the synthesis of substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines as potential anticancer agents that act via microtubule inhibition or HSP90 inhibition.<br>Microtubule-binding agents are effective against a broad range of tumors and lymphomas and have been common components of combination cancer-chemotherapy in the clinic. Despite the unparalleled success, drawbacks among microtubule-binding agents such as multi-drug resistance, dose-limiting toxicity, poor pharmacokinetic profile and high cost have supported the sustaining momentum in searching for novel agents of this class.<br>The research on microtubule-binding agents in this dissertation was initiated by an unexpected discovery. The lead compound, a 4-<italic>N</italic>-methyl-4'-methoxyaniline-substituted pyrrolo[2,3-<italic>d</italic>]pyrimidine, was found to inhibit the majority cancer cell lines in the NCI-60 panel at sub-micromolar concentration. The COMPARE analysis based on the activity profile indicated microtubule inhibition as the main mechanism of action of this compound, and was later confirmed through multiple assays. Further, the lead compound displaced 70% of [<super>3</super>H]colchicine from tubulin at a concentration of 5 μM, and was identified as a colchicine-site binder. The compound has also shown unabated or even increased activities against several drug-resistant cancer cell lines, especially the cell lines overexpressing P-glycoprotein or βIII-tubulin. In addition, the compound has favorable physicochemical properties such as high water solubility as its hydrochloride salt.<br>Based on the preliminary data and molecular modeling, a hypothesis on the relationship between binding affinity and the lowest-energy conformation of pyrrolo[2,3-<italic>d</italic>]pyrimidines was proposed. To test the hypothesis and search for compounds with improved potency, 38 pyrrolo[2,3-<italic>d</italic>]pyrimidine analogs in six series were designed and synthesized. The biological evaluations of these compounds are currently in progress at the time this dissertation is submitted.<br>HSP90 is one the molecular chaperones that assist the proper folding of the newly synthesized polypeptides and proteins. The majority of its client proteins are signal transducers with unstable conformations, which play critical roles in growth control, cell survival and development. The expressions of these proteins in normal cells were much less than cancer cell, making HSP90 a viable target for cancer chemotherapy. As of 2012, there are 16 HSP90 inhibitors in clinical trial, among which four are based on the purine-scaffold. All the compounds in clinical trials bind to or overlap with the ATP site on the N-terminal of HSP90.<br>The pyrrolo[2,3-<italic>d</italic>]pyrimidine scaffold is structurally close to purines. In the design of receptor tyrosine kinase (RTK) inhibitors, Gangjee et al. have shown that properly functionalized pyrrolo[2,3-<italic>d</italic>]pyrimidines bind to the ATP site and achieve high degrees of selectivity. This was partly attributed to the incorporation of substitution patterns that are impossible on the purine scaffold. Based on these previous findings and the established SAR of the two purine derivatives in clinical trials (<bold>PU-H71</bold> and <bold>BIIB021</bold>), 18 substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines in three series (in connection with this dissertation) were designed and synthesized. The biological evaluations of these compounds are currently in progress. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

<p>The tubulin homolog FtsZ provides the cytoskeletal framework for bacterial cell division. FtsZ is an essential protein for bacterial cell division, and is the only protein necessary for Z-ring assembly and constriction force generation in liposomes in vitro. The work presented here utilizes structural and genomic analysis methods to investigate FtsZ function for cell division with three separate questions: (1) What is the function of the C-terminal linker peptide in FtsZ? (2) Are there interacting proteins other than those of the divisome that facilitate FtsZ function? (3) Do lateral contact sites exist between protofilaments in the Z ring, resulting in an organized Z-ring substructure?</p><p>The FtsZ protein has an ~50 aa linker between the protofilament-forming globular domain and the C-terminal (Ct) membrane-tethering peptide. This Ct linker is widely divergent across bacterial species, and has been thought to be an intrinsically disordered peptide (IDP). We have made chimeras where we have swapped the <italic>Escherichia coli</italic> IDP for Ct linkers from other bacteria, and even for an unrelated IDP from human &alpha-adducin. Most of these substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter -any IDP appears to work (with some exceptions). Length, however, was important: IDPs shorter than 39 or longer than 89 aa's had compromised function. We conclude that the Ct linker of FtsZ functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA and ZipA at the membrane. As a worm-like-chain, the Ct linker will function as a stiff entropic spring linking the constricting protofilaments to the membrane. </p><p>Previous work from our laboratory found that mutant and foreign FtsZ that do not normally function for cell division can function upon acquisition of a second site suppressor mutation, somewhere in the <italic>E. coli</italic> genome. We expect that some mutant or foreign FtsZ are partially functional for division in <italic>E. coli</italic>. As such, these FtsZ require another mutation that further enables their function. These suppressing mutations may reveal proteins interacting with FtsZ and the divisome, that have previously been unknown. In the present study, we have identified, via whole genome re-sequencing, single nucleotide polymorphisms that allow 11 different foreign and mutant FtsZ proteins to function for cell division. While we see a trend toward mutations in genes related to general metabolism functions in the cell, we have also identified mutations in two genes, <italic>ispA</italic> and <italic>nlpI</italic>, that may be interacting more directly with the cell division mechanism.</p><p>Finally, we have devised a screen to identify mutations in FtsZ that may be involved in lateral bonding between protofilaments. There are presently two proposed models of FtsZ substructure: the scattered or the ribbon model. A major difference between these models is that the scattered model proposed no interaction between adjacent protofilaments in the Z ring, while the ribbon model suggests that adjacent protofilaments are bonded laterally to create an organized substructure of aligned protofilaments. Our screen was designed to identify complementary surface-exposed residues that may be involved in lateral bonding. We initially identified two lateral contact candidate residues: R174, and E250 and mutated them to abrogate FtsZ function. We also mutated L272, which is known to make contacts across the protofilament interface, to look for compensating mutations in these contact residues. Using the screen, we identified a number of secondary mutations in FtsZ that can complement these initial loss-of-function mutations. While this screen has not yielded strong candidates for lateral bonding partners, it has emerged as a high-throughput method for screening large libraries of mutant FtsZ proteins in order to identify compensating mutation pairs.</p> / Dissertation

Malaria has long affected the world both socially and economically. Annually, there are 1.5-2.7 million deaths and 300-500 million clinical infections (WHO, 1998). Several antimalarial agents (such as chloroquine, quinine, pyrimethamine, cycloguanil, sulphadoxine and others) have lost their effectiveness against this disease through drug resistance being developed by the malarial parasites (The- Wellcome-Trust, 1999). Although there is no hard-core evidence of drug resistance shown on the new antimalarial compounds (artemisinin and artesunate), induced resistant studies in animal models have demonstrated that the malarial parasites have capabilities to develop resistance to these compounds (Ittarat et al., 2003; Meshnick, 1998; Meshnick, 2002; Walker et al., 2000). Furthermore, a useful vaccine has yet to be developed due to the complicated life cycle of the malarial parasites (The- Wellcome-Trust, 1999). As such, the re-emergence of this deadly infectious disease has caused an urgent awareness to constantly look for novel targets and compounds. In this present study, Plasmodium falciparum (clone 3D7) was cultured in vitro in human red blood cells for extraction of total RNA which was later reverse transcribed into cDNA. The áI-, áII- and â-tubulin genes of the parasite were then successfully amplified and cloned into a bacterial protein expression vector, pGEX- 6P-1. The tubulin genes were then sequenced and analysed by comparison with previously published homologues. It was found that the sequenced gene of áItubulin was different at twelve bases, of which only six of these had resulted in changes in amino acid residues. áII- and â-tubulin genes demonstrated 100% sequence similarity with the published sequences of clone 3D7, but differences were observed between this clone and other strains (strains NF54 & 7G8) of â-tubulin. Nevertheless, the differences were minor in áI- and â-tubulins and there was greater than 99% homology. Subsequently, all three Plasmodium recombinant tubulin proteins were separately expressed and purified. Insoluble aggregates (inclusion bodies) of these recombinant tubulins were also refolded and have been tested positive for their structural characteristics in Western blot analysis. Both soluble and refolded recombinant tubulins of malaria were examined in a drugtubulin interaction study using sulfhydryl reactivity and fluorescence quenching techniques. Known tubulin inhibitors (colchicine, tubulozole-c and vinblastine) and novel synthetic compounds (CCWA-110, 239 and 443) were used as the drug compounds to determine the dynamics and kinetics of the interactions. In addition, mammalian tubulin was also used to determine the potential toxicity effects of these compounds. Similarities were observed with other published reports in the binding of colchicine with the recombinant tubulins, hence confirming proposed binding sites of this compound on the Plasmodium recombinant tubulins. Two synthetic compounds (CCWA-239 and 443) that have previously tested positive against P. falciparum in vitro were found to bind effectively with all three tubulin monomers, while displaying low binding interactions with the mammalian tubulin, thus indicating that these compounds have potential antimalarial activity. Therefore, this study has satisfied and fulfilled all the aims and hypotheses that have previously been stated.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

abstract: Alzheimer's Disease (AD) is the sixth leading cause of death in the United States and the most common form of dementia. Its cause remains unknown, but it is known to involve two hallmark pathologies: Amyloid Beta plaques and neurofibrillary tangles (NFTs). Several proteins have been implicated in the formation of neurofibrillary tangles, including Tau and S100B. S100B is a dimeric protein that is typically found bound to Ca(II) or Zn(II). These experiments relate to the involvement of S100B in Alzheimer's Disease-related processes and the results suggest that future research of S100B is warranted. Zn(II)-S100B was found to increase the rate at which tau assembled into paired helical filaments, as well as affect the rate at which tubulin polymerized into microtubules and the morphology of SH-SY5Y neuroblastoma cells after 72 hours of incubation. Zn(II)-S100B also increased the firing rate of hippocampal neurons after 36 hours of incubation. Together, these results suggest several possibilities: Zn(II)-S100B may be a key part of the formation of paired helical filaments (PHFs) that subsequently form NFTs. Zn(II)-S100B may also be competing with tau to bind tubulin, which could lead to an instability of microtubules and subsequent cell death. This finding aligns with the neurodegeneration that is commonly seen in AD and which could be a result of this microtubule instability. Ultimately, these results suggest that S100B is likely involved in several AD-related processes, and if the goal is to find an efficient and effective therapeutic target for AD, the relationship between S100B, particularly Zn(II)-S100B, and tau needs to be further studied. / Dissertation/Thesis / M.S. Applied Biological Sciences 2013

Diploid organisms including yeast, most animals, and humans, typically carry two copies of each gene. Variation above or below two copies can however sometimes occur. When gene copy number reduction from two to one causes a disadvantage, that gene is considered haploinsufficient (HI). In the first part of my work, I identified associations between Saccharomyces cerevisiae gene properties and genome-scale HI phenotypes from earlier work. I compared HI profiles against 23 gene properties and found that genes with (i) greater numbers of protein interactions, (ii) greater numbers of genetic interactions, (iii) greater gene sequence conservation, and (iv) higher protein expression were significantly more likely to be HI. Additionally, HI showed negative relationships with (v) cell cycle regulation and (vi) promoter sequence conservation. I exploited the aforementioned associations using Linear Discriminant Analysis (LDA) to predict HI in existing data and guide experimental identification of 6 novel HI phenotypes, previously undetected in genome-scale screenings. I also found significant relationships between HI and two gene properties in Schizosaccharomyces pombe, relationships that hold despite the lack of conserved HI between S. cerevisiae and Sz. pombe orthologue gene pairs. These data suggest associations between HI and gene properties may be conserved in other organisms. The relationships and model presented here are a step towards understanding HI and its underlying mechanisms. Increases in copy number can occur through gene duplication. When duplication produces two functional gene copies, both experience relaxed selection and rapid mutation. This sometimes leads to interesting evolutionary events such as gain of novel function (neofunctionalisation). Previous work shows an ancient ancestor of S. cerevisiae underwent whole genome duplication (WGD) followed by massive redundant gene loss. Interestingly some duplicate pairs show retention of both copies, including the pair TUB1 and TUB3. Existing sequence data shows that TUB3 has experienced a very high rate of evolution post-WGD, suggesting neofunctionalisation. To characterise TUB3, I have carried out experiments measuring fitness effects of varying TUB1, TUB2 and TUB3 copy number across many environments. In ethanol media, some TUB1 and TUB3 null mutants interestingly show severe defects. Other data suggest stress response, ethanol tolerance, protein degradation and/or regulatory roles, which may involve the regulatory Snf1p protein kinase complex.

Les kinésines sont des protéines moteur liées au cytosquelette de microtubules. Elles convertissent l’énergie provenant de l’hydrolyse de l’ATP en un travail mécanique. Leur fonction typique est de se déplacer le long du microtubule pour véhiculer des charges. La plupart des kinésines sont des dimères. Elles comprennent un domaine moteur, qui porte à la fois les sites de liaison du nucléotide et du microtubule, un domaine intermédiaire de dimérisation et une partie dite « queue » qui confère la spécificité des charges à transporter. Mon objectif est d’établir le mécanisme moléculaire à la base de la motilité, avec un intérêt particulier pour la détermination des variations structurales du domaine moteur de la kinésine le long de son cycle mécano-chimique. Au cours de ma thèse, mon objet d’étude principal a été la kinésine-1 humaine, encore appelée kinésine conventionnelle.J’ai étudié plus particulièrement deux aspects du cycle mécano-chimique de la kinésine-1, en combinant des approches de biologie structurale et l’étude de mutants. Les deux aspects concernent l’étude de la fixation de la kinésine-ADP au microtubule, conduisant à l’éjection du nucléotide et à une liaison forte de la kinésine au microtubule. Dans un premier temps, j’ai déterminé la structure du domaine moteur de la kinésine-1, dépourvue de nucléotide, et sous forme d’un complexe avec la tubuline. La tubuline est la protéine constitutive des microtubules. Cette structure était la donnée principale qui nous manquait dans le cycle structural de la kinésine. En comparant cette structure avec celle de la kinésine dans un état ATP, on peut rendre compte des changements de conformation de la kinésine selon le mouvement de trois sous-domaines du domaine moteur. Cette analyse explique notamment le lien entre la fixation de l’ATP et l’ouverture d’une poche hydrophobe distante de 28 Å du site du nucléotide. Cette cavité va accommoder le premier résidu du neck linker, conduisant à la stabilisation de ce peptide situé en partie C-terminale du domaine moteur. En s’ordonnant, le neck linker va faire avancer la charge ainsi que l’autre domaine moteur de la kinésine dimérique. Il lie ainsi la fixation de l’ATP au mouvement. L’étude de l’effet de mutations du neck linker montre aussi comment, réciproquement, le neck linker bloque la kinésine dans la conformation active pour l’hydrolyse de l’ATP. Ceci diminue la probabilité que l’ATP soit hydrolysé avant que l’étape mécanique se soit produite; cet aspect est essentiel pour rendre compte de la processivité de la kinésine-1.Ces données structurales suggèrent également comment la fixation de la kinésine-ADP au microtubule accélère l’éjection de l’ADP. Pour étudier cet aspect plus en détail, j’ai étudié l’effet de mutations sur la vitesse de largage de l’ADP. L’idée était de mimer à l’aide de mutations la fixation au microtubule. J’ai identifié ainsi deux séries de mutants qui présentent une vitesse accélérée de largage spontané de l’ADP, ce qui suggère deux voies pour interférer avec la fixation du nucléotide. J’ai ensuite déterminé la structure de deux de ces mutants dépourvus de nucléotide, ainsi que celle de la kinésine de départ également dans une forme apo, obtenue par digestion de l’ADP. En absence de microtubule, la kinésine dépourvue de nucléotide adopte une conformation soit à l’image de celle de la kinésine-ADP, ou proche de celle de la kinésine-apo liée à la tubuline. Dans un contexte naturel, seule la deuxième conformation est compatible avec la fixation au microtubule. L’ensemble de ces résultats suggère que le microtubule accélère l’éjection du nucléotide par un double mécanisme : en interférant avec la liaison du magnésium et en déstabilisant le motif P-loop de liaison du nucléotide. / Kinesins are a family of microtubule-interacting motor proteins that convert the chemical energy from ATP hydrolysis into mechanical work. Many kinesins are motile, walking along microtubules to fulfill different functions. Most kinesins are dimers, the monomer comprising a motor domain, a dimerizing stalk domain, and a tail domain. The motor domain contains both the nucleotide-binding site and the microtubule-binding site. I am interested in the molecular mechanism of kinesin's motility. In particular I want to establish the structural variations of the kinesin motor domain along with the mechanochemical cycle of this motor protein. During my thesis, I have focused my work on the human kinesin-1, also named conventional kinesin, which is the best characterized kinesin.I have studied two aspects of the kinesin mechanochemical cycle, by combining structural and mutational approaches. Both aspects rely on the binding of ADP-kinesin to a microtubule, which leads to the release of the nucleotide and to a tight kinesin-microtubule association. First I determined the crystal structure of nucleotide-free kinesin-1 motor domain in complex with a tubulin heterodimer, which is the building block of microtubule. This structure represented the main missing piece of the structural cycle of kinesin. Three subdomains in the kinesin motor domain can be identified through the comparison of my structure with ATP-analog kinesin-1-tubulin structure. The relative movements of these subdomains explain how ATP binding to apo-kinesin bound to microtubule triggers the opening of a hydrophobic cavity, 28 Å distant from the nucleotide-binding site. This cavity accommodates the first residue of the “neck linker”, a short peptide that is C-terminal to the motor domain, allowing the neck linker to dock on the motor domain. The docking of the neck linker is proposed to trigger the mechanical step, i.e. the displacement of the cargo and the stepping of the dimeric kinesin. By studying mutants of the neck linker, I have shown that, reciprocally, this peptide locks kinesin in the ATP state, which is also the conformation efficient for ATP hydrolysis. Doing so, it prevents the motor domain from switching back to the apo-state. It prevents also an untimely hydrolysis of ATP, before the mechanical step has occurred. These features are required for movement and processivity.Second, these structural data also suggest how the binding of ADP-kinesin to tubulin enhances nucleotide release from kinesin. To further study this step of the kinesin cycle, I studied the effect of kinesin-1 mutations. These mutations were designed in isolated kinesin to mimic the state when kinesin is bound to a microtubule. I identified two groups of mutations leading to a high spontaneous ADP dissociation rate, suggesting that there are two ways to interfere with ADP binding. Then I determined the crystal structures of the apo form of two mutants as well as that of the nucleotide-depleted wild type kinesin. It showed that apo-kinesin adopts either and ADP-like conformation or a tubulin-bound apo-like one. In the natural context, the second one is stabilized upon microtubule binding. Overall, the mutational and structural data suggest that microtubules accelerate ADP dissociation in kinesin by two main paths, by interfering with magnesium binding and by destabilizing the nucleotide-binding P-loop motif.

Tubulin and elastin-like polypeptides (ELPs) both form large protein structures which can be thermodynamically evaluated using isothermal titration calorimetry and differential scanning calorimetry. ELPs are thermos-responsive biopolymers that undergo phase separation and form coacervates when heated. This project assesses the liquid-liquid phase separation of an ELP sequence derived from tropoelastin with a SynB1 cell-penetrating peptide attached to the N-terminus in conjunction with the chemotherapeutic drug doxorubicin. Microtubules (MTs) are a dynamic cellular structure formed of tubulin alpha/beta-heterodimers and are responsible for several important cellular processes, making them a viable target for anti-cancer drugs. There has been extensive research done to identify new ligands that show selective binding to microtubules. Ruthenium (II) polypyridyl complexes (RPCs) have been found to promote the polymerization of tubulin into microtubules. ITC has been used to determine the binding affinity of [Ru(II)(Ph2phen)3]2+ (RPC2).

No description available.