Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...

Eukaryontische Zellen besitzen ein Zytoskelett, ein zelluläres Netzwerk aus Biopolymeren. Unter diesen Biopolymeren sind die Mikrotubuli weitgehend konserviert. Diese aus Tubulin aufgebauten Filamente sind dynamisch und wechseln zwischen Phasen des Wachstums und der Schrumpfung. Die genauen Mechanismen, die die dynamische Instabilität der Mikrotubuli bestimmen, werden noch erforscht. Die Allgegenwart von Mikrotubuli wirft die Frage auf, wie sie in verschiedenen thermischen Umgebungen konservierte Funktionen ausführen können. Um dieser Fragestellung nachzugehen, habe ich verwandte Froscharten mit unterschiedlich temperierten Lebensräumen untersucht: Xenopus laevis (16-22 °C), Xenopus borealis (19-23 °C) und Xenopus tropicalis (22-30 °C). Um zu untersuchen, ob sich die biochemischen Eigenschaften von Tubulin und die Dynamik der Mikrotubuli bei den drei Arten an die Temperatur angepasst hat, habe ich die Methoden der Tubulin-Affinitätsreinigung und die temperaturgesteuerte TIRF-Mikroskopie zur Rekonstitution der Mikrotubuli-Dynamik kombiniert. Dabei habe ich festgestellt, dass bei einer Temperatur von 25°C die Wachstumsgeschwindigkeit der Mikrotubuli im Bezug zur thermischen Nische der einzelnen Arten negativ korreliert. Die Verwendung der Arrhenius-Gleichung zum Vergleich der Aktivierungsenergie der Mikrotubuli-Polymerisation für jede Spezies ergab, dass die freie Energie des Tubulins umso höher ist, je kälter die thermische Nische der Spezies ist. Die Mikrotubuli von X. laevis und X. borealis zeigten eine längere Lebensdauer und wurden häufiger zerstört als die von X. tropicalis. Die Tubuline von X. laevis und X. borealis sind phosphoryliert, im Gegensatz zu X. tropicalis. Die Ergebnisse zeigen, dass sich Xenopus Tubulin und die Dynamik der Mikrotubuli an die Temperatur angepasst haben. Kalt lebende Arten kommen mit der niedrigeren Energie des Milieus zurecht, durch verbessertes Wachstum und Stabilität. / Eukaryotic cells hold a cytoskeleton, a cellular network of biopolymers. Among the filaments of the cytoskeleton, microtubules are widely conserved. Built from tubulin, those filaments are dynamic, alternating between phases of growth and shrinkage. The biochemical properties of tubulin shape the dynamic behavior of microtubules, which is crucial for many cellular processes. The precise mechanisms determining microtubule dynamic instability are still under investigation. The ubiquity of microtubules raises the question of how they can perform conserved functions within various thermal environments. To address this, I turned to closely related frog species living at different temperatures, Xenopus laevis (niche: 16-22°C), Xenopus borealis (19-23°C) and Xenopus tropicalis (22-30°C). To probe whether the biochemical properties of tubulin and microtubule dynamics adapted to temperature across those three species, I combined tubulin affinity purification and temperature-controlled TIRF microscopy of in vitro reconstitution of microtubule dynamics. I found that at 25°C, the microtubule growth velocity inversely correlates with the thermal niche of each species. Adjusting temperature to each species’ endogenous condition modulates the growth rate differences across species. Using the Arrhenius equation to compare the activation energy of microtubule polymerization for each species suggested that the colder the thermal niche of the species, the higher the free energy of its tubulin. Microtubules from the cold-adapted species X. laevis and X. borealis have longer lifetimes and rescue more often than those of X. tropicalis, both at 25°C and at each species’ endogenous condition. X. laevis and X. borealis tubulins are phosphorylated, contrary to X. tropicalis. My results show that Xenopus tubulin and microtubule dynamics have adapted to temperature. Cold-living species cope with the lower energy of the milieu by facilitating growth and stability.

No description available.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction Normal cerebral cortex development depends on extensive neuronal migration during embryogenesis, permitting the formation of accurate synaptic circuits and a highly ordered laminar neocortex. The motility of a migrating neuron is achieved by a dynamic microtubule cytoskeleton that alternates between states of stabilization/lengthening and destabilization/shortening. This dynamic instability of the microtubule cytoskeleton is controlled by numerous microtubule-stabilizing and -destabilising proteins that bind directly to microtubules. Autophagy (“self-eating”), a major bulk intracellular degradation system, involves the fusion of autophagosomes with lysosomes, followed by proteolysis and recycling of cellular constituents. Like neuronal migration, autophagy is a microtubule-dependent process. The dynamic microtubule network serves as a track for autophagosomes to be transported to the lysosomes. WDR47 is a protein that is expressed in the brain during development, but of which the function is largely unknown. Novel interactions have recently been identified between Reelin and WDR47 and between the microtubule-destabilising protein superior cervical ganglion 10 (SCG10) and WDR47. These findings suggest that WDR47 may be regulating microtubule-dependent processes such as neuronal migration and autophagy. We hypothesize that WDR47 may play a role in regulating neuronal migration and/or autophagy, and that this regulation may be mediated by a tubulin stability-regulating role of WDR47. Aims and Methods Our aims are to assess the cellular localization of WDR47 in GT1-7 cells and to determine whether WDR47 is able to influence neuronal migration, filopodia extension, surface adhesion, ultra-structure, autophagy, tubulin stability, and tau or SCG10 protein levels. GT1-7 neuronal cells were cultured under normal conditions and transfected with WDR47 siRNA for 24 hours, followed by western blot verification of the knock-down. A 36 hour neuronal in vitro cell migration assay was performed and images of the wound were captured every 6 hours; the migration distances and the wound areas for the different time points were measured and analysed. A 24 hour migration assay was performed, capturing images every hour, and the direction of migration was determined. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to analyse neuronal surface morphology and ultra-structure. Western blot analysis of SCG10, acetylated α-tubulin, Tau, LC3, and Sequestosome 1/p62 (SQTM1) protein levels was performed. Super-resolution structured Illumination microscopy (SR-SIM) three-dimensional (3-D) imaging of WDR47-YFP transfected cells, confocal microscopy of LC3 and acetylated tubulin, co-localization analysis of WDR47 and acetylated tubulin, and fluorescence recovery after photo-bleaching (FRAP) analysis were performed. Results WDR47 siRNA treatment significantly reduced the average migration distance and the migration velocity, resulted in fewer filopodia-like extensions as well as perturbed surface adhesion of migrating neurons, and lead to an increased presence of endoplasmic reticulum (ER) structures as well as an expanded nuclear envelope. LC3-II protein levels were significantly lower with WDR47 siRNA treatment, but were significantly increased with WDR47 siRNA treatment in conjunction with Bafilomycin A1 treatment, indicating increased autophagic flux. SCG10 protein levels were significantly decreased with WDR47 siRNA treatment. SR-SIM and confocal microscopy of WDR47 siRNA treated cells revealed a robust presence of highly convoluted acetylated tubulin in the perinuclear region as well as decreased LC3 fluorescence signal. Confocal microscopy revealed co-localization of WDR47 with acetylated tubulin. - Discussion and Conclusion: The results suggest that WDR47 is involved in regulating neuronal migration, neuronal surface adhesion and filopodia formation, microtubule dynamics, and likely also autophagic flux. Taken together, we propose that WDR47 is regulating microtubule dynamics by facilitating assembly of microtubule-regulating proteins such as SCG10, thereby affecting microtubule-dependent processes such as neuronal migration and autophagy. / AFRIKAANSE OPSOMMING: Inleiding Normale serebrale korteks ontwikkeling is hoogs afhanklik van neuronale migrasie tydens embriogenese, en is belanrik vir die vorming van akkurate sinaptiese netwerke en 'n hoogs geordende laminêre neokorteks. Die vermoё van 'n neuron om te migreer berus op 'n hoogs dinamiese mikrotubulien sitoskelet wat verleng/stabiliseer of verkort/destabiliseer soos tubulien-eenhede begevoeg of verwyder word. Hierdie dinamiese onstabiliteit van die mikrotubulien sitoskelet word beheer deur verskeie mikrotubulien-stabiliserende en - destabiliserende proteïene wat direk bind aan mikrotubuliene. Autofagie ("self-eet"), 'n grootmaat intrasellulêre degradasie stelsel, behels die fussie van autofagosome met lisosome, gevolg deur proteolitiese afbraak van sellulêre organelle en proteine. Soos neuronale migrasie is autofagie 'n mikrotubulien-afhanklike proses. Die dinamiese mikrotubulien netwerk dien as 'n spoor vir die vervoer van autofagosome na lisosome. WDR47 is 'n proteïen wat voorkom in die brein tydens ontwikkeling, maar waarvan die funksie grootliks onbekend is. Interaksies was onlangs geïdentifiseer tussen beide Reelin en WDR47 en die mikrotubulien-destabiliserende proteïen SCG10 en WDR47. Hierdie bevindinge dui daarop aan dat WDR47 n rol speel in die regulering van tubulienstabiliteit en sodoende mikrotubulien-afhanklike prosesse. Ons veronderstel dat WDR47 'n rol kan speel in die regulering van neuronale migrasie en/of autofagie en dat hierdie regulasie moontlik afhanklik is van 'n tubulien-stabiliteit-regulerende rol van WDR47. - Doelwitte en Metodes: Ons doelwitte is om die sellulêre lokalisering van WDR47 in GT1-7 neurone te evallueer en om te bepaal of WDR47 n effek het op neuronale migrasie, oppervlak adhesie en filopodia formasie, ultra-struktuur, autofagie, tubulien-netwerke en -stabiliteit, en Tau of SCG10 proteïenvlakke. GT1-7 neuronale selle is gekweek onder normale omstandighede en vir 24 uur getransfekteer met WDR47 siRNA, gevolg deur verifikasie met Western-blot analise. 'n 36 uur neuronale in vitro sel migrasie toets is uitgevoer en fotos van die wond is elke 6 uur geneem. Die migrasie afstande en die wondareas vir die verskillende tydpunte is gemeet en ontleed. 'N 24-uur-migrasie toets is uitgevoer, 'n foto van die wond is elke uur geneem, en die rigting van migrasie is bepaal. Skandering elektronmikroskopie (SEM) en transmissieelektronmikroskopie (TEM) is uitgevoer om neuronale oppervlakmorfologie en ultrastruktuur te observeer. Western blot analise van SCG10, geasetieleerde α-tubulien, Tau, LC3 en Sequestosome 1/p62 (SQTM1) proteïenvlakke is uitgevoer. Super-resolusie gestruktureerde verligting mikroskopie (SR-SIM) driedimensionele (3-D) beelding van WDR47-YFP getransfekteerde selle, konfokale mikroskopie vir visualisering van LC3 en tubulien, co-lokalisering analise van beide WDR47 en LC3 en WDR47 en tubulien, asook fluorescentie hersteling na foto-bleek (FRAP) analise is uitgevoer. Resultate Die gemiddelde migrasie-afstand en die migrasiesnelheid (μm/min) het beduidend afgeneem met WDR47 siRNA behandeling. SEM analise van WD47 siRNA-behandelde neurone het minder filopodia en veranderde oppervlak adhesie vertoon, en TEM analise het 'n verhoogde teenwoordigheid van endoplasmiese retikulum (ER) strukture, en 'n uitgebreide kernmembraan vertoon. LC3-II proteïenvlakke was beduidend laer met slegs WDR47 siRNA behandeling, maar beduidend hoёr met WDR47 siRNA behandeling in samewerking met Bafilomycin A1 behandeling. Hierdie resultate dui aan op toeneemende autofagie met WDR47 siRNA behandeling. Verder, beduidend laer vlakke van SCG10 proteïenvlakke is waargeneem met WDR47 siRNA behandeling. SR-SIM en konfokale mikroskopie van WDR47 siRNA behandelde selle het 'n robuuste teenwoordigheid van hoogs buigende geasetieleerdetubulien in die area rondom die nukleus, 'n afgeneemde LC3 Bespreking en Gevolgtrekking Die resultate dui daarop aan dat WDR47 betrokke is by die regulering van neuronale migrasie, filopodia vormasie, oppervlak adhesie, mikrotubuliendinamika, en waarskynlik ook autofagie. Ons stel voor dat WDR47 mikrotubuliendinamika afekteer deur die regulering van proteïene soos SCG10, en sodoende mikrotubulienafhanklike prosesse soos neuronale migrasie en autofagie fasiliteer.

<p>Primary cilia are evolutionarily conserved, acetylated microtubule-based organelles that transduce mechanical and chemical signals. Primary cilium assembly is tightly controlled and its deregulation causes a spectrum of human diseases. Formation of primary cilium is a collaborative effort of multiple cellular machineries, including microtubule, actin network and membrane trafficking. How cells coordinate these components to construct the primary cilia remains unclear. In this dissertation research, we utilized a combination of cell biology, biochemistry and light microscopy technologies to tackle the enigma of primary cilia formation, with particular focus on isoform-specific roles of non-muscle myosin II family members. We found that myosin IIB (Myh10) is required for cilium formation. In contrast, myosin IIA (Myh9) suppresses cilium formation. In Myh10 deficient cells, Myh9 inactivation significantly restores cilia formation. Myh10 antagonizes Myh9 and increases actin dynamics, permitting pericentrosomal preciliary complex formation required for cilium assembly. Importantly, Myh10 is upregulated upon serum starvation-induced ciliogenesis and this induction requires Atat1/Mec-17, the microtubule acetyltransferase. Our findings suggest that Atat1/Mec17-mediated microtubule acetylation is coupled to Myh10 induction, whose accumulation overcomes the Myh9-dependent actin cytoskeleton, thereby activating cilium formation. Thus, Atat1/Mec17 and myosin II coordinate microtubules and the actin cytoskeleton to control primary cilium biogenesis.</p> / Dissertation

This dissertation describes an introduction, background and research progress in the areas of multitargeted single agents and tubulin inhibitors in cancer chemotherapy and selective Toxoplasma gondii TS inhibitors for the treatment of toxoplasmosis.&lt;br&gt; Tubulin inhibitors are important antitumor agents that disrupt microtubule dynamics. Thymidylate synthase (TS) inhibitors prevent cell division by interfering with de novo thymidylate synthesis. Antiangiogenic agents target tumor angiogenesis crucial for tumor growth and metastasis. Under normal circumstances, angiogenesis is typically limited to tumor cells and is mediated by receptor tyrosine kinases (RTKs). Combination chemotherapies of RTK inhibitors with cytotoxic agents that target either TS or tubulin have shown significant promise and several preclinical and clinical studies with such combinations are in progress. Multitargeted single agents with dual antiangiogenic and cytotoxic mechanisms could avoid the major limitations associated with cancer chemotherapy: multidrug resistance and dose limiting toxicities. This dissertation focuses on the design and synthesis of pyrimido[4,5-b]indoles and furo[2,3-d]pyrimidines as potential single agents with dual antiangiogenic and cytotoxic activities. These efforts led to the identification of structural features that are necessary for inhibition of RTKs and/or tubulin polymerization. Novel synthetic strategies were developed for efficient synthesis of 2,4-diamino-5-thioaryl-pyrimido[4,5-b]indoles and 4-anilino-5-methyl-furo[2,3-d]pyrimidines.&lt;br&gt; Taxanes and vinca alkaloids are widely used tubulin inhibitors in cancer chemotherapy. However, their clinical use is compromised by two major mechanisms of drug resistance: the overexpression of Pgp and bIII-tubulin. This dissertation describes the design and synthesis of pyrimido[4,5-b]indoles as tubulin inhibitors that circumvent Pgp and bIII-tubulin mediated resistance. This work identified the structural features crucial for tubulin inhibition for the pyrimido[4,5-b]indole scaffold.&lt;br&gt; Infection by Toxoplasma gondii can lead to toxoplasmosis in immune compromised patients such as organ transplant, cancer and AIDS patients. Current therapy involving combination of sulfadiazine and pyrimethamine is limited by drug resistance and treatment failures. The thymidylate synthase‒dihydrofolate reductase enzyme is important for thymidylate synthesis in T. gondii, and hence can be targeted to treat T. gondii infection. TS is highly conserved across species and selectivity for tgTS over human TS is significantly more challenging. The present work provides an efficient synthesis of 2-diamino-4-oxo-5-thioaryl-pyrimido[4,5-b]indoles as selective tgTS inhibitors. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

<p>FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?</p><p> One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.</p><p> The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.</p> / Dissertation

Oxymonads are a group of heterotrophic flagellates living in low oxygen environment. These protists inhabit mainly the gut of xylophagous insects (cockroaches, termites), with an exception of the genus Monocercomonoides, which was described from the intestinal contents of many vertebrates. On the basis of molecular data, Oxymonadida are classified into the supergroup Excavata (Cavalier-Smith, 2002; Simpson et al., 2006, Hampl et al. 2009). This thesis was focused on the diversity of genus Monocecomonoides from the morphologically simplest family Polymastigidae. The main goal of our work was to gather sequence data from strains isolated from a wide spectrum of hosts. We have obtained 26 partial sequences of the gene for the SSU rDNA in total, of which two belonged to another oxymonad, apparently genus Oxymonas. Our phylogenetic analysis indicated that the representatives of the genus Monocercomonoides form one group, however with a low bootstrap support. On the basis of published data about the presence of non-canonical genetic code in some oxymonads (Keeling and Leander, 2003; de Koning et al., 2008), we decided to explore this rare phenomenon in representatives of the genus Monocercomonoides. For this part of the study we gathered 9 partial sequences of α-tubulin gene. In these sequences we have not...

The genus Geosmithia contains 11 described and several tens of undescribed species of fungi living nearly exclusively in galleries of subcorticolous insects, especially bark beetles. In this work, a phylogenetic analysis of the genus was made using DNA sequences of four protein-coding genes, namely TEF-1, RPB2, Mcm7 and Tsr1. The analysis has confirmed that ecological strategies of these fungi (such as association with conifers or broad leaved trees or symbiosis with ambrosia beetles) have evolved several times in this genus. 51 species are recognized based on the obtained phylogenetic tree according to Genealogical Concordance Phylogenetic Species Recognition. I have also tested utility of the above mentioned genes to serve as "barcode" for identification of closely related Geosmithia species.

O câncer é a segunda maior causa de mortes no mundo, sendo os tumores de mama os mais prevalentes e letais entre as mulheres. Apesar de vasta quimioterapia disponível, os tratamentos apresentam problemas como alta toxicidade e resistência. Dentre os tumores de mama, o subtipo triplo negativo (TNBC) apresenta o pior prognóstico e a maior limitação de tratamentos. O presente trabalho de doutorado visa o desenvolvimento de novos candidatos para tratamento de tumores de mama triplo-negativos. Fármacos que têm como alvo a proteína tubulina estão entre as terapias anticâncer mais bem-sucedidas e representam a primeira linha de tratamento para tumores do tipo TNBC. Neste contexto, foram desenvolvidas acridinonas que inibem a polimerização da tubulina e são capazes de impedir os principais mecanismos de resistência aos fármacos desta classe. O composto líder não interage com a bomba de efluxo glicoproteína-P, além de ser igualmente potente contra a linhagem celular superexpressando a tubulina &beta;III, uma isoforma clinicamente relevante. O mecanismo de ação revelou a interação desses compostos com o sítio da colchicina da proteína alvo. Os compostos apresentaram valores de IC50 entre 10 e 12.000 nM contra a linhagem tumoral TNBC MDA-MB-231. Em contrapartida, não foi observada citotoxicidade na linhagem normal de fibroblastos humanos (HFF1). Ensaios de imunofluorescência reforçaram a ação seletiva dos compostos, mostrando que os mesmos perturbaram a rede de microtúbulos nas células MDA-MB-231, mas não nas células HFF1. As substâncias também inibiram a migração celular e a angiogênese in vitro. Os enantiômeros do composto líder foram separados, levando a identificação de um eutômero 10 vezes mais potente contra as células tumorais e 2 vezes mais potente contra a tubulina quando comparado com a mistura racêmica. Um candidato a fármaco, eficaz e seguro, deve apresentar um balanço favorável entre a sua potência e seus parâmetros farmacocinéticos. Dessa forma, o metabolismo e a farmacocinética das acridinonas bioativas foram investigados. Em geral, os compostos foram metabolicamente estáveis, mas requerem otimização da solubilidade e permeabilidade para o desenvolvimento de fármacos administrados por via oral. A avaliação do composto líder revelou propriedades promissoras que justificam a sua consideração em modelos pré-clínicos de prova de conceito. O objetivo é a geração de candidatos a novos fármacos moduladores da tubulina com ação anticâncer. / Cancer is the second most common cause of death globally, being the breast tumors the leading cause of death in women. The number of anticancer medicines grows yearly but still exhibits problems such as high toxicity and resistance. The breast cancer has a number of subtypes and the one presenting the poorest prognosis and biggest therapeutic limitation is called triple negative breast cancer (TNBC). This PhD work aims to develop new small molecules as candidates to the treatment of the triple negative breast cancer. Drugs that target the protein tubulin are among the most successful anticancer therapies and represent the first line treatment to TNBC tumors. In this context, we developed a series of acridinones as tubulin inhibitors that can circumvent common resistance mechanisms to tubulin modulators. The lead compound did not interact with the P-glycoprotein and presented same effectiveness against cell lines overexpressing a clinically relevant tubulin isotype (&beta;III). In this work, we determined the mechanism of action of these compounds, that bind to the colchicine site in the tubulin. The compounds presented IC50 cytotoxicity values between 10 &ndash; 12000 nM against the TNBC cell line MDA-MB-231, with no cytotoxicity against a normal fibroblast cell line (HFF1). Immunofluorescence studies reinforced the compounds selectivity showing they disrupted the microtubules network on MDA-MB-231 cells but not on the HFF1 ones. The substances also inhibited cell migration and angiogenesis in vitro. The enantiomers of the lead compound were purified, and we identified an eutomer 10-fold more potent against the tumor cells and 2-fold more potent against the tubulin when compared to the racemic mixture. The metabolism and pharmacokinetics of the compounds were also studied aiming the development of agents with a favourable balance between potency and pharmacokinetic parameters, essential feature of an effective and safe drug. In general, the compounds were metabolically stable but need an improvement in the solubility and permeability properties to be developed as oral drugs. In summary, the lead compound presents promising properties to be assessed in preclinical proof of concept studies for further development as next-generation antitubulin drugs.