Rozprawy doktorskie na temat „Human carbonic anhydrase”

Pharmaceutical Sciences
Ph.D.
Carbonic anhydrases (CAs, EC 4.2.1.1) are a class of metalloenzymes that catalyze the hydration of CO2 under physiologic conditions and are involved in many physiological and pathological processes. Modulation of CA activity, particularly CA inhibition is exploited pharmacologically for the treatment of many diseases such as cancer, glaucoma, edemas, mountain sickness. CA activation has been less frequently investigated till recently. Genetic deficiencies of several CA isozymes are reported in the literature and reflect the important role of carbonic anhydrases in human physiology and homeostasis. Activation of CA isozymes in brain have been correlated recently with spatial learning and memory. Based on these premises, activators of CA isozymes have the potential to alleviate mild dementias and to act as potential nootropic agents. In chapter 3, continuing our long-term interests towards the development of potent and selective CAAs, we carried out X-ray crystallographic studies with a small series of pyridinium histamine derivatives, previously developed as CAAs by our group. This study revealed important insights into the binding of this class of activators into the active site of CA II isozyme. A potent pyridinium histamine CAA 25i was successfully crystallized with CA II isozyme and was found to bind into the hydrophobic region of the active site, with two binding conformations being observed. This is one of the very few X-ray crystal structures of a CAA available. Based on the findings of this X-ray crystallographic study and building on our previously developed ethylene bis-imidazole CAAs, we advanced a novel series of lipophilic bis-imidazoles. Enzymatic assays carried out on purified human CA isozymes revealed several low nanomolar potent activators against various brain-relevant CA isozymes. Bis-imidazole 30e was found to be a nanomolar potent activator for CA IV, CA VA and CA IX. Due to their conjugated structure, these CAAs were also fluorescent and therefore were fully characterized in terms of photophysical properties, with several representatives proving to display very good fluorophores. The very good activation profile against several different CA isozymes, along with excellent fluorescence properties recommend these compounds as great molecular tools for elucidation of role of CA isozymes in brain physiology, as well as towards improvement of memory and learning. Focusing on inhibition of CA isozymes, it must be stressed that over the last decade a clear connection had been established between the expression of CA IX and CA XII and cancer. Since cancer is the second most common cause of death in the world, we explored the possibility to kill cancer cells via inhibition of different CA isozymes present in cancer cells. The membrane bound carbonic anhydrase IX (CA IX) isozyme represents a particularly interesting anticancer target as it is significantly overexpressed in many solid tumors as compared to normal tissues. In malign tissues this CA isozyme was found to play important role in pH homeostasis and promotes tumor cell survival, progression and metastasis. Thus, CA IX represents a potential biomarker and an appealing therapeutic target for the detection and treatment of cancer. CA IX can be targeted either through the development of small or large molecular weight, potent, and selective inhibitors or through the development of CA IX targeted drug delivery systems for selective delivery of potent chemotherapeutic agents. Building on these premises, in this dissertation, we also revealed our continuing efforts towards the development of potent and selective CA IX inhibitors along with their translation into the development of CA IX targeted drug delivery systems. In chapter 4, we designed a series of small molecular weight (MW) ureido 1,3,4-thiadiazole sulfonamide derivatives employing the “tail approach”, through the decoration of established sulfonamide CA inhibitor warheads with different tail moieties via ureido linker. The generated CAIs were tested against tumor associated CA IX and CA XII isozymes and off-target cytosolic isozymes CA I and CA II, and were revealed to be moderate to highly selective and nanomolar, even sub-nanomolar, potent CA IX inhibitors. Several potent pan-inhibitors were also identified in this section. We assessed these CAIs for their in vitro cell killing ability using MDA-MB 231 breast cancer cell line expressing CA IX and CA XII. The most efficient CAI proved to be ureido-1,3,4-thiadiazole-2-sulfonamide 69, which showed subnanomolar potency against purified human CA IX and CA XII isozymes, with good selectivity against CA I and CA II, and consistent, statistically significant cancer cell killing. In Chapter 5, continuing our efforts towards the development of potent and selective CA IX inhibitors, we designed, synthesized, characterized and evaluated a new series of PEGylated 1,3,4-thiadiazole-2-sulfonamide CAIs, bearing different PEG backbone length. We increased the PEG size from 1K to 20K, in order to better understand the impact of the PEG linker length on the in vitro cell killing ability against CA IX expressing cancer cell lines and also against a CA IX negative cell line. In vitro cell viability assays revealed the optimum PEG linker length for this type of bifunctional bis-sulfonamide CAIs in killing the tumor cells. The most efficient PEGylated CAI was found to bis-sulfonamide DTP1K 91, which showed consistent and significant cancer cell killing at concentrations of 10−100 μM across different CA IX and CA XII expressing cancer cell lines. DTP1K 91 did not affect the cell viability of CA IX negative NCI-H23 tumor cells, thus revealing a CA IX mediated cell killing for these inhibitors. In chapter 6, we decided to further explore the possibility of using CA IX as a targeting epitome for the development of a gold nanoparticle-based drug delivery system. We translated the oligoEG- and PEGylated CAI conjugates into efficient targeting ligands for gold nanoparticle decoration along with chemotherapeutic agent doxorubicin (Dox), in a novel multi-ligand gold nanoplatform designed to selectively release the drug intracellularly, in order to enhance the selective tumor drug uptake and tumor killing. We were successful in developing compatible CAI- and Dox- ligands for efficient dual functionalization of gold nanoparticles. Our optimized, CA IX targeted gold nanoplatform was found to be very efficient towards killing HT-29 tumor cells especially under hypoxic conditions, reducing the hypoxia-induced chemoresistance, thus confirmed the potentiating role of CA IX as a targeting epitome.
Temple University--Theses

The genetic ability to feel the bitter taste of thioureas, such as PROP, varies greatly among individuals influencing the choice of food and body composition. Sensitive and non-sensitive individuals were defined respectively as “ Taster ” and “ No Tasters ”. The term “ Super Tasters ” is used to distinguish individuals who perceive PROP as most bitter to those defined as “ Medium Tasters ” who perceive the bitter taste moderately. The sensitivity to PROP is associated with the haplotypes ( PAV and AVI ) receptor gene TAS2R38, and may be associated with polymorphisms of the gene gustina ( CA6 ). The gustina is a zinc dependent enzyme present in human saliva implicated in the development of taste buds. The aim of this work was to analyze the association between sensitivity to PROP , the polymorphism rs2274333 (A / G) gene gustina, zinc and salivary polymorphisms of TAS2R38 and BMI . In 75 volunteers aged between 21 and 28 years were determined by BMI and Zn ² + salivate. The sensitivity to PROP was determined by evaluation of the intensity of the sensation evoked by suprathreshold solutions and determining the threshold of perception. Molecular analysis of the gene and gustina receptor gene TAS2R38 were performed by means of PCR, PCR -RFLP and sequencing of fragments obtained . The average values of the concentration of zinc salivary and BMI were higher in individuals defined as “ No Tasters ” than those determined in the “Super Tasters ”. The low taste sensitivity to PROP of “ No Tasters ” was strongly associated with the G allele of the gene polymorphism of gustina and the variant of the TAS2R38 AVI, while the high sensitivity of the “Super Tasters ” is strongly associated allele A gene gustina all'aplotipo PAV and the TAS2R38. Moreover, while the A allele of the gene of gustina is found to be more important for the perception of low concentrations of PROP, the variant of the TAS2R38 PAV is most important result for the evaluation of the intensity of the sensation evoked by high concentrations of PROP. These data show that the sensitivity to PROP is inversely related to BMI and Zinc salivary and directly associated with the gene dimorphism gustina is assumed that might influence the function of the protein. In addition, these new findings explain 6 how the combination of gene gustina and TAS2R38 genotype may modulate the phenotype of sensitivity to PROP providing an additional tool for the evaluation of human eating behavior and nutritional status.

Amyotrophic Lateral Sclerosis (ALS) is one of the most common motor neuron diseases with a crude annual incidence rate of ~2 cases per 100,000 in European countries, Japan, United States and Canada. The role of Carbonic Anhydrase 1 (CA1) in ALS pathogenesis is completely unknown. Previous unpublished results from Dr. Jian Liu have shown in the spinal cords of patients with sporadic amyotrophic lateral sclerosis (SALS) there is a significant increased expression of CA1 proteins. The purpose of this study is to examine the effect of CA1 expression in mammalian cells, specifically, whether CA1 expression will affect cellular viability and induce apoptosis. To further understand whether such effect is dependent upon CA1 enzymatic activity, three CA1 mutants (Thr199Val, Glu106Ile and Glu106Gln) were generated using two-step PCR mutagenesis. Also, a fluorescence-based assay using the pH-sensitive fluorophore Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) to measure the anhydrase activity was developed. The assay has been able to circumvent the requirement of the specialized equipment by utilizing a sensitive and fast microplate reader and demonstrated that three mutants are enzymatically inactive under the physiologically relevant HCO3- dehydration reaction which has not been tested before by others. The data show that transient expression of CA1 in Human Embryonic Kidney 293 (HEK293), African Green Monkey Kidney Fibroblast (COS7) and Human Breast Adenocarcinoma (MCF7) cell lines did not induce significant changes to the cell viability at 36hrs using the Water Soluble Tetrazolium-8 (WST8) assay. Wild-type CA1 significantly reduced cell viability in HEK293 using a virally transduced inducible stable expression system at 96hrs and 144hrs of protein induction whereas out of the two mutants used only Thr199Val induced significant toxicity at 144hrs. Wild-type CA1 has also been found to protect COS7 cells against doxycycline-induced toxicity at 96hrs and 144hrs of protein induction whereas no protective effect was seen by the mutants. Using flow cytometry analysis the results has shown wild-type CA1 expression significantly increased Caspase-3 activation and its downstream molecule Poly (ADP-Ribose) Polymerase 1 (PARP-1) cleavage at 96hrs whereas Glu106Ile only significantly increased Caspase-3 activation. In conclusion, this study marks the first time where CA1 expression has shown to directly cause significant apoptotic toxicity in HEK293 cells and protect against doxycycline-induced toxicity in COS7 cells. Although the implication of this study in ALS requires further investigation, the results here suggest in healthy cells increased levels of CA1 expression may cause onset of toxicity, whereas when cells undergo stress, increased CA1 expression can be protective to prevent further loss in cell viabilities. Despite numerous previous studies that have examined CA1 as potential diseases marker, these results represent for the first time in understanding the effect of CA1 in mammalian cells.

The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO₂-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

Abstract The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues of the human body by catalyzing the reversible reaction CO2 + H2O ⇔ HCO3- + H+. Carbonic anhydrase isoenzyme VI (CA VI) is secreted into the human saliva by the serous acinar cells of the parotid and submandibular glands. The present work was undertaken in order to gain an understanding of the physiological role of CA VI in the oral cavity. CA VI concentrations were compared with other salivary characteristics and with the clinical dental status of the subjects. Saliva samples were collected under strictly controlled conditions from 209 young, healthy men and their CA VI concentrations determined by means of a specific time-resolved immunofluorometric assay. Salivary secretion rate, pH, buffering capacity, α-amylase activity level and counts of lactobacilli and mutans streptococci were also determined. Salivary CA VI concentrations showed positive correlations with salivary secretion rate (r = 0.20, p = 0.003) and amylase activity level (r = 0.46, p < 0.001), but not with pH, buffering capacity, or counts of mutans streptococci or lactobacilli. Salivary CA VI concentration, pH and buffering capacity correlated negatively with the number of decayed, missing or filled teeth (DMFT index). The correlation between salivary CA VI concentration and DMFT index was closest in the subjects with poor oral hygiene. No correlation was found between salivary secretion rate or amylase activity and the DMFT index. The location of CA VI in the enamel pellicle, a thin layer of proteins on dental surfaces providing a protective interface between the tooth surface and the external environment, was demonstrated in samples of extracted teeth using immunostaining with anti-CA VI antibody. Immunostaining for salivary α-amylase, which was used as a positive control, produced virtually the same staining patterns. The presence of CA VI in the natural enamel pellicle was confirmed by Western blotting of pellicle proteins. Histochemical staining of enamel pellicle formed in vitro showed that the bound enzyme retains its CA activity. To determine whether CA VI is transferred into the circulation, blood and saliva samples were collected from four healthy male volunteers at 3-h intervals throughout a 24-h period and assayed for CA VI concentration. CA VI was present in all the serum samples, although its concentration was about 22 times lower than in the saliva. The presence of CA VI in serum was confirmed using a sensitive Western blotting method. Western blotting also showed that serum CA VI is associated with IgG, which may protect the enzyme from proteolytic degradation or target it to sites that do not contain CA VI. The present results suggest that salivary CA VI is not involved in regulation of the actual pH or buffering capacity of the saliva, but it does seem to have a specific role in the oral cavity. High salivary concentrations of CA VI appear to be associated with low caries experience. Since active CA VI is located in the enamel pellicle, it may function locally in the microenvironment of the dental surfaces and accelerate the neutralization of the acid metabolic products of bacterial plaque.

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.

Funding Agencies|UK National Service for Computational Chemistry Software||UK National Supercomputer Service Hector||Marie Curie Fellowships (FP7 of EU)||

Carbonic anhydrases (CAs) are metalloenzymes that catalyze the conversion between carbon dioxide and bicarbonate. Their inhibition can be applied for treatment of different diseases, such as glaucoma, cancer, obesity, epilepsy, osteoporosis, etc. There are nearly 30 small molecule ligands that are used as drugs for carbonic anhydrase related diseases. In this work interaction between recombinant human carbonic anhydrases I, II, VII, IX, XIII and sulfonamide ligands was analysed. Stability of selected carbonic anhydrases was evaluated in different experimental conditions. Oligomeric structure of anticancer target CA IX was determined. Using carbonic anhydrases as model proteins, the application range of thermal shift assay was extended. Binding parameters of 40 new compounds to human carbonic anhydrases were measured. The binding thermodynamics of sulfonamide ligands to CA XIII was analyzed and intrinsic binding parameters, independent of the experimental conditions and linked protonation reactions, were determined.
Karboanhidrazės (CA) yra metalofermentai, katalizuojantys virsmus tarp anglies dioksido ir bikarbonato. Jų slopinimas gali būti taikomas gydyti tokias skirtingas ligas kaip glaukoma, vėžys, nutukimas, epilepsija, osteoporozė ir kt. Šiuo metu yra beveik 30 mažamolekulinių junginių, kurie naudojami kaip vaistai, su padidėjusiu karboanhidrazių aktyvumu susijusioms ligoms gydyti. Darbe tirta rekombinantinių žmogaus karboanhidrazių I, II, VII, IX ir XIII sąveika su sulfonamidiniais ligandais. Įvertintas tirtų baltymų stabilumas skirtingomis eksperimentinėmis sąlygomis, nustatyta priešvėžinio taikinio CA IX oligomerinė būsena. Modeliniais baltymais naudojant karboanhidrazes, praplėstos terminio poslinkio metodo taikymo ribos. Išmatuoti 40 naujų susintetintų junginių sąveikos su karboanhidrazėmis termodinaminiai parametrai, išanalizuota CA XIII sąveikos su sulfonamidiniais slopikliais termodinamika, atskiriant tikruosius, nuo eksperimento sąlygų ir susijusių reakcijų nepriklausančius jungimosi parametrus.

In this thesis, a polypeptide binder concept is illustrated. By conjugation to a set of sixteen polypeptides, a small binding molecule can evolve into a polypeptide binder with increased affinity and selectivity. The concept was applied to 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) and acetazolamide (AZM) for development of high affinity binders targeting human C-reactive protein (CRP) and human carbonic anhydrase (HCA) II and IX respectively. In addition, peptididic macrocycles were developed as inhibitors of lysine specific demethylase 1 (LSD1). CRP is a well-known biomarker of inflammation in humans and binders recognizing it are therefore of large interest as medical diagnostics. Until now, phosphocholine (PCh) and derivatives are the only known small molecule binders for CRP, but they have low μM affinity and bind CRP in a Ca2+ dependent manner. The small molecule DQ was designed as a CRP binder that is structurally unrelated to PCh. Its polypeptide conjugate, 4-C25l22-DQ, was demonstrated as a strong, Ca2+ independent binder for CRP, and had an affinity approximately three orders of magnitude higher than DQ itself. HCA IX is a protein that is interesting for diagnosis of cancer. AZM is a small molecule inhibitor of HCAs with a dissociation constant of 38 nM for HCA II and 3 nM for HCA IX. Interestingly, polypeptide conjugate 4-C10L17-AZM displayed stronger binding to both HCA II (KD 4 nM) and HCA IX (KD 90 pM). This result provided evidence that the binder concept can be applied also for small molecules which already have high affinity for their protein receptors. LSD1 is an enzyme that regulates the methylation of Lys 4 of histone 3 via a PPI-like interaction and which is of therapeutic interest in certain cancers. Based on the structures of two peptidic ligands bound to LSD1, we sequentially prepared truncated, mono-substituted and macroyclic peptides in order to develop reversible inhibitors of LSD1. Some stapled cyclic peptides bound to LSD1 with 10-fold higher affinity than the corresponding linear parent peptide. Changing the staple into a lactam further improved the binding potency and the best lactams inhibited the enzymatic activity of LSD1 at low μM Ki values.

Stabilization of proteins is of great interest for the biotechnological society, industrial as well as research areas. Proteins with high stability are more suitable as reagents, easier to handle, store, transport and use in industrial processes. One way to stabilize a protein is to introduce a disulfide bridge into the structure by protein engineering. In this report the formation of a disulfide bridge between engineered cysteines in non ideal conformations in human carbonic anhydrase has been investigated. The disulfide bridge is not formed when the protein is in its native state. It is shown that when the protein is exposed to mild concentrations of urea in the presence of DTTox the disulfide bridge is formed. Also upon refolding in vitro, in a non oxidative environment, disulfide bridges are formed. This observation is worth to notice, since the disulfide bridge does not form to any appreciable extent when the protein is expressed and folded in vivo in Escherichia coli.

Les interactions protéine - protéine sont au centre de nombreux processus biologiques, et représentent des cibles thérapeutiques pertinentes pour le traitement de certaines maladies. La conception de molécules antagonistes visant à inhiber ces interactions requiert la reconnaissance spécifique d’une des surfaces protéiques impliquées. Les foldamères de type oligoamides de quinoline constituent de bons candidats. Leur production et leur fonctionnalisation sont relativement aisées. Ils adoptent des structures hélicoïdales semblables à celles rencontrées dans les protéines. Grâce à différentes techniques biophysiques (CD, SPR, diffraction des rayons X), nous montrons que ces molécules sont aptes à reconnaître une surface protéique. Deux protéines cibles ont été choisies : l’interleukine 4 et l’anhydrase carbonique humaine II.La stratégie retenue dans ce travail (ancrage du foldamère à la protéine via un bras espaceur) nous a permis d’obtenir des informations structurales sur les interactions foldamère – protéine avant toute optimisation de ces interactions. La première structure 3D d’un complexe foldamère de quinoline complexée à une protéine est décrite
Protein-protein interactions are a central issue in biological processes and represent relevant therapeutic targets for the treatment of diseases. The design of antagonistic molecules directed towards the disruption of these interactions requires the specific recognition of protein surfaces. Quinoline oligoamide foldamers present all the properties to reach that point. They are easily synthesized and fold into helices (similar to α helices) which can be decorated. Thanks to biophysical studies (CD, SPR, RX diffraction), we demonstrate that these molecules are able to recognize protein surfaces. Two proteins have been studied: the human interleukin 4 and the human carbonic anhydrase II.The applied strategy (keeping the foldamer close to the protein surface via a linker) allowed us to gather structural information about foldamer protein interactions before any strong binding is established. The first crystal structure between a protein and an aromatic amide foldamer is reported

Les protéines étant au coeur d’un grand nombre de processus biologiques, elles sont des cibles thérapeutiques largement convoitées. Les foldamères, notamment les oligoamides aromatiques, présentent une structure bien définie, prévisible, stable en solution et à l’état solide. Ajouté à cela, leur taille moyenne en fait de bons candidats pour la reconnaissance de surfaces de protéines, grâce à leurs chaînes latérales protéinogènes. Cette thèse présente les différentes étapes de leur conception, de la synthèse de la brique constitutive à l’obtention d’un foldamère fonctionnalisé grâce à la synthèse en phase supportée. La stratégie d’investigation des interactions entre un foldamère et une protéine est détaillée. L’originalité réside dans le fait que le foldamère est ancré directement à la protéine et le dichroïsme circulaire sert de méthode de screening. L’analyse structurale des hits permet de générer de nouveaux foldamères dans le but d’améliorer les interactions avec la protéine : c’est une stratégie itérative. Cette approche est appliquée premièrement à l’anhydrase carbonique humaine II, protéine modèle qui sert de preuve de principe pour cette approche ; puis à des protéines d’intérêt thérapeutique plus important : l’interleukine 4 et la cyclophiline A. Enfin, une étude concernant l’introduction de flexibilité au sein de foldamères de quinolines est présentée
Since proteins are at the basis of many biological processes, they are widely studied as therapeutic targets. Aromatic oligoamide foldamers have a very well defined structure, predictable and stable both in solution and solid state. Because of their medium size, they appear as potent candidates for protein surface recognition thanks to their proteinogenic side chains. This manuscript presents the different steps of their design, from the scaffold’s synthesis to obtaining a functionalized foldamer, thanks to solid phase synthesis. The strategy to investigate protein/foldamer interactions will be detailed. Its originality lies in the fact that the foldamer is anchored to the protein. Circular dichroism has been used as a screening method to detect foldamer/protein interactions. Structural analysis of the hits will allow the design of new foldamers with the objective of enhancing foldamer/protein interactions: it is an iterative strategy. This approach has been applied firstly to human carbonic anhydrase II (HCA). This protein is used as a model system and proof of concept before moving to more therapeutically relevant proteins; interleukin 4 and cyclophilin A. Finally, a study on introducing flexibility in quinoline foldamers is presented

This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.

Within a short timeframe, the CuI-catalysed 1,3-dipolar cycloaddition (1,3-DCR) of an organic azide to a terminal acetylene to form a 1,4-disubstituted-1,2,3-triazole, has emerged as a powerful synthetic transformation in combinatorial chemistry, organic synthesis and bioconjugation research. This synthetic methodology, now known as click chemistry, has had an appreciable impact in the drug discovery and biotechnology sectors and has shown broad scope and compatibility with small molecule and polymeric substrates. The application of this powerful synthetic transformation, specifically in carbohydrate based drug discovery and glycobiology is a recent and emerging trend. Chapter one of this thesis is a review of the current literature concerning the use of click chemistry in carbohydrate based drug discovery and glycobiology. Several examples have appeared within the literature highlighting the potential of click chemistry for rapidly generating structurally diverse neoglycoconjugates, ranging from small molecule drug leads to multivalent constructs, as well as a bioconjugation strategy for labelling cell-surface glycoconjugates. The review aims to be exhaustive in its coverage, with emphasis on future perspective. This thesis presents the investigation of click chemistry as a synthetic tool in carbohydrate chemistry, and its application for generating novel carbohydrate based enzyme inhibitor libraries for lead discovery and optimisation purposes. Chapter two describes the utility of click chemistry and the glycosyl triazole moiety in synthetic carbohydrate chemistry. The reaction is well suited to the synthesis of mimetics of complex oligosaccharides and glycoconjugates, owing to the mild ambient nature and remarkable regio- and stereo- selectivity. The transformation was therefore interrogated under conditions typically encountered in carbohydrate chemistry, including glycosylation reactions and protective group manipulations. The study represents the first exhaustive investigation into the stability of the triazole moiety under these conditions as well as the synthetic utility of the CuI-catalysed 1,3-DCR as a potential orthogonal transformation in carbohydrate chemistry and an adjunct to existing methods. The first aspect of the study aimed to examine the stability of the glycosyl triazole moiety under conditions employed in protective group chemistry and the compatibility of the transformation with pre-installed functional groups. Using click chemistry, the triazole moiety could be introduced onto the carbohydrate scaffold in the presence of a wide range of protective functional groups. In addition, the 1,2,3-triazole moiety was indeed shown to be a robust entity that is compatible with essential protecting group manipulations and glycosylation chemistry - an important outcome with respect to its potential utility as an additional tool for the synthesis of oligosaccharide/glycoconjugate mimetics, which are often heavily reliant on orthogonal reaction sequences. Next, the utility of the reaction with respect to solvent and catalyst conditions was examined. The reaction was performed in different organic and aqueous solvents in the presence of two different CuI-catalyst systems. It was shown that the reaction is reasonably insensitive to the nature of the solvent or aqueous co-solvent and the catalyst system. Reaction times and yields displayed little variation with respect to the solvent and catalyst system. In all cases, the 1,4-disubstituted 1,2,3-glycosyl triazole model compound was isolated in high yields and required minimal purification. The work also amply demonstrated, in a proof-of-concept manner, the powerful scope of the reaction for preparing structurally diverse neoglycoconjugates in high yield and purity. Several artificial glycomimetics were prepared using a suite of glycosyl azides through the facile 1,3-DCR to a series of acetylenes. Chapter three presents an extensive study into the preparation and biological activity of glycoconjugate benzene sulfonamides as a novel class of carbonic anhydrase (CA) inhibitor. The conjugation of carbohydrate “tails” to a benzene sulfonamide pharmacophore provides access to CA inhibitors which are neutral, water-soluble and features high chiral density and polyfunctionality that may be exploited for tissue delivery applications and to survey active site architectures in order to impart isozyme selectivity. Glycoconjugate benzene sulfonamides could also display compromised plasma membrane permeability allowing for the selective targeting of tumour associated isozymes with extracellular catalytic domains. Glycoconjugate benzene sulfonamides have received little attention as CA inhibitors, and this work represents the first comprehensive study in the area. By utilising a novel “click-tailing” strategy developed in our laboratory, a panel of structurally diverse carbohydrate “tails” were appended to the primary arylsulfonamide (ArSO2NH2) pharmacophore. A panel of azido sugars and propargyl glycosides were reacted with acetylene- and azide-functionalised benzene sulfonamide scaffolds, respectively, and subsequently evaluated for their inhibition of human carbonic anhydrase (hCA) isozymes hCA I, II, IX, XII and XIV in vitro. In this manner, a total of 50 glycoconjugate benzene sulfonamides belonging to three libraries were prepared and assessed for their inhibition of human cystolic isozymes hCA I, II and transmembrane isozymes hCA IX, XII and XIV. Selective inhibition among CA isozymes is challenging owing to conservation of active site topology within this enzyme family, yet the design of selective CA inhibitors is necessary for the development of efficacious and safe CA-based therapeutics which are void of side effects arising from systemic CA inhibition. Many of the glycoconjugate benzene sulfonamides exhibited a non-clustered in vitro inhibition profile, demonstrating that the carbohydrate tail was a powerful structural element able to distinguish isozyme selectivity. A significant outcome of this study was the discovery of several potent and selective CA inhibitors of the tumour-associated transmembrane isozyme, hCA IX, and the physiologically dominant cytosolic isozyme, hCA II. Chapter four of this thesis explores the synthetic utility of click chemistry for the solution-phase synthesis of N-alkylated azasugar libraries. To date, click chemistry has seen limited application for the synthesis and screening of natural product-based libraries. To the best of my understanding, this work represents the first example of the use of click chemistry for the generation of azasugars containing structurally diverse N-alkyl substituents as potential glycosidase and glycosyltransferase inhibitors. By employing the click chemistry methodology, various synthetically accessible aliphatic and aromatic azides were conjugated to the acetylene-functionalised 6- and 7-membered ring N-propynyl azasugar scaffolds using click chemistry, thus providing expedient access to N-methylene triazole-substituted azasugars in a single, high yielding step. The work demonstrates the applicability of the reaction for generating not only the structural diversity deemed necessary for distinguishing inhibitory potency and selectivity, but also a powerful means of tuning the physicochemical properties of the azasugar for in vivo targeting and lead optimisation purposes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Full Text

The development of small molecule affinity-based chemical probes as research tools for studying the role of carbonic anhydrases (CAs) in their wider biological context has become an active field of research owing to an increasing awareness of the therapeutic relevance of this enzyme family, particularly in diseases such as glaucoma (CA II) and solid tumors (CA IX, CA XII). High CA isozyme selectivity, low nonspecific labeling, and efficient labeling yield are the characteristics of an ideal chemical probe, however achieving an effective balance of all three properties is challenging. A panel of chemical probes for two-step labeling of CA II or CA IX has been designed to study the impact of probe structural features on the efficiency and specificity of CA-selective labeling when in a challenging environment, including protein mixtures, cell lysates, or live cells. The panel comprised Generation 1 probes (P1 and novel probes P2–P5), Generation 2 linear PAL probes (P6 and novel probes P7–P15), and Generation 3 branched PAL probes (novel probes P16–P20).
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
Full Text

Part A: Structure-Based Drug Design for Carbonic Anhydrases In humans there are twelve carbonic anhydrase (CA) isozymes that possess catalytic activity for the reversible hydration of carbon dioxide (Supuran, & Scozzafava, 2007). Carbonic anhydrases (CAs) underpin vital physiological and pathological processes and are so pharmaceutical targets for a variety of diseases. The recent findings in CA research were the validation of transmembrane human CA IX and human XII proteins as targets for cancer chemotherapy. Studies have shown that the specific targeting of CA IX (or XII) can lead to an effective anti-cancer therapy, especially for hypoxic tumours. Chapter one of this thesis is the introduction to carbonic anhydrases and the catalytic and inhibition mechanisms of different CA families. It has appeared within the literature, highlighting the involvement of CAs in a wide range of physiological functions, which makes them notable as targets for clinical inhibitors (Supuran & Scozzafava, 2007; Liljas, Hakansson, Jonson & Xue, 1994; Hilvo et al, 2008). This chapter introduces the classical recognition moiety needed for the inhibitor compounds in order to recognise and bind to the active site of CA, which is the aromatic or heteroaromatic sulfonamide moiety (R-SO2NH2). They operate through coordination of the sulfonamide anion to the active site zinc cation, replacing thez inc-bound hydroxyl anion, thereby impeding the endogenous reaction. Typically, 'tail' groups are appended to the aromatic sulfonamide anchor with the aim of improving potency and optimise d pharmacokinetic properties of CA inhibitors. Varying tail moieties can be appended to th e aromatic sulfonamide anchor via a triazole linker. Chapter two is a brief description of the objectives of this thesis. This chapter tries to convey an idea about the proteins involved in the thesis and reasons as to why these enzymes were investigated, with detailed descriptions presented in the relevant chapters. Chapter three presents a thorough study of the X-ray crystallographic structures of different CA II- inhibitor complexes. The three-dimensional structures of these CA inhibitor compounds with human CA II enabled us to understand their interactions with the active site residues of CA II, and thus to determine the structural features of th e ligands responsible for their weak or strong CA inhibition.T he interactions of all of these compounds with CA II were compared to the interactions and properties of topirama te with CA II active site residues. Topiramate is a low nanomolar inhibitor of human CA I I, and a well-established anti-epileptic drug. This is to compare the active site binding properties of the new CA ligands with those of topiramate when inside the human CA II active site. To obtain a preliminary idea on the stability of different CA II: ligand complexes, thermal denaturation studies of these complexes were performed using circular dichroism (CD) spectroscopy. Since the recent findings in CA research has shown the specific targeting o f transmembrane CA IX (or XII) leading to an effective anti-cancer therapy, ou r collaborators started working on the aspect to differentiate the inhibition of cancer-related , transmembrane CAs such as CA IX, CA XII from cytosolic CAs like CA I, CA II. Th e strategy was to attach a carbohydrate moiety to the high affinity zinc-binding aromat ic sulfonamide CA moiety leading to the formation of sulfonamide glycoconjugates with a sugar-aromatic-SO2NH2 motif. The -SO2NH2 zinc binding moiety of the glycoconjugates plays a key role in CA enzyme recognition and is essential for efficacy. The sugar tail is responsible for the high-water solubility of the compound and this hydrophilic group impairs the ability of the sulfonamide glycoconjugates to passively diffuse through lipid membranes, and this facilitates a selective or preferential inhibition of transmembran e CAs over cytosolic CAs, which would help in treating cancer. The stereochemical and structural variations of the carbohydrate moiety provide the opportunity for exploring th e differences among different CA active site architecture, thus yielding a neutral and wate r soluble CA inhibitor that has excellent potential as an isozyme selective inhibitor. Three different aromatic sulfonamides carrying a thio, sulfinyl or sulfonyl glucoside triazole tail moiety on the benzenesulfonamide CA pharmacophore have been cocrystallised with human CA II. Additional sugar-containing derivatives of similar topology have been co-crystallised with CA II to assess the effects of length variation and acetylation. Compounds different from the typical aromatic sulfonamide CA inhibitors were also co-crystallised with CA II, where the classic aromatic moiety of th e zinc-binding sulfonamide CA inhibitors is absent from these compounds, and instead a hydrophilic monosaccharide or disaccharide moiety has been introduced directly to th e primary sulfonamide group to get sugar-SO2NH2 motif. The fourth set of CA inhibitors used for co-crystallisation was sulfamate compounds (R-OSO2NH2). Sulfamates, like sulfonamides, are a group of strong CA inhibitors. While direct interactions between th e compounds and the protein were identified only in few cases, the current structure s provide clues as to where and how to extend the compounds in order to increase direc t interactions, and thus obtain an isozyme-specific inhibitor with improved pharmacologic al properties. Chapter four explores two other CA isozymes; human CA IX and carbonic anhydras e from Plasmodium falciparum. This chapter also present the attempts to successfully express and purify these proteins. Part B: Membrane Interactions of Human Visinin-like Protein-1 (VILIP-1) VILIPs are part of the subfamily of neuronal calcium sensor (NCS) protein. All members of NCS protein family are EF-hand proteins, and they share the characteristic feature of N-terminal myristoylation as well as the calcium-myristoyl switch. When calcium levels elevate, NCS proteins undergo the calcium-myristoyl switch which is the central mechanism of their involvement in cellular calcium signalling. Previous studies have shown that the membrane association of proteins by a myristoyl group alone is weak and requires other interactions between the protein and phospholipid on the membrane for stability. In addition to increasing the strength of the protein-membrane association, protein-phospholipid interactions also help to target proteins to different subcellullar domains (Braunewell et al, 2010). Chapter one provides a background to NCS proteins, particularly VILIP-1. The chapter highlights the functional and structural aspects of the proteins in this family. Chapter two presents the objectives of this part of the thesis. The aims include expression and purification of VILIP-1 proteins (unmyristoylated, myristoylated and VILIP-1 mutant S6A/K7A), protein folding experiments of these proteins in the absence and presence of calcium ions, and the monolayer adsorption experiments using Langmuir surface film balance. Chapter three explains the expression and purification details of the recombinant VILIP-1 proteins; unmyristoylated VILIP-1, myristoylated VILIP-1 and VILIP-1 mutant S6A/K7A. Ser6 and Lys7 are two of the main N-terminal residues found to be involved in forming interactions with membrane phospholipids, which in turn, help in protein-membrane association. These two residues have been replaced using the neutralcharged alanine to investigate the importance and the effects on the protein-membrane association in their absence. Chapter four of this thesis explores the thermal stability of VILIP-1 proteins. Four separate experiments were performed; unmyristoylated VILIP-1 in the absence of calcium, unmyristoylated VILIP-1 in the presence of calcium, myristoylated VILIP-1 in the absence of calcium, and myristoylated VILIP-1 in the presence of calcium. Quaternary structure of VILIP-1 mutant S6A/K7A protein in solution was also determined using SEC-MALLS. Chapter five experimentally determines the postulated interaction of VILIP-1 with different PIP derivatives. Studies have shown VILI...
Thesis (Masters)
Master of Philosophy (MPhil)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective. The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid. This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins.

O fungo Aspergillus fumigatus é o segundo maior causador de infecções fúngicas invasivas em pacientes imunocomprometidos e a principal espécie causadora da aspergilose invasiva, doença de alta taxa de mortalidade que atinge principalmente os pulmões e que pode se disseminar pelo organismo. Durante o processo de infecção, o fungo precisa adaptar-se ao organismo do hospedeiro e um dos obstáculos encontrados é a mudança na concentração de dióxido de carbono (CO2), que, de 0,033% no ambiente, chega a até 6% no interior do hospedeiro. As anidrases carbônicas são enzimas envolvidas na hidratação reversível do CO2 e já foram apontadas como importantes na virulência de patógenos como Plasmodium falciparum, Mycobacterium tuberculosis, Helicobacter pylori, Cryptococcus neoformans e Candida albicans. Esse trabalho teve como objetivo avaliar o papel da enzima anidrase carbônica no desenvolvimento e virulência do fungo A. fumigatus, que apresenta quatro homólogos desta enzima (cafA, cafB, cafC e cafD). Para isso, foram utilizadas linhagens de A. fumigatus com os homólogos da enzima deletados (?cafA, ?cafB, ?cafC, ?cafD e ?cafA?cafB) e a linhagem selvagem (?akuBku80), da qual foram originadas as mutantes. Foram realizadas avaliações fenotípicas da estrutura dos conidióforos das diferentes linhagens, determinação da sensibilidade frente a diferentes agentes estressantes (antifúngicos, promotores de apoptose, estresse iônico, nitroativo, oxidativo, e de parede celular) e determinação da expressão gênica global em diferentes concentrações de CO2. Foi verificado que a deleção de cada um dos homólogos da anidrase carbônica de A. fumigatus não interfere na estrutura dos conidióforos deste fungo. Por outro lado, a deleção induziu alteração da sensibilidade do fungo frente a alguns compostos estressantes (ácido acético e peróxido de hidrogênio). Ainda, a análise da expressão gênica revelou um gene envolvido na adaptação do fungo ao aumento da concentração de CO2, o gene cipC, que não apresenta homólogos nas células de mamíferos. Este gene foi caracterizado neste trabalho por meio de sua deleção na linhagem selvagem (?akuBku80) de A. fumigatus e avaliação fenotípica microscópica e de sensibilidade a agentes estressantes (antifúngicos, promotores de apoptose, estresse iônico, nitroativo, oxidativo, e de parede celular). A deleção do gene não interferiu na estrutura do fungo, porém aumentou sua sensibilidade a alguns compostos (calcoflúor e menadiona). Foram realizados, ainda, testes de virulência em modelo animal utilizando-se o mutante ?cipC, os quais revelaram que a deleção deste gene atenua a virulência do fungo. Assim, foi possível concluir que as anidrases carbônicas não são relevantes para o desenvolvimento e virulência de A. fumigatus; porém, este fungo modifica a expressão de seus genes de modo a adaptar-se às variações na concentração atmosférica de CO2. O gene cipC está envolvido nesse processo de adaptação e é importante para o desenvolvimento do fungo e sua virulência, tornando-se um alvo para o estudo de novas terapias para o tratamento da aspergilose invasiva.
The fungus Aspergillus fumigatus is the second cause of fungal infections in immunocompromised patients and it is the main specie which causes invasive aspergillosis, a disease with high mortality rate that mainly affects the lungs and it can spread through the body. During the infectious process, the fungus must adapt to the host and one of the obstacles is the drastic change of the carbon dioxide (CO2) concentration, which is 0.033% in the environment and until 6% inside the host. The carbonic anhydrases are enzymes which are involved in the reversible hydration of carbon dioxide and they have been pointed as important in the virulence of pathogens such as Plasmodium falciparum, Mycobacterium tuberculosis, Helicobacter pylori, Cryptococcus neoformans and Candida albicans. This work aimed to evaluate the role of the enzyme carbonic anhydrase in the development and virulence of the fungus A. fumigatus, which has four homologues of this enzyme (cafA, cafB, cafC e cafD). Therefore, strains, which have the homologues of the enzyme deleted (?cafA, ?cafB, ?cafC, ?cafD and ?cafA?cafB) were used in parallel with the wild strain (?akuBku80), which originated the mutant ones. We did structure phenotypic evaluations of the different strains of conidiophores, sensibility determination against different stressors (antifungal agents, apoptosis, ionic, nitrosative, oxidative, and cell wall stress promoters) and global gene expression determination at different carbon dioxide concentrations. It was verified that the carbonic anhydrases homologues deletion of A. fumigatus did not interfere on the n structure (conidiophore) of this fungus, in the tested conditions. On the other hand, the deletion caused a change in sensibility of the fungus against some stressors (acetic acid and hydrogen peroxide). The gene expression experiments showed a gene involved in the adaptation to the increase of CO2 concentration, the cipC gene. This gene does not have homologues in the mammalian cells. The cipC gene was characterized in this work by its deletion in the A. fumigatus wild strain (?akuBku80) and microscopic phenotypic evaluation and sensibility tests against stressors (antifungal agents, apoptosis, ionic, nitrosative, oxidative, and cell wall stress promoters). The gene deletion did not interfere on the fungus conidiophore structure but increase its sensibility to some compounds (calcoflúor white and menadione). Virulence tests in animal model using the ?cipC mutant were done and they showed that the deletion of this gene attenuates the fungus virulence. In conclusion, the carbonic anhydrases are not relevant to development and virulence of the fungus, which modifies the gene expression to adapt to the variations of atmospheric CO2 concentration. Besides, the cipC gene seems to be involved in this adaptation process. Moreover, the cipC gene showed to be important to the development of the fungus and its virulence, which makes the gene a target for the study of new therapies for the treatment of invasive aspergillosis.

The Carbonic Anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes expressed in almost all living organisms. Being one of the main actors in pH regulation and in the maintenance of proper concentrations of CO2, the dysruption of the activity of such enzymes, by means of adequate modulators, is a validated strategy for the treatment of human affecting pathologies and for the eradication of etiological agents (i.e. pathogenic bacteria, fungi and protozoa). In addition, CA-based biotechnological applications (i.e. CO2 capture) may benefit from modulation of the enzymatic activity. CA Inhibitors (CAIs) have been extensively investigated over the time, and have been validated for the management of hypertensive glaucoma, systemic hypertension, epilepsy, obesity related diseases and recently neuropathic pain, inflammation and hypoxic tumors. Although CA activators (CAA) traditionally lacked interests, currently a repurposing of such compounds is underway with promising results as potential agents for the management of memory deficits related to neurodegenerative diseases. In this Thesis work, an introductive overview on CAs as the main biological targets (Chapter 1) and three distinctive projets (Chapters 2-4) are reported ranging from synthetic chemistry to enzyme biology and spctrophotometry. The first one (Chapter 2) concerns the synthesis and evaluation of new CAIs with Carbon Monoxide (CO) releasing properties for the management of RA. The reported compounds have been fully characterized, their CA inhibitory properties along with the CO releasing effect were assessed. In particular, a spectrophotometric assay was properly set with slight modifications of the protocols reported in the literature. This allowed to obtain a precise and quantitative evaluation of CO released over time from our compounds. The pain refief effect of the designed CAI-CORMs was also evaluated in a rat model of RA with very promising results. The second project (Chapter 3) was aimed to synthetize a small series of CAI-AZT hybrids and to evaluate them as Telomerase Inhibitors, thus with possible antitumoral applications. The compounds synthetized have been profiled in vitro on seven CA isoforms (i.e. I, II, Va, VB, VII, IX and XII). The effects of our compounds on Telomerase Activity have been also determined showing a low-medium inhibition potency. Two promising derivatives have been identified with good IC50 and IC90 values. Co-crystallyzation of selected compounds in adduct with hCA II has been performed and their binding modes were determined. The third project (Chapter 4) was entirely carried out during my six-months visiting student experience at the University of Poitiers in France, and it concerns the synthesis in Superacid medium of new mono and di-fluorinated diamines as CAIs. Insertion of one or more C-F bonds is a validated strategy in Medicinal Chemistry. Fluorine insertion can modulate pharmacokinetic and pharmacodynamic properties of the compounds, thus being an attractive tool in the design of bioactive compounds. Another unrelated project included in this Chapter, is the synthesis of enantiopure fluorinated tricyclic scaffolds obtained by means of a diastereoselective approach.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes widespread in all life kingdoms genetically classified in 7 unrelated classes (i.e. α-, β-, γ-, δ-, ζ-, η- and θ). These enzymes catalyse a very simple and essential physiological reaction, which is the carbon dioxide hydration to afford bicarbonate and protons. So far, 16 different α-CA isoforms were isolated and characterized in mammals. In many tissues CAs are concomitantly present in a variety of isoforms, which differ for their kinetics, structural properties as well as cellular and tissutal abundancy. To date, human (h) CAs are well established therapeutic targets to treat a hypertension, glaucoma. New proof-of-concepts are therapeutic applications for the treatment of epilepsy, obesity related pathologies and neuropathic pain. In the last years CA inhibitors (CAIs) were validated for the treatment of hypoxic tumors. Within the scope of this Thesis, we report new and more isoform selective modulators of CAs expressed in humans and/or in pathogenic organisms with the intent to pave the ways to the treatment of pathologies by means of innovative approaches. The current Thesis is composed of seven chapters, each one dealing with the drug design, synthesis as well as in vitro kinetic assay of new CA modulators: (i) Novel inhibitors against hCAs (ii) Potential anticancer drugs targeting primarily hCA IX and XII that are predominantly expressed in tumor cells. (iii) New class of agents for the prevention of diabetic cerebrovascular pathology (which probably target the mitochondrial isoforms hCA VA and/or hCA VB). (iv) Potential drug leads for the treatment of different neurological disorders such as antiepileptic or neuropathic pain (probably targeting hCA II and VII). (v) Agents that target various CAs from pathogenic microorganisms such as bacteria and protozoa. (vi) New activators of hCAs (vii) Kinetic activation studies on no human expressed CAs such as the α-, β-, γ-, δ-, ζ- and η-classes.

博士
國防醫學院
生命科學研究所
96
Carbonic anhydrase III (CAIII) is distinguished from the other members of the CA family by low carbon dioxide hydratase activity, resistance to the CA inhibitor acetazolamide, and a predominant expression in the liver of males. In our animal study, the CAIII expression was over-expressed in the foci of altered hepatocytes (FAH) and was consistently down-regulated in hepatocellular carcinoma. We also found that the expression of CAIII was slightly increased in early stage of most HCC (63%), but it became significantly weaker in late stages of HCC. Ectopic overexpression of CAIII in SK-Hep1 cells resulted in increased anchorage-independent growth and invasiveness. Compared with the control animals, the metastasis nodules of lung increased in the population of pooled CAIII transfectants. And siRNA-mediated silencing of CAIII expression decreased the invasive ability of SK-Hep1 cells. Furthermore, CAIII transfectants showed elevated focal adhesion kinase (FAK), Src activity and MMP-3 protein expression. Silencing of FAK expression in CAIII transfectants led to suppression of HCC cell invasion. More importantly, the CAIII transfectants acidified the culture medium at an accelerated speed than the control cells did. Intracellular pH, determined by pH-sensitive bis-carboxyethyl-carboxyfluorescein (BCECF) probe, was lower in CAIII transfectant cells than vector only control cells. The number of migrated and invasive cells was significantly increased in cultured medium of pH 6.8 than that in pH 7.4 condition. Western blot also showed that SK-Hep1 cells cultured at pH 6.8 had higher phospho-FAK, phospho-Rac-1/cdc42 and vimentin expression than that at pH 7.4. Base on these results, we suggest that the CAIII-promoted invasive ability of HCC cells may probably be mediated through, at least in part, the FAK signaling pathway via intracellular and/or extracellular acidification.

博士
中山醫學大學
生化暨生物科技研究所
94
Cytosolic carbonic anhydrases (CAs), including CAI, CAII and CAIII are presented in normal hepatocytes. This study was aimed to investigate the expression status of CAs in hepatocellular carcinomas (HCC) and breast cancer and the role of tumor progression. The activity, protein expression pattern and messenger RNA of cytosolic CA were analyzed by CA activity analysis, Western blotting and RT-PCR in 60 human hepatocellular carcinomas and 60 human breast cancer surgical specimens. The result shows that in each of 60 HCC, CA activity and protein expression in tumor area is significantly lower than that of paired adjacent normal tissues (P<0.01), and mRNA expressions in tumor areas are also reduced (P<0.001). Furthermore, the immunohistochemical studies have further confirmed this reduction of CAI, CAII and CAIII protein expression in tumor areas. There is a statistically significant reduction in the expression of cytosolic CAII in poorly differentiated cancer (P < 0.001). Furthermore, in 60 breast cancer patients, the result showed that CA activity and CA II protein expression in tumor area was significantly higher than that of paired adjacent normal tissues (P < 0.01), while the CA I and CA III protein expression was lower (P < 0.001). Furthermore, further linear regression analysis has showed that the tumor size positively correlated with CA activity in tumor tissue samples (R=0.48, P<0.05). In addition, further statistical analysis for clinic pathological parameters revealed that CA activity was significantly increased in patients with metastasis (P <0.05). CA activity in tumor tissue might reflect the severity of invasion of breast cancer and various CA inhibitors might be selectively used as potential anti-metastasis agents according to tumor size.

Paramagnetic Nuclear Magnetic Resonance (NMR) is developing to aid the characterization of paramagnetic molecules, whose paramagnetic centers changes the spectroscopic proprieties of said molecules. These paramagnetic centers can be exploited to overcome some troublesome aspects of NMR, such as sensitivity, by increasing the number of experiments. To aid this development, we used human Carbonic Anhydrase II (hCAII), which is a model protein that contains zinc(II) in its active center, which is diamagnetic. hCAII is an enzyme capable of interconverting carbon dioxide to bicarbonate, making it one of the most important proteins in life. Several comprehensive studies have structurally and functionally characterized hCAII making it an excellent model protein. Furthermore, the metal ion in the active center can be substituted by other transition metals ions (see chapter 1), such as cobalt(II) (see chapter 3), nickel(II) (see chapter 4) and copper(II) (see chapter 5), which are paramagnetic that will help answering different problems described in this thesis. The ion cobalt(II), explored in chapter 3, can induce considerable changes on the NMR observables and is useful to understand the interactions of ligands with proteins. For this we used cobalt(II)-hCAII and used NMR, Electron Paramagnetic Resonance (EPR) and X-ray crystallography to characterize the interaction of thiocyanate under high concentrations with the hCAII. The addition of 500 mM of sodium thiocyanate changes the dynamics of the protein without changing the protein structure. Solid-state NMR (SSNMR) is another field of NMR where the methodological and practical aspects are currently under development to reach better sensitivity and resolution. We proposed the usage of nickel(II)-hCAII as a paramagnetic molecule to increase the amount of tools in SSNMR, which is explored in chapter 4. The nickel(II) ion is capable of breaking the dipolar bath by changing the frequency of the nuclei close to the paramagnetic center, thus increasing the resolution and sensitivity of the SSNMR experiments. Furthermore, in parallel we discovered that hCAII is capable of binding two nickel(II) ions, one in the active center, as expected and described in literature, and one in the N-terminal site of the protein, a novel discovery. The description of the paramagnetic effects, such as the Pseudocontact Shifts (PCS), in the NMR observables have been subjected to debate, where different treatments of theoretical equations were clashing. The experimental proof to determine which equation holds true is fully described in chapter 5. For this, we developed copper(II)-hCAII to acquire NMR and EPR data under the same conditions, and determine which equations describe better the PCS. The data interpretation from different techniques (both NMR and EPR) led us to conclude that the original treatment from Kurland and McGarvey equation is the correct one.

Carbonic Anhydrases (CAs, EC 4.2.1.1) are a family of monomeric Zinc metalloenzymes that catalyze the reversible hydration of CO2. The relevant role of this class of enzymes is the regulation of a broad range of physiological functions (gluconeogenesis, lipogenesis, and ureagenesis).[1] The family of human carbonic anhydrases (hCAs) comprises 15 different α-carbonic anhydrase isoforms: CA I-IV, CA VA and CA VB, CA VI-VII, CA IX, CA XII- XV and CARPs CA VIII, CA X, CA XI). In the last years, the attention toward this class of metalloproteins is in continuous growth because it was discovered their involvement in different diseases such as cancer,[2] glaucoma,[3] obesity,[4] and epilepsy.[5] Among the hCAs, the hCA VII is one of the least investigated cytosolic CA isoforms; it presents a limited distribution, being mainly expressed in the cortex, hippocampus, and thalamus regions within the mammalian brain where it is involved in generating neuronal excitation and seizures. Moreover hCA VII is expressed in other organs including the stomach, duodenum, colon, liver and skeletal muscle of mice. [2, 5-7] The hCA VII is currently considered to be involved in the mechanism of GABAergic generated seizures. [5, 8] Recently, its involvement in neuropathic pain control has been proposed by a mechanism which is not completely known. [6, 7, 9]Thereby, the investigation on hCA VII could also represent an interesting tool for the design of new pain killers useful for therapeutic applications. Several CA inhibitors such as acetazolamide (AAZ) and topiramate (TPM) have a long history as anticonvulsants, but their molecular targets and mechanisms of action at the neuronal network level are still poorly understood.[10] In the last years in the Department ChiBioFarAm – medicinal chemistry laboratories of the University of Messina, a collection of small molecules acting as carbonic anhydrase inhibitors had been discovered. The most active inhibitors displayed ki values in the nanomolar range. Unfortunately, some of them showed poor selectivity toward the more druggable isoforms. These inhibitors possess the sulfonamide portion as key chemical portion that binds the Zinc2+ ion that is located at the bottom of a deep cleft of catalytic site (Figure 1).[11] The other moieties of these compounds control the major or minor interactions with the hydrophilic and/or hydrophobic regions of catalytic pocket. The degree of this pattern of contacts might be considered responsible of the inhibitory potency as well as selectivity toward specific CA isoforms. During the three years doctoral course we chose to introduce new chemical moieties and modify these sulfonamides to obtain the new compounds. Specifically, we planned the synthesis of new compounds to improve inhibitory effects and isoforms selectivity. Therefore, we planned the synthesis of a series of new sulfonamides bearing the quinoline and isoquinoline skeleton that has been variously decorated through the introduction of chemical fragment as hydrophilic/hydrophobic anchoring groups. This series of novel hCAIs were in vitro tested against the hCA I, hCA II, hCA VII, hCA IX, hCA XII and hCA XIV. Structural and computational studies have been performed to explain the mechanism of inhibitory properties. Furthermore, in vivo studies were carried out to find new potential therapeutics for the treatment of brain pathologies.

This Thesis is focused on the development of novel inhibitors targeting the tumour-related carbonic anhydrase isoforms and on the development of small molecule ligands targeting the human tyrosinase and tyrosinase related protein 1. The carbonic anhydrases (CAs) are a superfamily of metalloenzymes that catalyse the reversible hydration of carbon dioxide (CO2) into bicarbonate (HCO3-) and protons (H+). The human CAs (hCAs), belonging to the α family, are involved in several functions and pathological conditions. Particularly, hCA IX and hCA XII, known as tumour-related isoforms, are selectively upregulated in solid tumours. Though a large number of inhibitors have been developed to date, only two of them are currently in clinical trials highlighting the urgent need of novel and selective inhibitors targeting hCA IX and hCA XII. Thus, the aim of the project was the design, synthesis and SAR analysis of two libraries of compounds based on the 2-(benzylsulfinyl)benzoic acid scaffold and on the saccharin and acesulfame nucleus. Considering the first library of compounds, the chemical modifications attempted on the 2-(benzylthio)benzamide, obtained after a lead optimization of the 2-(benzylsulfinyl)benzoic acid , led to 15 derivatives that appeared to be effective exclusively against hCA IX, though less active than their lead compound. Considering the second library of compounds, the different strategies applied on the saccharin and acesulfame scaffold led to 60 derivatives that exhibited different inhibitory profile mostly related to the presence of moieties of conformational freedom. The molecular modelling studies proposed as inhibition mechanism the anchoring to zinc-bound water molecule. Human tyrosinase (hTYR) and tyrosinase-related protein 1 (TYRP1) are closely related proteins involved in the synthesis of melanin and selectively upregulated in melanoma. Thus, the aim of the project was to develop and evaluate small molecule ligands specific to hTYR and TYRP1 using previously collected data elaborated from the DNA encoded chemical libraries (DELs) and the multivalent approach applied to an alkyne derivative of Thiamidol™. The ligands identified from the DELs after a screening performed on hTYR and TYRP1 resulted active in the micromolar range. The multimerization of an alkyne derivative of Thiamidol™ led to multimeric structures of different valence. among them the tetrameric structure showed the best binding profile though worse than the one of the TA99, a murine antibody targeting TYRP1.

碩士
國立中正大學
化學所
95
Carbonic anhydrase (CA) is a zinc-containing metalloenzyme that is widespread in nature and catalyzes the reversible hydration of CO2 to HCO3- and H+. CA is expressed in a number of isoforms (CA I-XIV) with varying degrees of enzymatic activity and participates in various physiological/pathological processes in human body. Inhibition of the zinc metalloenzyme human CAII by sulfonamides has the potential to treat clinically important diseases such as glaucoma, osteoporosis and most recently cancers. In this study, a new class of human CAII inhibitors with conformationally constrained core structure was designed to increase its binding affinity. Totally, Sixteen quinoxalinone-based sulfonamide inhibitors were synthesized in 2~3 steps with the total isolated yields of 5-89%. The activity (Ki) of these inhibitors toward human CAII were screened spectrometrically and determined by the Lineweaver-Burk plots. The inhibition constants obtained were in the range of 370-8200 nM. In this study, the conformationally constrained inhibitors were demonstrated to be more potent in the inhibition of human CAII, as compared to the conformationally relaxed inhibitors. In addition, the rigidity of the fused aromatic ring appears to play important role in enzymatic inhibition. Ionic liquids are solvents systems that consist of cation and anions at ambient temperature. We previously reported the new ionic liquid, [b-3-C-im][NTf2], which is not only chemically stable, but also has a low melting point, low water content and dynamic viscosity. In this work, this new ionic liquid was applied to the Claisen rearrangement reaction，in conjunction with focused microwaves. Under the optimized conditions, the Claisen rearrangement reaction was achieved in 3 minutes. Moreover, the Lewis acid (MgCl2) was introduced to decrease the reaction temperature (250℃→200℃) successfully, and with good overall isolated yield.

博士
中山醫學大學
生化暨生物科技研究所
99
Part: I Carbonic anhydrase (CA) is an enzyme with zinc metal ions, including a number of different isoforms, and its main function lies within the catalytic acid cells and maintain the cell''s internal balance. CA II, CA IX and CA XII are considered to affect the cell''s internal balance of carbonate ions and maintain the normal extracellular pH. A previous study conducted with 60 breast cancer patients revealed a significant increase of CA II protein expression and CA activity in in tumor (P &lt;0.001), as well as mRNA and protein expression levels. Furthermore, a significant correlation was found between CA II activity and tumor size while CA IX and CA XII are involved in the process of tumor development. Although there were extensive results indicating the involvement of CA II and CA IX in the cancer cell invasion and migration, similar studies for CA XII is less and insufficient. Previous studies have revealed that CA XII expression is an indicator for breast cancer cell invasion and useful for tracking surgical outcomes. Therefore, the aim of this study was to explore the impact of CA II, CA IX and CA XII on the extent of invasion and mobility in different breast cancer cells. Furthermore, the related pathways were alsoexplored. In the study with breast cancer cells of different levels of invasion and mobility, results from RNA interference technology showed that the CA XII RNA interference has the most significant inhibition capability for the invasion and migration of MDA-MB-231 cells. Further studies revealed that such inhibition is via an inhibition of phosphorylation of p38. In the final in vivo experiments, CA XII-knockdown MDA-MB-231 cells were inoculated into mice to show that the tumor size and animal body weight were significantly reduced in CA XII-knockdown mice. Take together, these results confirmed that CA XII indeed play an important role in the invasion and migration of MDA-MB-231 breast cancer cells. Part: II Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.

Carbonic anhydrases (CAs. EC 4.2.1.1) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and proton. In humans, 15 isozymes have been described with different subcellular localization. Indeed, CA I-III, VII, and XIII are cytosolic, CA IV, IX, XII and XIV are membrane-bound, CA VA and CA VB are mitochondrials, and CA VI is secreted. Human CA isozymes are extensively distributed in several tissues and organs where, as modulators of pH and ion transport, they take part in a variety of physiological and pathological processes. As a result, in the last years many of these enzymes have become important therapeutic targets for pharmaceutical research. However, given the high degree of sequence and structure similarity among the different isoforms, to date most of the CA-directed drugs developed lack of selectivity and consequently present many side-effects. X-Ray crystallography is one of the most useful instruments in the structure-based drug design of selective molecules able to interact with a target enzyme. Indeed, there are several examples in the literature where knowledge of the crystallographic structure of an enzyme allowed the design of molecules able to interact with specific residues, thus regulating enzyme biological activity. This is why in recent years there has been an extensive research effort focusing on the crystal structure resolution of all catalytically active alfa-CA isoforms, with the result that most of the human CA isoforms have been so far structurally characterized. As a part of a general research project based on the structure-based drug design of isoform-selective CA inhibitors (CAIs), my Ph.D thesis has been focused on the study of the biochemical and structural features of two cytosolic CA isoforms, namely hCA VII and hCA I, which among the 15 human isoforms have been poorly investigated. hCA VII was the sole cytosolic isozyme, that at the beginning of this Ph.D thesis was not yet structurally characterized. This enzyme, similarly to hCA II, is a very efficient catalyst for hydration of carbon dioxide, being 10-50 times more active compared to other two cytosolic isoforms, hCA I and hCA XIII. However, in contrast to hCA II which is widely spread in human tissues, CA VII has a more limited distribution, being localized mainly in some brain tissues of humans and rats, in stomach, duodenum, colon, liver and skeletal muscle of mice. In the first part of my Ph.D thesis a complete biochemical characterization of this enzyme was carried out. Interestingly, these studies highlighted the capability of two reactive cysteines to be S-glutathionylated during the purification procedures. Since S-glutathionylation was reported in vivo also for another cytosolic CA isozyme, namely CA III, and was associated to a protective response to oxidative stress, this phenomenon was investigated in details also for CA VII. Such studies showed that Cys183 and Cys217 were involved in adduct formation. These reactive cysteines were mutagenized and the corresponding double mutant (C183S/C217S) was expressed. The native enzyme, its double mutant and the S-glutathionylated adduct were fully characterized for their CO2 hydration, esterase and phosphatase activity. These kinetic studies indicated that the modification of Cys183 and/or Cys217 by glutathione does not have a relevant impact on the active site of the enzyme, causing rather small differences in its specific activity. Moreover, an important observation was that hCA VII was highly effective as esterase and phosphatase, compared to other cytosolic CA isoforms, such as CA I, II, III and XIII. These findings seem to indicate that the observed S-glutathionylation, if present in vivo, is not involved in the regulation of the enzyme catalytic activity but rather, as observed for hCA III, can help hCA VII to function as an oxygen radical scavenger to protect cells from oxidative damage. Further in vivo studies are currently underway to investigate this issue. The X-ray crystallographic structure of a hCA VII mutated form in complex with a classical sulfonamide inhibitor, namely acetazolamide, was also solved. A detailed comparison of the obtained structure with those already reported for other CA isozymes provided novel insights into the catalytic properties of this protein family and offered the basis for a mutagenesis approach aimed at determining the contribution of the active site single residues to the enzyme catalytic efficiency. Moreover, on the basis of the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme-specific CA inhibitors have been obtained. hCA I was one of the first members of the CA family to be identified. Even though the 3D structure of this enzyme was first characterized already in 1975, only few structural studies on complexes formed with different inhibitors have been reported so far. Since these studies are fundamental for the drug design of isoform-selective inhibitors, part of this thesis has been dedicated to the structural characterization of a complex that hCA I forms with topiramate (TPM), which is a molecule of pharmacological interest for the treatment of epilepsy. The analysis of the structure of the complex showed that, upon binding of the inhibitor, the active site of hCA I undergoes a profound reorganization, which has never been observed in any other CA/inhibitor complex and might therefore be useful in designing CA inhibitors. Moreover, the comparison with hCA II/TPM and hCA VA/TPM complex structures, previously investigated, showed that a different H-bond network together with the movement of active site residues to accommodate the inhibitor may account for the difference of inhibition constants of TPM towards different CA isozymes. These data may be helpful in the design of CAIs selective for various isozymes.