La propagation de bactéries à Gram négatif multirésistantes aux antibiotiques représente un problème de santé publique majeur urgent à résoudre car le risque d’un retour à l’ère pré-antibiotique est réel. Parmi les modes de résistance existant, la production de métallo-B-lactamases (MBLs) responsables de l’inactivation des B-lactamines, la famille d’antibiotiques la plus utilisée, représente un challenge thérapeutique.Les travaux décrits dans ce manuscrit concernent la synthèse, la caractérisation et l’évaluation biologique de composés construits autour d’un cœur 2,4-dihydro-3H-1,2,4-triazole-3-thione substitués en deux positions. En se basant sur des études de criblage in silico et des études cristallographiques ayant permis d’identifier ce noyau comme un bon candidat dans le développement d’inhibiteurs de MBLs, la synthèse de différentes séries d’analogues a été entreprise afin d’identifier de nouveaux inhibiteurs pouvant potentiellement atteindre les tests cliniques.Dans un premier temps, une série de composés 4-amino-1,2,4-triazole-3-thione substitués en position 5 a été préparée en suivant des voies de synthèse classiques. Différentes séries ont ensuite été développées en introduisant une diversité structurale et fonctionnelle en position 4. Ces composés ont ensuite été testés sur des enzymes représentatives des 3 sous-classes de MBLs et les plus intéressants ont été évalués sur bactéries résistantes recombinantes.Afin de réaliser une évaluation rapide des produits synthétisés au sein du laboratoire, une méthode de criblage à moyen débit en plaque 96 puits sur cinq MBLs représentatives a été mise au point et validée grâce à l’appui de nos collaborateurs spécialistes des MBLs. / The spread of multiresistant Gram negative bacteria is a growing threat to public health and the risk of return to the pre-antibiotic era is real. Among existing resistance modes, the production of metallo-B-lactamases (MBLs) responsible of the inactivation of B-lactams, the most used family of antibiotics, represents a therapeutical challenge.This manuscript describes the synthesis, characterization and biological evaluation of compounds built on a 2,4-dihydro-3H-1,2,4-triazole-3-thione scaffold substituted on two positions. Based on previous in silico screening and crystallographic studies, which identified this structure as a good candidate for MBLs inhibition, several series have been developed to found new inhibitors that could potentially be amenable to clinical development.First, 1,2,4-triazole-3-thione compounds substituted at position 5 have been prepared following classical pathways. Then, several series have been developed where the structural and functional diversity was introduced at position 4. Compounds have been tested on representative MBLs of the three sub-classes and the most interesting ones on recombinant resistant bacteria.To perform a rapid screening of compounds in the laboratory, a method of medium throughput screening inhibition tests on five MBLs performed in 96-wells plate has also been developed and validated during this study with the help of our collaborators specialists of MBLs

The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform computational molecular docking in our efforts to develop new inhibitors against CTX-M. The goal of this project was to determine the hydrogen bonding network and proton transfer process at different stages of the reaction pathway as well as develop novel inhibitors against CTX-M β-lactamases. The results I have obtained from the project have elucidated the catalytic mechanism of CTX-M β-lactamase in unprecedented detail and facilitated the development of novel inhibitors for antibiotic drug discovery. The first aim of the project focused on developing high affinity inhibitors against class A β-lactamase using a structure-based drug discovery approach, which ultimately led to the identification of CTX-M9 inhibitors with nanomolar affinity. Compound design was based on the initial use of computational molecular docking results along with x-ray crystal structures with known inhibitors bound in the active site. In addition, chemical synthesis was used to build and extend the existing inhibitor scaffold to improve affinity to CTX-M9 and related serine β-lactamases. Through a fragment-based screening approach, we recently identified a novel non-covalent tetrazole-containing inhibitor of CTX-M. Structure-based design was used to improve the potency of the original tetrazole lead compound more than 200-fold with the use of small, targeted structural modifications. A series of compounds were used to probe specific binding hotspots present in CTX-M. The designed compounds represent the first nM-affinity non-covalent inhibitors of a class A β-lactamase. The complex structures of these potent compounds have been solved using high resolution x-ray crystallography at ~ 1.2-1.4 Å, which provides valuable insight about ligand binding and future inhibitor design against class A β-lactamases. Specifically, the first aim of the project was to use ultra-high resolution x-ray crystallography to study β-lactamase catalysis. Through the use of ultra-high resolution x-ray crystallography with non-covalent and covalent inhibitors, I was able to structurally characterize the critical stages of the enzyme mechanism. Here we report a series of ultra-high resolution x-ray crystallographic structures that reveal the proton transfer process for the early stages of the class A β-lactamase catalytic mechanism. The structures obtained include an a 0.89 Å crystal structure of CTX-M β-lactamase in complex with a recently-developed 89 nM non-covalent inhibitor, and a 0.80 Å structure in complex with an acylation transition state boronic acid inhibitor. Nearly all the hydrogen atoms in the active site, including those on the ligand, polar protein side chains and catalytic water, can be identified in the unbiased difference electron density map. Most surprisingly, compared with a previously determined 0.88 Å apo structure determined under the same conditions, the hydrogen-bonding network has undergone a series of reshuffling upon the binding of the non-covalent ligand. Two key catalytic residues, Lys73 and Glu166, appear to have both changed from a charged state to being neutral. Interestingly, structural evidence suggests the presence of a low barrier hydrogen bond (LBHB) shared between Lys73 and Ser70. These unprecedented detailed snapshots offer direct evidence that ligand binding can alter the pKa's of polar protein side chains and their affinities for protons. Such effects can be a common mechanism utilized by enzymes to facilitate the proton transfer process of a reaction pathway. They also have important implications for computational modeling of protein-ligand interactions. Ultra-high resolution x-ray crystallography allowed us to determine the hydrogen atom positions for key active site residues involved in catalysis. As a result, the ability to characterize the hydrogen bonding network led to the determination of the specific proton transfer process that occurs during the reaction stages of the CTX-M β-lactamase mechanism. Overall, the results from this project demonstrate the effectiveness of using ultra high resolution x-ray crystallography as a useful tool to study enzyme catalysis as well as develop and discover novel inhibitors.

<p>Acute otitis media (AOM) is a common disease in childhood and is one of the most common causes for outpatient antibiotic treatment. The major aetiological agents of AOM have varied over the decades. Now the three most common pathogens are <i>Streptococcus pneumoniae</i>, <i>Haemophilus influenzae</i> and <i>Moraxella catarrhalis</i>. The resistance patterns of these organisms have also varied from the beginning of the antibiotic era to the situation we have today with an increasing incidence of penicillin-resistant <i>S. pneumoniae</i> and a moderate to high frequency of beta-lactamase production in <i>H. influenzae</i> and <i>M. catarrhalis</i>. In Sweden we have continued to use the Scandinavian treatment policy of penicillins as the first-line antibiotic treatment of AOM, which has been implemented with good results in the past. The question is if this policy will continue to have acceptable treatment results.</p><p>In order to investigate aspects of treatment, protection and structural changes in AOM, an animal model was used.</p><p>Amoxicillin treatment of AOM caused by <i>H. influenzae</i> was studied. Amoxicillin treatment was shown to shorten the duration of the infection and to reduce the morphological changes normally observed after an untreated AOM. The influence of antibiotic treatment on recurrent AOM was evaluated. Amoxicillin treatment did not lead to less protection against reinfection. Abstaining from antibiotics did not improve the levels of serum IgG antibodies. The IgG levels were significantly higher in treated animals after rechallenge. AOM caused by <i>H</i>. <i>influenzae</i> with a non-beta-lactamase-mediated resistance to beta-lactams was investigated and it was observed that during amoxicillin treatment the chromosomal changes mediating resistance were possibly advantageous for the bacterium. In cultures from children with AOM, there is sometimes growth of several bacteria. The possibility of a sheltering effect of beta-lactamase-producing <i>H. influenzae</i> on a penicillin-sensitive <i>S. pneumoniae</i> in a mixed infection was investigated, and amoxicillin was shown to eradicate the pneumococci from the middle ear despite the presence of beta-lactamase. An increasingly cultured bacterium in nasopharynx and in AOM is <i>M. catarrhalis</i>. It is now beta-lactamase-producing in almost 100% of cases and is thus not eradicated by penicillins. An animal model of AOM caused by beta-lactamase-producing <i>M. catarrhalis</i> was established to study the course of this infection with the possibility of evaluating aspects of virulence between AOM pathogens. The AOM observed was a self-limiting disease.</p><p>The results obtained in this study in a rat model support the continuing use of penicillins as first-line drugs in the treatment of AOM. Penicillins are not sufficient to treat all causative agents, but the majority of pathogens including the most virulent bacteria are eradicated from the middle ear. </p>

Enterobacteriaceae belong to the normal enteric flora in humans and may cause infections. Escherichia coli is the leading urinary tract pathogen with septicaemic potential, whereas Klebsiella pneumoniae causes opportunistic infections and often outbreaks in hospital settings. Beta-lactams are the first choice for treatment of infections caused by Enterobacteriaceae, and might be destroyed by extended-spectrum beta-lactamases, ESBLs. ESBLs hydrolyse all beta-lactams except cephamycin and carbapenems, and constitute a large heterogeneous group of enzymes with different origins. The phenotypic and molecular characteristics of a K. pneumoniae strain causing a major outbreak at Uppsala University Hospital between 2005 and 2008 were described. The strain was multiresistant and produced CTM-M-15, a common ESBL type in Europe. Due to the lack of obvious epidemiological links between patients, a case-control study was performed, which identified risk factors for the acquisition of the outbreak strain in urine cultures. The complex chain of transmission facilitated by patient overcrowding and the interventions applied to curb the outbreak, was revealed in the subsequent study. In the final study, the genetic background of the observed increase in ESBL-producing E. coli isolates during the K. pneumoniae outbreak was explored. The utility of six typing methods in epidemiological investigations of a local outbreak with ESBL-producing E. coli was compared. The increase of ESBL-producing E. coli isolates was not secondary to the K. pneumoniae outbreak. Twentytwo per cent belonged to the epidemic O25b-ST131 clone and only a limited number of infections were caused by nosocomial transmission. ESBL-producing Enterobacteriaceae are a challenge to clinical microbiology laboratories and infection control teams. To investigate their dissemination, typing methods need to be continuously adapted to the current situation. Proper hand disinfection and structural key problems such as over-crowding, under-staffing, lack of single rooms and bathrooms must be adressed to limit transmission.

In this study, the effects of bioprocess design parameters on &amp / #946 / -lactamase production were systematically investigated using wild type Bacillus species. For this purpose, the research programme was carried out in mainly four parts. Initially, potential &amp / #946 / -lactamase producers were screened and Bacillus licheniformis ATCC 25972, a constitutive &amp / #946 / -lactamase producer, was selected. Next, the effects of bioprocess medium components, i.e., carbon sources (glucose, fructose, sucrose, citric acid and glycerol), inorganic nitrogen sources ((NH4)2HPO4 and NH4Cl) and organic nitrogen sources (yeast extract, peptone and casamino acids), were investigated in agitation and heating rate controlled laboratory scale bioreactors. Thereafter, by using the designed medium, the effects of bioprocess operation parameters, i.e., pH and temperature, on &amp / #946 / - lactamase activity were investigated in order to achieve a higher &amp / #946 / -lactamase production. Among the investigated bioprocess conditions, the highest &amp / #946 / - lactamase activity was obtained as 275 U cm-3, in the medium with 10.0 kg m-3 glucose, 1.2 kg m-3 (NH4)2HPO4, 8.0 kg m-3 yeast extract and the salt solution, at pH0=6.0, T=32&deg / C, N=200 min-1, which was 7.9 fold higher than the activity obtained in the reference medium. Finally, using the optimum bioprocess parameters obtained in laboratory scale experiments, the fermentation and oxygen transfer characteristics of the bioprocess were investigated in 3.0 dm3 pilot scale bioreactor, having temperature, pH, foam and stirring rate controls, at Q0/V=0.5 vvm and N=500 min-1 oxygen transfer conditions. The variations in &amp / #946 / - lactamase activity, cell, glucose, amino acid and organic acid concentrations with the cultivation time / the oxygen uptake rate and the liquid phase mass transfer coefficient values were determined. Throughout the bioprocess, overall oxygen transfer coefficient (KLa) varied between 0.008-0.016 s-1 / oxygen uptake rate varied between 0.001-0.003 mol m-3 s-1. Furthermore, rate limiting step analysis was performed / the yield and maintenance coefficients for the bioprocess as well as the kinetic parameters for &amp / #946 / -lactamase were determined.

Acute otitis media (AOM) is a common disease in childhood and is one of the most common causes for outpatient antibiotic treatment. The major aetiological agents of AOM have varied over the decades. Now the three most common pathogens are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. The resistance patterns of these organisms have also varied from the beginning of the antibiotic era to the situation we have today with an increasing incidence of penicillin-resistant S. pneumoniae and a moderate to high frequency of beta-lactamase production in H. influenzae and M. catarrhalis. In Sweden we have continued to use the Scandinavian treatment policy of penicillins as the first-line antibiotic treatment of AOM, which has been implemented with good results in the past. The question is if this policy will continue to have acceptable treatment results. In order to investigate aspects of treatment, protection and structural changes in AOM, an animal model was used. Amoxicillin treatment of AOM caused by H. influenzae was studied. Amoxicillin treatment was shown to shorten the duration of the infection and to reduce the morphological changes normally observed after an untreated AOM. The influence of antibiotic treatment on recurrent AOM was evaluated. Amoxicillin treatment did not lead to less protection against reinfection. Abstaining from antibiotics did not improve the levels of serum IgG antibodies. The IgG levels were significantly higher in treated animals after rechallenge. AOM caused by H. influenzae with a non-beta-lactamase-mediated resistance to beta-lactams was investigated and it was observed that during amoxicillin treatment the chromosomal changes mediating resistance were possibly advantageous for the bacterium. In cultures from children with AOM, there is sometimes growth of several bacteria. The possibility of a sheltering effect of beta-lactamase-producing H. influenzae on a penicillin-sensitive S. pneumoniae in a mixed infection was investigated, and amoxicillin was shown to eradicate the pneumococci from the middle ear despite the presence of beta-lactamase. An increasingly cultured bacterium in nasopharynx and in AOM is M. catarrhalis. It is now beta-lactamase-producing in almost 100% of cases and is thus not eradicated by penicillins. An animal model of AOM caused by beta-lactamase-producing M. catarrhalis was established to study the course of this infection with the possibility of evaluating aspects of virulence between AOM pathogens. The AOM observed was a self-limiting disease. The results obtained in this study in a rat model support the continuing use of penicillins as first-line drugs in the treatment of AOM. Penicillins are not sufficient to treat all causative agents, but the majority of pathogens including the most virulent bacteria are eradicated from the middle ear.

Metalloproteins account for at least half of all known proteins. Metal ions often facilitate chemical that are energetically and/or kinetically challenging. Metal ion-dependent proteins are responsible for a myriad of essential biological functions, including respiration, biosynthesis of essential amino acids, nitrogen fixation, oxygen transport, photosynthesis and metabolisms (e.g. glycolysis and citric acid cycle). Not surprisingly, a growing number of disorders (e.g. various cancers, phenylketonuria, Wilson’s disease) are associated with mutations in metalloenzymes. A general introduction of the importance of metals in biology is presented in chapter 1. This thesis is aimed at obtaining a greater understanding of the structure and function of three metalloenzymes, ketol acid reductoisomerase (KARI), purple acid phosphatase (PAP) and metallo β lactamase (MβL). Chapter 2 examines the structure and dynamics of plant KARI. KARI is an enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. KARI is a binuclear Mg2+ enzyme that catalyses the conversion of 2-acetolactate (AL) into (2R)-2,3-dihydroxy-3-isovalerate or 2-aceto-2-hydroxybutyrate into (2R, 3R)-2,3-dihydroxy-3-methylvalerate in the presence of NADPH. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn2+-(phospho) ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg2+-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate (IpOHA) is a predicted transition-state analog. These studies demonstrate that the enzyme is consisted of two domains, N- domain and C- domain, with the active site at the interface of these domains. In this chapter, the structures of the rice KARI-Mg2+ and rice KARI-Mg2+-NADPH complexes were determined to 1.55 and 2.8 Å resolutions, respectively. Comparisons of all the available plant KARI structures have revealed several major differences. Firstly, the N-domain is rotated up to 15o relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ~ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg2+ complex are disordered. In the rice KARI-Mg2+-NADPH complex and spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures have thus, led to the proposal of an induced-fit mechanism. In this proposed induced-fit mechanism, (i) substrate enters the active site, (ii) active site is closed during catalysis, and (iii) the opening of active site facilitates product release. PAP is also a binuclear metalloenzyme and is capable of utilizing a heterovalent active site to hydrolyse a broad range of phosphomonoester substrates. Chapter 3 examines the catalytic mechanism of PAP based on several new crystal structures. The red kidney bean PAP structure in complex in sulphate was determined to 2.4 Å. This sulphate-bound structure provides insight into the pre-catalytic phase of its reaction cycle. This stucture demonstrates the significance of an extensive hydrogen-bonding network in the second coordination in initial substrate binding and orientation prior to hydrolysis. Most importantly, the two metal ions, Fe3+ and Zn2+, are five-coordinate in this structure, with only one nucleophilic μ-hydroxide present in the metal-bridging position. In combination with kinetic, crystallographic and spectroscopic data, all PAP structures form the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general. To date, no reliable method for producing recombinant PAP at levels suitable for structural biology have been reported. Natural sources are the only way so far to obtaining PAP in a large quantity. Attempts to produce active and recombinant PAP from Mycobacterium marinum using bacterial are found in chapter 4. In brief, in combination with Nus fusion tag, Rosetta (DE3) strain and lower temperature (e.g. 25oC), expression of soluble and mycobacterial PAP becomes possible. However, this soluble protein is non-functional and thus, switching into other expression system (e.g.algal sytem) is the only approach to obtain soluble and functional protein. In algal expression system, human PAP was attempted. Preliminary results indicate that some PAP activity was observed when expressed in algal system. Chapter 5 focuses on the investigation of metallo β lactamase (MβL) from Klebsiella pneumoniae (Kp-MβL). This enzyme requires one or two Zn2+ ions for catalysis. Kinetic properties of Kp-MβL for the hydrolysis of various β-lactam substrates (e.g. benzyl-penicillin, cefoxitin, imipenem and meropenem) were investigated and the role of the metal ions in catalysis was also examined. Kinetic data demonstrate that Klebsiella pneumoniae MβL can degrade a broad spectrum of β-lactam antibiotics, with a high preference for cephems and carbapenems. Kinetic data from pH dependence studies has revealed that catalysis of benzyl-penicillin and meropenem is preferred at acidic pH. The kcat vs pH profile demonstrates that catalysis is enhanced by protonation, thus it is likely that the relevant group is responsible for the donation of a proton to the product or leaving group. In this case, a doubly Lewis activated, bridging hydroxide molecule has been speculated. A single protonation event (pKa ~7) is also observed in kcat/Km vs pH profile. Since benzyl-penicillin does not have an acidic moiety in this pH range, this event is likely to be associated with the free enzyme. His 79 and 139 have been speculated to enhance substrate binding. In contrast, catalysis of both cefoxitin and imipenem is favoured at alkaline pH, leading to the proposal that a terminally bound water is likely to form a nucleophile. A bell-shaped pH profile for kcat/Km is observed for cefoxitin and imipenem substrates. pKa of ~ 9-9.5 is likely to be associated with Lys161, which enhances substrate binding. In Chapter 6, a novel MβL from Serratia proteamaculans (Spr-MβL) is investigated. This chapter includes expression, purification and preliminary characterization of this MβL using steady-state kinetics. Expression of this enzyme in Rosetta (DE3) plysS E. coli strain yields only a small amount of soluble enzyme (1 mg/ 6 L culture). To improve the amount of soluble protein, Spr-MβL was subjected to several rounds of in vitro evolution. About two-fold gain in solubility was achieved by this method along with a five-fold increase in β-lactamase activity. Further rounds of directed evolution are now planned. The kinetic behaviour for Spr-MβL-catalysed the hydrolysis of three β-lactam substrates, penicillin, cefoxitin and imipenem were also studied. Kinetic data suggest that a water molecule bridging the two Zn2+ ions is the likely nucleophile in the reaction with penicillin while the reaction-initiating nucleophile is likely to be a terminally bound hydroxide in the reaction with cephalothin and imipenem (Chapter 6). In summary, this project has led to a better understanding of the structures of KARI and PAP prior to catalysis. This project has also aided in the understanding of catalytic mechanism of MβLs and the role the metal ions play. The knowledge gained will facilitate the development of new chemotherapeutics and herbicides.

In the first part of my thesis I focus on mapping problems associated with antibiotic therapy and subsequent development of antibiotic resistance. Tracking resistance is based primarily on data collection and evaluation of the results set sensitivity from around the world. Antibiotic resistance is a natural phenomenon that can be observed in the evolution of microbes as one of the mechanisms of adaptation to new conditions in the environment. For this work I have chosen the following research questions. Do the incidence of ESBL strains in the České Budějovice Hospital a.s. increase over time? Are these values comparable to those achieved in another region, namely in Moravian hospitals the University Hospital of Olomouc, Ostrava University Hospital and Regional University Hospital of T. Bata in Zlin? The data collection I made in collaboration with the laboratory technicians and doctors at Hospital?s Bacteriology Laboratory in České Budějovice. Bacteries tested for the detection of ESBL production originated from biological materials, witch came from patients of hospital in České Budějovice. The first objective was to compare the results achieved in the České Budějovice Hospital in the period of 2007 to 2012. If we look at the total number of ESBL strains that have been isolated since 2007, values have upward trend. While in 2007 there were only 64 strains a year later, the number more than doubled. In 2010, the value soared to 281 tribes and in the year 2012, the number was 321 tribes. The incidence of ESBL strains in 2007 increased about five times. In the long term we can say the numbers have increasing tendency and the range of each species in the production of ESBL has significantly changed. In 2007, it was K. pneumoniae strains that dominated the statistics, but over time the strains of E. coli came forefront. Values of 2012 suggest that the presence of ESBL strains of K. pneumoniae is again almost equal to the number of E. coli strains. The second objective was to compare the results of the 2012 with study of the Prevalence of ESBL-positive Enterobacteriaceae in large Moravian hospitals. In the general overview of ESBL producers values in Hospital České Budějovice (5.23%) are comparable to those in Ostrava (4.9%) and in Zlín (4.3%). Number of strains in the Hospital in Olomouc (11.8%) is about twice as high as the numbers in České Budějovice. In this comparison the České Budějovice Hospital is one of the hospitals with a lower incidence of ESBL producers. The České Budějovice Hospital is below the national average, which originate from an elaborate system of care for patients with colonization or infection with ESBL strains, and from therapy control system using antibiotic center. These results may serve to the Hospital in České Budějovice for statistical purposes, and also for proposals for improving patient care. In the discussion, I pointed out the danger of the spread of resistant strains of bacteria in the community and also the associated risks that mentioned bacteria mean for patients injured in mass accidents or disasters. In these cases, number of infections including ESBL producers can penetrate through open wounds into the affected body. Unlike conventional sensitive bacteria those strains are resistant to commonly used antibiotics and thereby endanger the lives of people affected by the accident.

Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, inhibitors have with the binding pocket, optimization efforts can be focused on maximizing interactions with “hot spots” within the pocket, thus leading to larger, high affinity inhibitors. A similar optimization technique can also be applied to known inhibitors. By examining the interactions of a known inhibitor with the binding site, new compounds can be designed to target “hot spots” in the binding pocket using the known inhibitors core structure as a starting point. The affinity of the newly designed compounds can then be compared to the affinity of the original inhibitor, and further rounds of optimization can be carried out. While simple in design, there are many challenges associated with structure based drug design studies, and there is no guarantee novel inhibitors will be found, but ultimately, it is an extremely powerful methodology that results in a much higher hit rate than other, similar, techniques. The work herein describes the use of structure based drug design to target several different proteins involved in bacterial antibiotic resistance, and a protein that has been implicated in the development of Alzheimer’s disease. The goal of the first project was to design a new PBP inhibitor based upon an existing scaffold, and to better understand the binding mechanism and molecular interactions between penicillin binding proteins and their inhibitors. PBPs are a group of proteins that catalyze the last steps of bacterial cell wall formation, and are the targets of the β-lactam antibiotics. Two compounds were designed which conjugated a ferrocene or ruthenocene group to 6-aminopenicillinic acid, and their antibiotic properties were tested against a range of bacterial strains. To get a better understanding of how the 6-APA organometallic compounds interacted with the PBP active site, a CTX-M-14 β-lactamase model system was used for X-ray crystallographic studies. CTX-M-14 was chosen as its active site shares many key catalytic features with PBPs, and it easily, and reproducibly, yields crystals capable of diffracting to sub-atomic (< 1.0 Å) resolution. I determined a 1.18 Å structure of 6-APA-Ru in complex with CTX-M-14 E166A β-lactamase and was able to gain unprecedented details of the interactions of the ruthenocene group with the CTX-M active site. This structure also revealed that the compound bound in the CTX-M active site was actually the decarboxylated and hydrolyzed product, which was the first time a decarboxylated product had been captured in the CTX-M active site. A second, 0.85 Å, structure of CTX-M in complex with 6-APA-Ru was determined and shed light on how the hydrogen bonding network in the CTX-M active site changes in response to the 6-APA-Ru product binding. A final, 1.30 Å, structure captured the carboxylated and hydrolyzed 6-APA-Ru product in complex with CTX-M, which was the first time the carboxylated product had been captured in the CTX-M active with the catalytic Ser70 residue intact. The results show the potential of the ruthenocene group in improving antibiotic potency, and help to better elucidate the changes that occur in the CTX-M active site upon inhibitor binding, while at the same time, telling us what changes could occur in the active site of PBPs. The next project was focused on novel inhibitor discovery against several different PBPs. PBPs have been successfully inhibited by β-lactam antibiotics for decades, but the alarming rise of bacteria resistant to these antibiotics has placed increased urgency on the discovery of novel PBP inhibitors. A fragment based molecular docking approach was employed to virtually screen millions of small compounds for interactions with the targeted active sites, and then high scoring compounds were selected for visual inspection and inhibitory testing. Virtual screening was first done against Staphylococcus aureus monofunctional transglycosylase, a type of PBP. MTG provided a good binding pocket for virtual screening, but proved challenging to purify and crystallize. However, through great effort MTG crystals were eventually obtained. After repeated rounds of virtual screening against MTG, multiple compounds were selected for inhibition testing, and testing is currently ongoing. Virtual screening was also done against Pseudomonas aeruginosa PBP5 and PBP1a. Purification and crystallization of these proteins proved to be easier than MTG, and both yielded diffraction quality crystals. The final project focused on virtual screening against a protein implicated in the development of Alzheimer’s disease, Slingshot Phosphatase 1. The brains of AD patients have been found to contain elevated levels of active Cofilin, and these elevated levels of active Cofilin may lead to the overproduction of amyloid β. Aβ overproduction, and its resulting accumulation, is believed to be one of the pathways that lead to AD symptoms. Cofilin is activated when it is dephosphorylated by SSH1, and inhibiting this activation may decrease the production of Aβ and the development of AD symptoms. There is no known structure of SSH1, so to perform virtual screening a SSH1 homology model was constructed using the homolog SSH2 as a starting point. Virtual screening was then performed using the SSH1 homology model and many compounds were selected for inhibition testing. Initial testing found several compounds that could prevent Cofilin dephosphorylation at levels > 10μM. However, three compounds were found to be exceptionally active, and could prevent Cofilin dephosphorylation at both 1 and 10 μM. One of these three compounds was tested directly against purified SSH1 and found to inhibit its activity, and reduce Aβ production. Crystallization of purified SSH1, and SSH2, was attempted in order to get complex structures with the three best compounds. SSH2 crystals were obtained which diffracted to 1.91 Å, and several initial hits were found for SSH1. Optimization of crystals for both proteins is currently ongoing. The SSH1 inhibitor, along with the two other highly active compounds, provides an excellent starting point for the development of highly potent SSH1 inhibitors.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa multiresistant are responsible for high morbidity and mortality, failure of drug therapy, increased hospital stay and consequently the financial impact on the health system. However, while the occurrence of these bacteria to configure a public health problem, numerous studies reveal that it is scarce information about the resistance genes present in multi-drug resistant bacteria. This reality associated with the negative impact of these on society, justifies the importance of detecting the resistance genes of A. baumannii and P. aeruginosa multiresistant and clinically characterize patients from a public hospital in Sergipe. It is an analytical prospective cohort study and a quantitative approach. The collection of clinical data of patients was carried out through a specifically designed form. Strains of A. baumannii were subjected to PCR for identification of resistance genes (blaIMP, blaVIM, blaSIM, bla OXA-51, blaOXA-58, blaOXA-23 and blaOXA-24) and P. aeruginosa the blaSPM genes, blaVIM, blaIMP, blaKPC. Descriptive analyzes were performed, the Chi-square and Fisher exact tests, with 5% significance level. The software used was R version 3.1.2. The sample consisted of 119 patients. Of the 43 patients with P. aeruginosa isolates, 33 were male (76.7%) with mean age of 46.2 years. Twenty-eight were admitted to the ICU (65.1%) and 13 (30.2%) diagnosed with head trauma (TBI). Of the 76 patients with isolates of A. baumannii, 59 (77.6%) were male, mean age of 44.4 years. Fifty patients (65.8%) were from the ICU and 18 (23.7%) diagnosed with TBI. The median number of days of hospitalization was statistically significant between bacteria. Among the isolation sites, there is urine to P. aeruginosa, with 16 samples (37.2%) and tracheal aspirate for A. baumannii with 32 (42.1%) strains. A urinary catheter was the most used device in patients with isolates of A. baumannii (93.4% - 71) and the central venous catheter in patients with P. aeruginosa (93% - 40). All patients with P. aeruginosa isolates made use of carbapenems and 98.6% (75) of A. baumannii. It found statistically significant differences between bacteria in the use of aminoglycosides, 3rd generation cephalosporins and tigecycline. In P. aeruginosa was no significant difference in the use of oxacillin and cephalosporins of 1st and 3rd generations and polymyxin in the various sectors of the hospital. All the samples of A. baumannii and P. aeruginosa were susceptible to colistin, ranging between MIC <= 0.5 and 2. The majority (55.8% - 24) patients with P. aeruginosa and A. baumannii (52.6% - 40) died. Among the 76 strains of A. baumannii, 56 (73.6%) had concomitant both blaOXA-51 and blaOXA-23 genes. Among the 43 strains of P. aeruginosa, 28 (65.1%) had the blaSPM gene. It was concluded that the A.baumannii was more frequent than P. aeruginosa. There was a significant predominance of Pseudomonas and Acinetobacter in the urine in tracheal aspirates. Carbapenems was widely used throughout the hospital stay of patients with Acinetobacter and Pseudomonas isolates in different hospital departments. Most Pseudomonas strains showed blaSPM resistance gene and Acinetobacter blaOXA-23 genes and blaOXA-51 concurrently. The mortality of patients with Acinetobacter and Pseudomonas was greater than 50%. / As infecções causadas por Acinetobacter baumannii e Pseudomonas aeruginosa multirresistentes são responsáveis pela alta morbidade e mortalidade, falência da terapia medicamentosa, aumento do período de internação e consequentemente impacto financeiro no sistema de saúde. Todavia, embora a ocorrência destas bactérias se configure um problema de saúde pública, inúmeros estudos revelam que é escasso o conhecimento acerca dos genes de resistência presentes nas bactérias multirresistentes. Essa realidade associada ao impacto negativo destas na sociedade, justifica a importância de detectar os genes de resistência de A. baumannii e P. aeruginosa multirresistentes e caracterizar clinicamente os pacientes de um hospital público de Sergipe. Trata-se de um estudo analítico de coorte prospectiva e abordagem quantitativa. A coleta dos dados clínicos dos pacientes foi realizada através de um formulário especificamente elaborado. As cepas de A. baumannii foram submetidas à técnica PCR para identificação dos genes de resistência (blaIMP, blaVIM, blaSIM, bla OXA-51, blaOXA-58, blaOXA-23 e blaOXA-24) e em P. aeruginosa os genes blaSPM, blaVIM, blaIMP, blaKPC. Foram realizadas análises descritivas, os testes de Qui-Quadrado e Exato de Fisher, com nível de significância de 5%. O software utilizado foi o R versão 3.1.2. A amostra foi constituída de 119 pacientes. Dos 43 pacientes com isolados de P.aeruginosa, 33 eram do sexo masculino (76,7%), com idade média de 46,2 anos. Vinte e oito estavam internados na UTI (65,1%) e 13 (30,2%) com diagnóstico de trauma crânio encefálico (TCE). Dos 76 pacientes com isolados de A. baumannii, 59 (77,6%) era do sexo masculino, média de idade de 44,4 anos. Cinquenta pacientes (65,8%) eram procedentes da UTI e 18 (23,7%) com diagnóstico de TCE. A mediana de dias de internamento foi estatisticamente significante entre as bactérias. Dentre os sítios de isolamento, destaca-se a urina para P. aeruginosa, com 16 amostras (37,2%) e o aspirado traqueal para A. baumannii com 32 (42,1%) cepas. A sonda vesical foi o dispositivo mais usado nos pacientes com isolados de A. baumannii (93,4% - 71) e o cateter venoso central nos pacientes com P. aeruginosa (93% - 40). Todos os pacientes com isolados de P.aeruginosa fizeram uso dos carbapenêmicos e 98,6% (75) dos A. baumannii. Foi encontrado diferença estatisticamente significante entre as bactérias quanto ao uso dos aminoglicosídeos, cefalosporinas de 3ª geração e tigeciclina. Em P. aeruginosa houve diferença significativa no uso da oxacilina e cefalosporinas de 1ª e 3ª gerações e polimixina nos diversos setores do hospital. Todos as amostras de A. baumannii e P. aeruginosa apresentaram sensibilidade à colistina, com variação da MIC entre < = 0,5 e 2. A maioria (55,8% - 24) dos pacientes com P. aeruginosa e A. baumannii (52,6% - 40) foram a óbito. Dentre as 76 cepas de A. baumannii, 56 (73,6%) apresentaram concomitantemente os dois genes blaOXA-51 e blaOXA-23. Dentre as 43 cepas de P. aeruginosa, 28 (65,1%) apresentaram o gene blaSPM. Concluiu-se que o A.baumannii foi mais frequente do que a P.aeruginosa. Houve predomínio significante da Pseudomonas na urina e do Acinetobacter na secreção traqueal. Os carbapenêmicos foi amplamente utilizado ao longo da internação dos pacientes com isolados de Acinetobacter e Pseudomonas nos diversos setores do hospital. A maioria das cepas de Pseudomonas apresentaram gene de resistência blaSPM e Acinetobacter os genes blaOXA-23 e blaOXA-51 concomitantemente. A mortalidade dos pacientes com Acinetobacter e Pseudomonas foi superior a 50%.