To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton. The PG synthases that build this structure are called penicillin-binding proteins (PBPs). Since they are the targets of penicillin and related antibiotics, the structures and in vitro biochemical functions of the PBPs have been extensively studied. However, the in vivo functions of the PBPs and the factors they work with to build the PG meshwork remain poorly understood. PBPs work in the context of multicomponent complexes organized by cytoskeletal elements. A major outstanding question has been whether or not these complexes contain factors required for PBP function. I addressed this using Escherichia coli as a model system by taking advantage of the synthetic lethal phenotype resulting from simultaneous inactivation of the major PG synthases: PBP1a and PBP1b. Using a screen for mutants synthetically lethal with the inactivation of PBP1b, I identified LpoA as a factor required for PBP1a function. A colleague in the lab performed the analogous screen for mutants synthetically lethal with the inactivation of PBP1a and identified LpoB as a factor required for PBP1b function. We showed that the Lpo factors are outer membrane lipoproteins that form specific trans-envelope complexes with their cognate PBPs in the inner membrane and that LpoB can stimulate the activity of PBP1b in vitro. Our results reveal unexpected complexity in the control of PBP activity and indicate that they likely receive regulatory input from the outer membrane in addition to cytoskeletal elements in the cytoplasm. To investigate the role of LpoB in morphogenesis further, I took a genetic approach that has identified PBP1b* variants capable of functioning in vivo in the absence of LpoB. Preliminary characterization of these variants indicates that LpoB has cellular functions in addition to PBP1b activation and that LpoB may be important for coordinating the two different catalytic activities of PBP1b. Future study of these mutants is likely to uncover important insights into PBP function and their control by the Lpo factors. These insights may open new avenues for the development of novel therapeutics that target the PBPs.

In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate. / Chemistry and Chemical Biology

The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Histone deacetylase (HDAC) inhibition is an emerging novel therapeutic strategy in cancer therapy. HDAC inhibitors (HDACi) have shown ability to block angiogenesis and cell cycling, as well as initiate differentiation and apoptosis. In fact, suberoylanilide hydroxamic acid (SAHA) is the first in the class of HDACi approved by the FDA for the treatment of cutaneous T cell lymphoma. On the other hand, there is a sustained interest in the use of gold nanoparticles (AuNPs) for various cancer diagnostic and therapeutic applications - bioimaging, drug delivery, and binary therapy techniques such as photodynamic and photothermal therapies. This interest in AuNPs is facilitated by favorable attributes such as ease of fabrication, bioconjugation and biocompatibility, and unique optical and electronic properties. However, HDACi- and AuNPs- based antitumor agents are plagued with problems common to all chemotherapeutic agents such as lack of selectivity, which often results in systemic toxicity. Therefore, availability of a methodology to selectively deliver AuNPs and HDACi to cancer cells will significantly improve their therapeutic indices and lead to the identification of novel agents for use in diagnostic imaging and targeted cancer therapy applications.

Made available in DSpace on 2014-12-17T14:14:05Z (GMT). No. of bitstreams: 1 KattyaGHS.pdf: 407113 bytes, checksum: 7bf14e459b09e873eacee8a4b0b41b74 (MD5) Previous issue date: 2006-08-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Investigations in the field of pharmaceutical analysis and quality control of medicines require analytical procedures with good perfomance characteristics. Calibration is one of the most important steps in chemical analysis, presenting direct relation to parameters such as linearity. This work consisted in the development of a new methodology to obtain calibration curves for drug analysis: the stationary cuvette one. It was compared to the currently used methodology, and possible sources of variation between them were evaluated. The results demonstrated that the proposed technique presented similar reproducibility compared to the traditional methodology. In addition to that, some advantages were observed, such as user-friendliness, cost-effectiveness, accuracy, precision and robustness. Therefore, the stationary cuvette methodology may be considered the best choice to obtain calibration curves for drug analyis by spectrophotometry / As investiga??es no campo do controle de qualidade e da an?lise de f?rmacos requerem procedimentos anal?ticos com boas caracter?sticas de desempenho. A calibra??o ? uma das etapas mais importantes na an?lise qu?mica, apresentando a rela??o direta aos par?metros tais como linearidades. Este trabalho consistiu no desenvolvimento de uma metodologia nova para obter curvas de calibra??o para a an?lise de f?rmaco: a cubeta estacion?ria. Foi comparado ? metodologia atualmente usada, e as fontes poss?veis da varia??o entre elas foram avaliadas. Os resultados demonstraram que a t?cnica proposta apresentou par?metros semelhantes ? metodologia tradicional. Al?m de algumas vantagens como facilidade na execu??o, baixo custo, exatid?o, precis?o e robustez. Conseq?entemente, a metodologia da cubeta estacion?ria pode ser considerada como metodologia de escolha para obter curvas de calibra??o para a an?lise de f?rmacos por espectrofotometria

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.

Orientador: Ronilson Agnaldo Moreno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T19:06:44Z (GMT). No. of bitstreams: 1 Boldrina_LucasWillianLeal_M.pdf: 4238858 bytes, checksum: f936f1b9588d396ee62a90e197d6665d (MD5) Previous issue date: 2011 / Resumo: O objetivo deste estudo foi avaliar a bioequivalência de Fenoximetilpenicilina comprimido (500.000 UI) da Aché Laboratórios S/A, Meracilina formulação teste e Pen-Ve-Oral®, produzido por Eurofarma Laboratórios Ltda., Brasil, em voluntários sadios de ambos os sexos. O estudo foi do tipo aberto, randomizado, cruzado, com 2 tratamentos, 2 seqüências, 2 períodos, com uma semana de intervalo entre as doses, nos quais os voluntários receberam em cada período a formulação teste e a formulação de referência. Uma única dose de cada formulação foi administrada a 26 voluntários sadios. A seqüência de tratamento foi determinada por uma lista de randomização gerada automaticamente pelo sistema SCPCM (Sistema de Controle de Pesquisas Cínicas de Medicamentos). As amostras de plasma foram coletadas num intervalo de 36 horas. As concentrações de Fenoximetilpenicilina foram analisadas por cromatografia líquida de alta eficiência acoplada a um detector UV-visível. A partir da curva da concentração de Fenoximetilpenicilina no plasma vs tempo foram obtidos os parâmetros farmacocinéticos: ASC0-t,, ASC0-inf, e Cmax. As médias geométricas da Meracilina e Pen-Ve-Oral® foram: 99.89% (90% CI = 94,62%; 105,46%) para ASC0-t 99.76 (90% CI = 94,09%; 105,78%) para ASC0-inf 101.11% (98.61% - 103.37% ) para Cmax,. Diante dos resultados encontrados de Cmax e ASC0-t e estando dentro do intervalo de confiança entre 80% e 125% proposto pela Agência Nacional de Vigilância Sanitária (ANVISA) e pelo Food and Drug Administration (FDA), conclui-se que a Meracilina - comprimidos (500.000 UI) é bioequivalente ao Pen-Ve-Oral®, de acordo com sua taxa de extensão e biodisponibilidade / Abstract: This study aimed to compare the bioequivalence between Phenoxymethylpenicillin tablets (500.000 UI), a test formulation Meracilina by Aché Laboratórios S/A, and the Pen-Ve-Oral® tablet formulation elaborated by Eurofarma Laboratórios Ltda., Brazil, in healthy human volunteers of both sexes. The study was carried out by using an open, randomized-crossover design, consisting of a two-period treatment, in which the volunteers received, in each period, the test formulation or the reference formulation, with a seven-day washout interval. A single dose of each formulation was administered to 26 healthy volunteers. The treatment sequence was determined by a randomization list, automatically produced by the Clinical Trial Medicine Control System. Plasma samples were obtained over a 36-hour period. Phenoxymethylpenicillin concentrations were analyzed by high pressure liquid chromatography and UV-visible detection (HPLC-UV). From the Phenoxymethylpenicillin plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained: ASC0-t, ASC0-inf, and Cmax. The mean of Meracilina/Pen-Ve-Oral® 500.000 UI percent geometric mean was 99.89% for AUC0-t, 100.86% for AUC0-? and 101.11% for Cmax. The 90% confidence intervals were 94.62 - 105.46%, 95.22 - 106.83% and 98.61 - 103.87%, respectively. Considering the results of Cmax. and AUC0-t within the confidence interval between 80% and 125% proposed by the Brazilian National Agency for Sanitary Surveillance (Anvisa) and for the US Food and Drug Administration (FDA), it was concluded that Meracilina tablet (500.000UI) by Eurofarma Laboratórios Ltda. is bioequivalent to Pen-Ve-Oral® tablet for both rate and extent of bioavailability / Mestrado / Farmacologia / Mestre em Farmacologia

Self-medication impacts both negatively and positively the health of people, which has become evident during the COVID-19 pandemic. The study aimed to assess the prevalence of self-medicated drugs used for respiratory symptoms, as COVID-19 preventive, for its symptoms or once tested positive. To determine the perception of symptom relief and demographic variables that promote self-medication in Peru. We performed a cross-sectional, analytical, multicenter study in 3792 study respondents on the use, the reason for use, and perception of relief after the use of six drugs during the quarantine period. An online questionnaire was developed, pretested and submitted to the general public. Multivariable logistic regression was used to ascertain factors that influence an individual's desire to self-medicate, associations were considered significant at p < 0.05 and using region (coast, mountain and jungle) as cluster group. The majority of respondents self-medicated with acetaminophen for respiratory symptoms and mainly because they had a cold or flu. It was observed that all the surveyed drugs (acetaminophen, ibuprofen, azithromycin, penicillin, antiretrovirals and hydroxychloroquine) were consumed for various symptoms including: fever, fatigue, cough, sneezing, muscle pain, nasal congestion, sore throat, headache and breathing difficulty. Over 90% of respondents perceived relief of at least one symptom. Multivariable logistic regression showed that older people have a higher frequency of antiretroviral self-medication, respondents who currently have a job had a higher frequency of penicillin self-medication, and that respondents from the Andes consumed less acetaminophen, while the ones from the rainforest consumed it more. There were significant percentages of self-medication, including drugs without sufficient scientific evidence. Age, region where one lived and job status were variables associated with self-medication frequency. Continuous awareness and sensitization about the risks of self-medication are warranted. / Revisión por pares

For a sustainable future, there is a call to increase the market share of bio-based technologies and materials. Microbial-based technologies have the potential and the ability to contribute substantively on many levels to global efforts to achieve sustainability. Development and utilization of microbial technologies is, however, an extensive process involving numerous steps, including the discovery of novel technologies and the development of industrially viable production systems. In the presented thesis, individual steps of microbial biotechnology development were addressed. In the first part of the study, a variety of methodological approaches were employed in order to study the effect of the anthropogenic activity (i.e., decades lasting production of penicillin G) on the structure of soil microbial communities. Moreover, both cultivable and non-cultivable fractions of populations were subjected to functional screening in order to unravel the biotechnological potential of the microorganisms in terms of production of enzymes involved in biotransformation of beta-lactam antibiotics: penicillin G acylase (PGA) and alpha amino acid ester hydrolase (AEH). Our results indicated that the impacted communities harbour a microbial community with increased diversity and richness. However, on the...