Thesis (Ph. D.)--School of Biology, Georgia Institute of Technology, 2005. Directed by Paul Edmonds. / Paul Edmonds, Committee Chair ; Steve Harvey, Committee Member ; Igor Zhulin, Committee Member ; Yury Chernoff, Committee Member ; Mostafa El-Sayed, Committee Member. Includes bibliographical references.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

No description available.

Graduation date: 1994

Thesis (Ph.D.)--McMaster University (Canada), 1991. / Source: Dissertation Abstracts International, Volume: 54-02, Section: B, page: 0835.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2004. / Title from second page of PDF document. Includes bibliographical references.

Tesis por compendio / [EN] About 10 % of all adverse drug reactions are due to allergies, with ß-lactam antibiotics causing the majority of the episodes. Although the actual incidence remains unknown, individuals suspected of being allergic to a drug end up being prescribed with other medications that are less effective, more expensive or harmful. Consequently, a correct diagnosis is key to reduce the derived economic costs and proceed to an adequate 'delabeling' of the population. At present, clinical approaches to diagnose allergies to ß-lactam antibiotics are based on in vivo and in vitro tests. These tests present limited clinical performances since they are invasive, dangerous, and provide false positives and/or negatives. Moreover, the diagnostic sensitivity is far from what is expected, possibly because the epitopes that cause the allergic episodes are still not well detected. In this respect, the preparation of antigens has commonly been determined by the direct attachment of antibiotics to carrier molecules through the formation of an amide bond between amino lysine groups of the carrier molecule and the carboxylate group of the antibiotic. Even so, specific IgE are barely detected with such antigens. This dissertation addresses the synthesis of haptens and the generation of antigens to ß-lactam antibiotics, which develop a more reliable in vitro diagnosis of allergies to these drugs. The evaluation of the antigens has been carried out by means of multiplex in vitro tests based on compact disc technology. This research begins by focusing on the synthesis and preparation of penicillin antigens. To this end, first, the effect of the incorporation of aliphatic spacer arms in the chemical structure of penicillin has been approached, considering the possibility that a better molecular recognition is obtained by moving the hapten away from the carrier protein. Thirteen haptens derived from benzylpenicillin and amoxicillin were synthesized in order to prepare antigens with human serum albumin. The evaluation of the antigens revealed that even though they were immunogenic and were detected by the raised IgG antibodies, they were not detected by specific IgE from allergic patients. Additionally, the next approach considered the cationization of the carrier proteins, human serum albumin and histone. The modification of carboxylate groups of the protein to amino groups allow for an increase of the molar hapten/protein ratio. This strategy led to the generation of five antigens, four of which (only those histone-based antigens), did increase the sensitivity of the assay. Concretely, specific IgE has been determined in sera from allergic patients at low concentrations (LOD = 0.07 IU/mL) with a diagnostic specificity of 100 % and a sensitivity of 60 and 31 % for benzylpenicillin and amoxicillin, respectively. That means a 60 % improvement in the diagnostic sensitivity when compared to the in vitro reference test. Subsequently, the idea of preparing minor antigens based on penicillin metabolites was approached. Penicilloic, penilloic, penicillic, and 6-aminopenicillanic acids, together with penicillamine, were therefore conjugated to the carrier proteins human serum albumin and histone. Except penilloic acid, the rest of antigens were selectively detected when testing a set of serum samples from allergic patients. The diagnostic specificity obtained was 100 %, 94 % in the case of penicillic acid, and the sensitivity was between 67 and 100 %. Another approach was focused on the production of antigens for other families of ß-lactam antibiotics. The generation of antigens for cephalosporins, carbapenems, monobactams or ß-lactam inhibitors is essential, since in vitro tests for the detection of allergies to these antibiotics are not commercially available. Therefore, the results obtained after the preparation of major and minor antigens for the antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem, and aztreonam were evaluated. / [ES] Alrededor del 10 % de las reacciones adversas a medicamentos son debidas a alergias, siendo los antibióticos ß-lactámicos los que más episodios alérgicos ocasionan. Aunque la incidencia real sigue siendo desconocida, los individuos sospechosos de presentar alergia a algún medicamento acaban siendo prescritos con otros medicamentos, menos efectivos, más caros o perjudiciales. Así pues, un correcto diagnóstico resulta clave para disminuir los costes económicos derivados y proceder a un adecuado 'desetiquetado' de la población. En la actualidad, las pruebas de diagnóstico de alergias a antibióticos ß-lactámicos se basan en ensayos in vivo e in vitro. Estos ensayos muestran bajas prestaciones, ya que son invasivos y peligrosos y proporcionan falsos positivos y/o negativos. Además, la sensibilidad diagnóstica está lejos de ser la esperada, posiblemente porque aún no se ha conseguido reconocer todos los epítopos causantes de los episodios alérgicos. En este sentido, la preparación de antígenos se ha basado hasta el momento, en mayor medida, en la unión directa de los antibióticos a las moléculas portadoras mediante la formación de un enlace amida entre los grupos amino de las lisinas de la molécula portadora y el grupo carboxilato del antibiótico. Aun así, las IgE específicas son vagamente detectadas con estos antígenos. En esta tesis se ha abordado la síntesis de haptenos y la generación de determinantes antigénicos a antibióticos ß-lactámicos con los que poder realizar un diagnóstico in vitro más fiable de alergias a estos fármacos. La evaluación de los mismos se ha llevado a cabo mediante ensayos in vitro multiplex basados en tecnología de disco compacto. Esta investigación comienza centrándose en la síntesis y preparación de antígenos de penicilina. Para ello, en una primera fase se ha estudiado el efecto de la incorporación de brazos espaciadores alifáticos en la generación de antígenos, considerando la posibilidad de que se obtenga un mejor reconocimiento molecular al alejar el hapteno de la proteína portadora. Se sintetizaron trece haptenos derivados de bencilpenicilina y amoxicilina con los que se prepararon antígenos con la proteína albumina de suero humano. La evaluación de los antígenos reveló que a pesar de ser suficientemente inmunogénicos y ser reconocidos por anticuerpos IgG de conejo, éstos no fueron reconocidos por IgE específicas de muestras de pacientes alérgicos. Así bien, por otro lado, la estrategia de cationización de las proteínas albumina de suero humano e histona fue abordada teniendo en cuenta que la modificación de grupos carboxilatos de la proteína a grupos amino aumenta la relación molar hapteno/proteína. Esta estrategia permitió la generación de 5 antígenos, 4 de los cuales (los antígenos de histona), esta vez sí, incrementaron la especificidad de la respuesta inmunológica obtenida, reconociendo IgE específicas. Concretamente, se han determinado IgE específicas en suero de pacientes alérgicos a bajas concentraciones (LOD = 0.07 IU/mL) con una especificidad diagnóstica del 100 % y una sensibilidad del 60 y 31 % para bencilpenicilina y amoxicilina, respectivamente, mejorando la sensibilidad un 60 % en comparación con el ensayo in vitro de referencia. A pesar de las mejoras obtenidas con las estrategias llevadas a cabo, se estudiaron otras vías no clásicas para la síntesis de nuevos haptenos con mayor diversidad química. Este enfoque se basa en la generación de antígenos en librerías químicas de compuestos con diversidad estructural para encontrar nuevos haptenos biológicamente activos. Dichas estrategias, hasta el momento, no han sido empleadas para la generación de antígenos y el análisis de muestras de suero de pacientes alérgicos. Con el fin de incorporar diversidad estructural, se sintetizaron, mediante la técnica combinatoria diversity-oriented synthesis, 22 compuestos de los precursores de las penicilinas y cefalosporinas, ácido 6-aminopenicilánico y ácido 7-amino-desacetoxicefalosporánico, respectivamente, y de los antibióticos amoxicilina y ampicilina. Su evaluación con el inmunoensayo in vitro basado en disco compacto ha demostrado que la incorporación de diversidad permite el reconocimiento de epítopos causantes de episodios alérgicos. Concretamente, se observó que estos antígenos eran capaces de detectar anticuerpos tipo IgG e IgE específicos procedentes de suero de conejos inmunizados y de suero humano de pacientes alérgicos, siendo especialmente selectivos los determinantes de amoxicilina y ampicilina. Concretamente, se obtuvo una sensibilidad diagnóstica del 79 % y una especificidad diagnóstica del 100 % / [CA] Al voltant del 10% de les reaccions adverses a medicaments són degudes a al·lèrgies, sent els antibiòtics ß-lactàmics aquells que més episodis al·lèrgics ocasionen. Encara que la incidència real continua sent desconeguda, els individus sospitosos de presentar al·lèrgia a algun medicament acaben sent prescrits amb altres medicaments, menys efectius, més cars o perjudicials. Així doncs, un correcte diagnòstic resulta clau per a disminuir els costos econòmics derivats i procedir a un adequat 'desetiquetatge' de la població. En l'actualitat, les proves de diagnòstic d'al·lèrgies a antibiòtics ß-lactàmics es basen en assaigs in vivo i in vitro. Aquests assaigs mostren baixes prestacions, ja que són invasius i perillosos i proporcionen falsos positius i/o negatius. A més a més, la sensibilitat diagnòstica està lluny de ser l'esperada, possiblement perquè encara no s'ha aconseguit reconéixer tots els epítopes causants dels episodis al·lèrgics. En aquest sentit, la preparació d'antígens s'ha basat fins al moment, en major mesura, en la unió directa dels antibiòtics a les molècules portadores mitjançant la formació d'un enllaç amida entre els grups amino de les lisines de la molècula portadora i el grup carboxilat de l'antibiòtic. Així i tot, les IgE específiques són vagament detectades amb aquests antígens. En aquesta tesi s'ha abordat la síntesi d'haptens i la generació de determinants antigènics a antibiòtics ß-lactàmics amb els quals poder realitzar un diagnòstic in vitro més fiable d'al·lèrgies a aquests fàrmacs. La seua avaluació s'ha dut a terme mitjançant assaigs in vitro multiplex basats en tecnologia de disc compacte. Aquesta investigació comença centrant-se en la síntesi i preparació d'antígens de penicil·lina. Per a això, en una primera fase s'ha estudiat l'efecte de la incorporació de braços espaiadors alifàtics en la generació d'antígens, considerant la possibilitat que s'obtinga un millor reconeixement molecular en allunyar l'haptè de la proteïna portadora. Es van sintetitzar tretze haptens derivats de bencilpenicil·lina i amoxicil·lina amb els quals es van preparar antígens amb la proteïna albúmina de sèrum humà. L'avaluació dels antígens va revelar que malgrat ser prou immunogènics i ser reconeguts per anticossos IgG de conill, aquests no van ser reconeguts per IgE específiques de mostres de pacients al·lèrgics. Així bé, d'altra banda, l'estratègia de cationització de les proteïnes albúmina de sèrum humà i histona va ser abordada tenint en compte que la modificació dels grups carboxilats de la proteïna a grups amino augmenta la relació molar hapten/proteïna. Aquesta estratègia va permetre la generació de 5 antígens, 4 dels quals (els antígens d'histona), aquesta vegada sí, van incrementar l'especificitat de la resposta immunològica obtinguda, reconeixent IgE específiques. Concretament, s'han determinat IgE específiques en sèrum de pacients al·lèrgics a baixes concentracions (LOD = 0.07 IU/mL) amb una especificitat diagnòstica del 100 % i una sensibilitat del 60 i 31 % per a bencilpenicil·lina i amoxicil·lina, respectivament, millorant la sensibilitat un 60 % en comparació amb l'assaig in vitro de referència. Malgrat les millores obtingudes amb les estratègies dutes a terme, es van estudiar altres vies no clàssiques per a la síntesi de nous haptens amb major diversitat química. Aquest enfocament es basa en la generació d'antígens en llibreries químiques de compostos amb diversitat estructural per a trobar nous haptens biològicament actius. Aquestes estratègies, fins al moment, no han sigut emprades per a la generació d'antígens i l'anàlisi de mostres de sèrum de pacients al·lèrgics. Amb la finalitat d'incorporar diversitat estructural, es van sintetitzar, mitjançant la tècnica combinatòria diversity- oriented synthesis, 22 compostos dels precursors de les penicil·lines i cefalosporines, àcid 6-aminopenicilànic i àcid 7-amino-desacetoxicefalosporànic, respectivament, i dels antibiòtics amoxicil·ina i ampicil·lina. La seua avaluació amb l'immunoassaig in vitro basat en disc compacte ha demostrat que la incorporació de diversitat permet el reconeixement d’epítops causants d'episodis al·lèrgics. Concretament, es va observar que aquests antígens eren capaços de detectar anticossos tipus IgG i IgE específics procedents de sèrum de conills immunitzats i de sèrum humà de pacients al·lèrgics, sent especialment selectius els determinants d’amoxicil·lina i ampicil·lina. Concretament, es va obtindre una sensibilitat diagnòstica del 79 % i una especificitat diagnòstica del 100 %. / This work was supported by the H2020 program (project COBIOPHAD, grant agreement No. 688448 awarded to A.M.), being an initiative of the Photonics Public Private Partnership; Agencia Estatal de Investigación Agencia Estatal de Investigación (CTQ2016-75749-R, FEDER) (PID2019-110713RB-I00, FEDER) awarded to S.M.; Generalitat Valenciana (PROMETEO/2020/094 awarded to A.M. & S.M.); program UPV-La FE 2019 (P105 VALBIOAL awarded to S.M & E. I-E.); and the National Institute of General Medical Sciences (GM-1R35GM127045 awarded to S.L.S.). E.P.M. was supported by a FPU fellowship from the Ministerio de Educación, Cultura y Deporte (FPU15/01738 and EST18/00360). B.K.H. was supported by a fellowship from the National Science Foundation (DGE1144152 and DGE1745303). The authors acknowledge the Instituto de Investigación Sanitaria La Fe, Valencia, Spain, which provided the samples from both allergic patients and controls. / Peña Mendizabal, E. (2022). Synthesis and Evaluation of Antigenic Determinants for ß-lactam Allergy Diagnosis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182979 / TESIS / Compendio

The synthesis of p-lactams has been of foremost importance since the discovery of penicillin by Sir Alexander Fleming, in 1928, and its susequent structure elucidation in 1945. Ethyl N-2-bromo-alkylcarbamates show considerable potential as precursors to p- lactams. In the past, p-lactams have been prepared by many methods, none of which have involved 2-3 bond formation. The proposed ring closure using ethyl N-2-bromoalkylcarbamate involves 2-3 bond formation, making this method of synthesis novel. This work describes two attempted methods of cyclisation. The first using a Grignard reagent, and the second, using abstraction of an acidic proton a to a phosphonate group. These methods of intramolecular cyclisation were based on analogous intermolecular additions, which are also described. The second method was also used to determine the general potential of ethyl N-bromo- alkylcarbamtes as precursors to other heterocyclic systems. / Thesis (M.Sc.)-University of Natal, Durban, 2001. / NRF & NRF/DEA & T.

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes are divided into three classes (A, C, and D), while there is only one class of metalloenzymes, class B. Clavulanic acid, sulbactam, and tazobactam are β-lactam-based BLIs that demonstrate activity against class A and C β-lactamases; however, they have no activity against the class A KPC and MBLs, NDM and VIM. Avibactam and vaborbactam are novel BLIs approved in the last two years that have activity against serine carbapenemases (e.g., KPC), but do not inhibit MBLs. The overall goals of this project were to use X-ray crystallography to study the catalytic mechanism of serine β-lactamases with β-lactam antibiotics and to understand the mechanisms behind the broad-spectrum inhibition of class A β-lactamases by avibactam and vaborbactam. This project also set out to find novel inhibitors using molecular docking and FBDD that would simultaneously inactivate serine β-lactamases and MBLs commonly expressed in Gram-negative pathogenic bacteria. The first project involved examining the structural basis for the class A KPC-2 β-lactamase broad-spectrum of activity that includes cephalosporins and carbapenems. Three crystal structures were solved of KPC-2: (1) an apo-structure at 1.15 Å; (2) a complex structure with the hydrolyzed cephalosporin, cefotaxime at 1.45 Å; and (3) a complex structure with the hydrolyzed penem, faropenem at 1.40 Å. These complex structures show how alternative conformations of Ser70 and Lys73 play a role in the product release step. The large and shallow active site of KPC-2 can accommodate a wide variety of β-lactams, including the bulky oxyimino side chain of cefotaxime and also permits the rotation of faropenem’s 6-alpha-1R-hydroxyethyl group to promote carbapenem hydrolysis. Lastly, the complex structures highlight that the catalytic versatility of KPC-2 may expose a potential opportunity for drug discovery. The second project focused on understanding the stability of the BLI, avibactam, against hydrolysis by serine β-lactamases. A 0.83 Å crystal structure of CTX-M-14 bound by avibactam revealed that binding of the inhibitor impedes a critical proton transfer between Glu166 and Lys73. This results in a neutral Glu166 and neutral Lys73. A neutral Glu166 is unable to serve as a general base to activate the catalytic water for the hydrolysis reaction. Overall, this structure suggests that avibactam can influence the protonation state of catalytic residues. The third project centered on vaborbactam, a cyclic boronic acid inhibitor of class A and C β-lactamases, including the serine class A carbapenemase KPC-2. To characterize vaborbactam inhibition, binding kinetic experiments, MIC assays, and mutagenesis studies were performed. A crystal structure of the inhibitor bound to KPC-2 was solved to 1.25 Å. These data revealed that vaborbactam achieves nanomolar potency against KPC-2 due to its covalent and extensive non-covalent interactions with conserved active site residues. Also, a slow off-rate and long drug-target residence time of vaborbactam with KPC-2 strongly correlates with in vitro and in vivo activity. The final project focused on discovering dual action inhibitors targeting serine carbapenemases and MBLs. Performing molecular docking against KPC-2 led to the identification of a compound with a phosphonate-based scaffold. Testing this compound using a nitrocefin assay confirmed that it had micromolar potency against KPC-2. SAR studies were performed on this scaffold, which led to a nanomolar inhibitor against KPC-2. Crystal structures of the inhibitors complexed with KPC-2 revealed interactions with active site residues such as Trp105, Ser130, Thr235, and Thr237, which are all important in ligand binding and catalysis. Interestingly, the phosphonate inhibitors that displayed activity against KPC-2, also displayed activity against the MBLs NDM-1 and VIM-2. Crystal structures of the inhibitors complexed with NDM-1 and VIM-2 showed that the phosphonate group displaces a catalytic hydroxide ion located between the two zinc ions in the active site. Additionally, the compounds form extensive hydrophobic interactions that contribute to their activity against NDM-1 and VIM-2. MIC assays were performed on select inhibitors against clinical isolates of Gram-negative bacteria expressing KPC-2, NDM-1, and VIM-2. One phosphonate inhibitor was able to reduce the MIC of the carbapenem, imipenem 64-fold against a K. pneumoniae strain producing KPC-2. The same phosphonate inhibitor also reduced the MIC of imipenem 4-fold against an E. coli strain producing NDM-1. Unfortunately, no cell-based activity was observed for any of the phosphonate inhibitors when tested against a P. aeruginosa strain producing VIM-2. Ultimately, this project demonstrated the feasibility of developing cross-class BLIs using molecular docking, FBDD, and SAR studies.

Semi-synthetic β-lactam antibiotics are the most prescribed class of antibiotics in the world. Chemical coupling of a β-lactam moiety with an acyl side chain has dominated the industrial production of semi-synthetic β-lactam antibiotics since their discovery in the early 1960s. Enzymatic coupling of a β-lactam moiety with an acyl side chain can be accomplished in a process that is much more environmentally benign but also results in a much lower yield. The goal of the research presented in this dissertation is to improve the enzymatic synthesis of β-lactam antibiotics via reaction engineering, medium engineering and data-drive protein engineering. Reaction engineering was employed to demonstrate that the hydrolysis of penicillin G to produce the β-lactam nucleus 6-aminopenicillanic acid (6-APA), and the synthesis of ampicillin from 6-APA and (R)-phenylglycine methyl ester ((R)-PGME), can be combined in a cascade conversion. In this work, penicillin G acylase (PGA) was utilized to catalyze the hydrolysis step, and PGA and α-amino ester hydrolase (AEH) were both studied to catalyze the synthesis step. Two different reaction configurations and various relative enzyme loadings were studied. Both configurations present a promising alternative to the current two-pot set-up which requires intermittent isolation of the intermediate, 6-APA. Medium engineering is primarily of interest in β-lactam antibiotic synthesis as a means to suppress the undesired primary and secondary hydrolysis reactions. The synthesis of ampicillin from 6-APA and (R)-PGME in the presence of ethylene glycol was chosen for study after a review of the literature. It was discovered that the transesterification product of (R)-PGME and ethylene glycol, (R)-phenylglycine hydroxyethyl ester, is transiently formed during the synthesis reactions. This never reported side reaction has the ability to positively affect yield by re-directing a portion of the consumption of (R)-PGME to an intermediate that could be used to synthesize ampicillin, rather than to an unusable hydrolysis product. Protein engineering was utilized to alter the selectivity of wild-type PGA with respect to the substituent on the alpha carbon of its substrates. Four residues were identified that had altered selectivity toward the desired product, (R)-ampicillin. Furthermore, the (R)-selective variants improved the yield from pure (R)-PGME up to 2-fold and significantly decreased the amount of secondary hydrolysis present in the reactions. Overall, we have expanded the applicability of PGA and AEH for the synthesis of semi-synthetic β-lactam antibiotics. We have shown the two enzymes can be combined in a novel one-pot cascade, which has the potential to eliminate an isolation step in the current manufacturing process. Furthermore, we have shown that the previously reported ex-situ mixed donor synthesis of ampicillin for PGA can also occur in-situ in the presence of a suitable side chain acyl donor and co-solvent. Finally, we have made significant progress towards obtaining a selective PGA that is capable of synthesizing diastereomerically pure semi-synthetic β-lactam antibiotics from racemic substrates.