Diidroorotato desidrogenases (DHODHs) catalisam a quarta e única reação de oxidorredução na via de síntese de novo de nucleotídeos de pirimidina, que envolve a oxidação do diidroorotato (DHO) a orotato (ORO) em um mecanismo dependente do cofator FMN e de aceptores de elétrons. A relevância de pirimidinas para a proliferação e manutenção celular determina a DHODH como um potencial alvo terapêutico. DHODHs são divididas em duas classes baseado em comparações estruturais e na sequência de resíduos: classe 1, a qual é subdividida em classe 1A e 1B, e classe 2. A proteína utilizada como modelo em nosso estudo sobre as DHODHs de tripanosomatídeos classe 1A é a enzima de Leishmania major (LmDHODH). As metas principais desse trabalho foram de contribuir para a compreensão sobre o mecanismo de ação das DHODHs de classe 1A e propor estratégias de inibição seletiva. Inicialmente, ensaios no estado estacionário e pré-estacionário foram realizados para caracterização do ciclo catalítico da LmDHODH e permitiram a análise das etapas redutivas e oxidativa de forma independente e sua comparação com o ciclo global. Foi também analisada a interação da LmDHODH com os produtos da reação por meio de ensaios de cinética, titulação espectral e titulação isotérmica calorimétrica. Após análises topológicas baseadas na estrutura cristalográfica da LmDHODH, diferentes mutantes foram construídos e caracterizados estruturalmente e cineticamente a fim de investigar a importância de alguns sítios na conformação protéica. Durante a etapa de busca de inibidores, bibliotecas químicas de compostos foram analisadas por meio de interferometria de biocamada e ensaio enzimático. Considerando os resultados obtidos, foi possível pela primeira vez identificar um mecanismo cinético cooperativamente positivo frente à ligação do DHO com coeficiente de Hill (h) próximo a 2. Além disso, nossos resultados mostram que o grau da cooperatividade na ligação do DHO é afetado pelo ORO. A comparação entre os parâmetros obtidos para o estado estacionário e pré-estacionário aliada aos estudos da interação da LmDHODH com os produtos da reação, sugerem que a liberação do ORO é a etapa limitante na velocidade global da reação, o que não é verdade para a enzima humana (DHODH classe 2). A partir das análises dos mutantes, foi possível correlacionar o comportamento cooperativo da enzima LmDHODH com a conversa cruzada entre os sítios-ativos mediada pela interface dimérica. Além disso, foi possível observar que a dinâmica conformacional do loop catalítico é extremamente relevante no mecanismo global da reação e existe uma correlação entre os rearranjos do loop catalítico e a interface dimérica. Nossos resultados fornecem uma importante contribuição para o entendimento dos detalhes do mecanismo catalítico adotado pelas DHODHs de classe 1A. Eles também revelam diferenças chave no mecanismo adotado pelas enzimas de classe 1A e classe 2, os quais podem ser explorados no desenvolvimento de inibidores potentes e seletivos contra a LmDHODH. No que se refere à busca por inibidores, alguns hits foram obtidos por meio da varredura da biblioteca de fragmentos do Drug Discovery Unit (DDU) utilizando interferometria de biocamadas. Além disso, um ensaio enzimático de larga escala (hightroughtput) foi desenvolvido para a LmDHODH e permitiu a triagem de alto desempenho nas bibliotecas de fragmentos e diversidade química. Hits foram validados por meio de ensaio de potência e bons inibidores foram encontrados em ambas as bibliotecas. As varreduras identificaram diferentes séries de compostos como pontos de partida para química medicinal e investigações estruturais. Dois hits identificados a partir da biblioteca de diversidade química apresentam excelentes valores de IC50, 260 nM e 1,07 ?M. / Dihydroorotate dehydrogenases (DHODHs) catalyze the fourth and only redox reaction in the de novo pyrimidine biosynthetic pathway, the oxidation of dihydroorotate (DHO) to orotate (ORO) through a mechanism dependent on the FMN cofactor and electron acceptors. The significance of pyrimidine for cell proliferation and maintenance determine DHODH as potential therapeutic target. DHODHs have been distinguished in two major classes based on sequence and structural comparisons: class 1, which is further divided into subclasses 1A and 1B, and class 2. The model protein in our studies on trypanosomatid class 1A DHODHs is the enzyme from Leishmania major (LmDHODH). The essential question in this study was to contribute for the understanding about the catalytic and inhibition mechanisms of class 1A DHODHs . First, steady-state and pre-steady state kinetic approaches have been used to characterize the catalytic cycle of LmDHODH and have allowed the comparison between the global cycle and each independent half-reaction. Also, the interaction of LmDHODH with the products of the reaction was analyzed by kinetic, spectral titration and isothermal titration calorimetry assays. After topographical analysis based on the LmDHODH crystallographic structure, different mutants were constructed and structurally and kinetically characterized. For inhibitor search, chemical libraries of compounds were screened by using biolayer interferometry binding and enzymatic assays. Based on our results, for the first time, steady-state kinetics analysis of class 1A allowed the observation of a positive cooperativity for DHO binding with a Hill coefficient (h) value close to 2. Moreover, our results show that the degree of cooperativity in DHO binding is affected by ORO. Comparison between steady and pre-steady-state parameters together with studies of interaction for LmDHODH with both products, suggests that ORO release is the rate-limiting step in overall catalysis with is not true to the human enzyme (class 2 DHODH). The hybrid approach used to characterize LmDHODH mutants allowed us to propose that the cooperative behavior is related with the cross-talk between the two active sites mediated by the dimer interface. Also, the conformational dynamics of the catalytic loop is very relevant in the global mechanism of the reaction and there is a correlation between catalytic loop rearrangement and dimer interface. Our results bring an important contribution for a better understanding of the detailed catalytic mechanism adopted by class 1A DHODHs. They also reveal key differences in the enzymatic mechanism adopted by class 1A and class 2 DHODHs, to be exploited in the development of potent and selective LmDHODH inhibitors. Regarding to inhibitors search, few hits were obtained by using screening of Drug Discovery Unit (DDU) fragment library with biolayer interferometry binding assay. Also, an end-point enzymatic assay was developed for LmDHODH and the optimisation carried out at the DDU provided a reliable HTP assay using both DDU\'s fragment library and small diversity set. Hits were validated using a potency assay with good inhibitors found using both libraries. The screens identified a number of series as starting points for medicinal chemistry and structural investigations. Two hits identified from small diversity set exhibit great IC50 values, 260 nM and 1.07 ?M.

O presente projeto representa uma extensão dos paradigmas que criamos com estudos de simulação molecular para compreender as frequentes mudanças de estrutura e função das Aldeído Desidrogenases (ALDHs) durante a evolução. As ALDHs formam uma superfamília de proteínas que catalisam a oxidação de vários aldeídos, mas as origens evolutivas das preferências pelos seus substratos são pouco conhecidas. Apesar de possuírem uma elevada identidade sequencial, duas destas ALDHs, a ALDH1 e a ALDH2, exibem distintos papéis funcionais de sinalização celular e detoxicação, respectivamente. Através de prévia análise computacional e logenética, identicamos que, curiosamente, as ALDH1s de organismos invertebrados, Branchiostoma oridae e Ciona intestinalis apresentam características estruturais mais semelhantes às de suas ALDH2s do que das ALDH1 típicas. Isto sugere que essas ALDH1s divergentes podem ter evoluído na direção da atividade de degradação de aldeídos pequenos e tóxicos, que parecem representar a função ancestral das ALDH2s eucarióticas. Nossa análise identicou três assinaturas de aminoácidos localizadas na área interna do canal de entrada do substrato (CES) que distinguem as ALDH1 das ALDH2. Desta forma, constatamos que as ALDH1s possuem um CES amplo e desobstruído, consistente com o fato destas enzimas catalisarem aldeídos de cadeia longa como o retinaldeído, que é um precursor de vias de sinalização por retinóides. Em contraste, as ALDH2s possuem o CES pouco volumoso e constrito, consistente com sua função na degradação de pequenos aldeídos tóxicos e reativos, como o acetaldeído. Neste projeto o nosso objetivo é analisar a correlação funcional e estrutural entre as ALDH1 e ALDH2 presentes em B. oridae e C. intestinalis para desvendar e compreender seus papéis funcionais e evolutivos em cordados. Especicamente, testaremos a hipótese de que as três assinaturas descritas constituem o núcleo fundamental da preferência por substratos, e que sua inversão por mutações sitio-dirigidas entre ALDH1 e ALDH2 modicará a preferência de substrato de acordo com a origem da assinatura. Se conrmado experimentalmente, este será um exemplo pioneiro de reversão evolutiva molecular, que terá impacto direto sobre as interpretações atuais sobre controversa lei de Dollo da irreversibilidade evolutiva. / This project represents an extension of the paradigms that we created from our molecular simulation studies to understand the frequent structure and function changes of aldehyde dehydrogenases (ALDH) during evolution. The ALDHs form a superfamily of proteins that catalyze the oxidation of several aldehydes, but the evolutionary origins of their substrate preference are unknown. Despite having a high sequence identity, two of these ALDHs, the ADLH1, and ALDH2, exhibit distinct functional roles of cellular signaling and detoxication, respectively. Through previous computational and phylogenetic analysis, we found that, interestingly, the ALDH1s of invertebrate organisms (Branchiostoma oridaeand Ciona intestinalis) show structural features more similar to their ALDH2s than the typical ALDH1. It suggests that these divergent ALDH1sevolved to provide small aldehydes detoxication pattern, what seems to represent the ancestral eukaryotic ALDH2s function. Our analysis also identied three aminoacidsignatures, located internally in the substrate entry channel (SEC), that distinguishes the ALDH1 from ALDH2. Thus, we nd that ALDH1s have a wide, open and unobstructed SEC, consistent with the fact that these enzymes catalyze bulky long-chainaldehydes, like retinaldehyde, a precursor of important signaling pathways. In contrast, the ALDH2s have a small and constricted SEC, consistent with the degradation function of small aldehydes, like the toxic metabolite acetaldehyde. In this project, our objective is to understand the functional and structural correlation between ALDH1 and ALDH2 found in B. oridaeand C. intestinalisto discover and comprehend their functional and evolutionary roles in chordates. Specically, we will test the hypothesis that the three signatures described above are the fundamental core of the substrate preference, based on the signature origin. If conrmed experimentally, this will be a pioneeringexample of evolutionary molecular reversion, impacting directly on the current interpretations of the controversial Dollos Law of Irreversibility.

Diante da biodiversidade de micro-organismos existentes na natureza e da necessidade de descobrir novos biocatalisadores para a síntese de bloco de construções quirais e de produtos químicos de alto valor agregado, o presente trabalho teve como objetivo realizar a bioprospeção de micro-organismos para aplicação em reações de oxidação enantiosseletiva de álcoois. Os micro-organismos foram obtidos pelo processo de isolamento induzido de amostras de solo/sedimento coletadas no Parque Estadual Turístico do Alto Ribeira (PETAR) e na Antártica. A partir das amostras coletadas (43 de solo e 13 de sedimento) foram isolados 130 microorganimos mesofílicos (isolados de amostras de solo do PETAR) e 232 microorganimos psicrofílicos/psicrotróficos (isolados de amostras de solo/sedimento da Antártica). Realizamos também a avaliação do espectro de atividade enzimática dos micro-organismos em reações de oxidação enantiosseletiva, e para isso empregamos derivados para substituídos do (R,S)-1-(fenil)etanol. Dentre os micro-organismos estudados, 15 psicrofílicos/psicrotróficos e 11 micro-organismos mesofílicos apresentaram álcool desidrogenases que catalisaram a oxidação do enantiômero (S) do álcool racêmico à sua correspondente cetona. Nesses casos, as linhagens possuem atividade de álcool desidrogenase em que o rendimento de obtenção da cetona foi > 10 % e excesso enantiomérico > 60 %. Por outro lado 6 micro-organismos mesofílicos apresentaram álcool desidrogenases que catalisam a oxidação do enantiômero (R) do álcool racêmico à sua correspondente cetona. Foi realizada a caracterização taxonômica para alguns micro-organismos através do sequenciamento do 16S rDNA. Dentre os micro-organismos caracterizados, destacaram-se as bactérias Flavobacterium sp., Arthrobacter sp., Acinetobacter sp., e o Bacillus sp. por apresentar excelente atividade enzimática. Flavobacterium sp. e a Arthrobacter sp. foram selecionadas para o estudo de otimização da reação de oxidação do (R,S)-1-(4- metilfenil)etanol. Nesse processo foram utilizadas as células ressuspensas em tampão fosfato em diferentes temperaturas (5-30 ºC) e tempos reacionais (24-72 horas). A partir desse estudo, observou-se que a Flavobacterium sp. possui uma excelente atividade enzimática a 10 ºC e a Arthrobacter sp. a 25 ºC. Foram determinadas curvas de crescimentos destas bactérias em 15, 20 e 25 ºC, em que ambas apresentaram crescimento ótimo a 25 ºC, indicando que estas bactérias são psicrotróficas / In the view of the microorganisms biodiversity in nature and the necessity to discover new biocatalysts for chiral synthesis of building blocks and high value-added chemical products, the present work was aimed at making the bioprospection of microorganisms for application in enantioselective oxidation reactions of alcohols. Microorganisms were isolated by enrichment technique from soil/sediment samples collected from the State Park of Alto Ribeira (PETAR) and Antarctic. 130 Mesophilic microorganisms were isolated from soil samples of PETAR, and 232 psychrophilic/psychrotrophic microorganisms from soil/sediment samples of Antarctic. We also evaluated the enzymatic activity of the microorganisms in enantioselective oxidation reactions, by using derivatives of substituted para (RS)-1- phenylethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotrophic and 11 mesophilic strains contain alcohol dehydrogenases that catalyze the (S)-enantiomer oxidation of racemic alcohols to its corresponding ketone. In these cases, the strains showed alcohol dehydrogenase activity in which the ketone yields were higher than 10 % and the enantiomeric excess >60 %. Moreover, 6 mesophilic microorganisms showed alcohol dehydrogenases that catalyze the R)-enantiomer oxidation of racemic alcohols to its corresponding ketone. It was performed taxonomic characterization for some microorganisms by sequencing the 16S rDNA. Among the characterized microorganisms, Flavobacterium sp., Arthrobacter sp., Acinetobacter sp. and Bacillus sp. showed excellent enzymatic activity. The Flavobacterium sp. and Arthrobacter sp. were selected for optimization study of oxidation of the (R,S)-1-(4-methyl-phenyl)ethanol. In this process, bacterial cells were resuspended in phosphate buffer at different temperatures (5-30 °C) and reaction times (24-72 h). From these studies, it was observed that the Flavobacterium sp. has an excellent enzymatic activity at 10 ºC and Arthrobacter sp. at 25 ºC. We determined the growth curves of these bacteria in 15, 20 and 25 ° C. Both strains showed optimum growth at 25 ° C, indicating that these bacteria are psychrotrophics.

Neste trabalho, foi avaliada a reatividade de compostos orgânicos contendo boro frente a reações catalisadas por enzimas. Diferentes exemplos de álcoois secundários quirais contendo boro foram acetilados enantiosseletivamente pela lipase de Candida antarctica (CALB). Todas as reações ocorreram com excelente enantiosseletividade (E >200) e ambos os produtos acetilados, bem como os álcoois remanescentes foram obtidos com alto excesso enantiomérico (ee >99%). Além da resolução cinética enzimática, foi avaliado também o processo de resolução cinética dinâmica quimio-enzimática, empregando complexos de rutênio como agentes de racemização. Após estabelecer as condições adequadas para a reação, foi possível obter o produto acetilado de configuração-(R) com 83% de conversão, mantendo a enantiosseletividade observada na reação de resolução cinética enzimática. Como extensão ao trabalho realizado com os álcoois contendo boro, foi estudada a resolução cinética de aminas primárias quirais contendo boro. Foram avaliadas diversas condições reacionais para alcançar a máxima resolução cinética destas aminas via acilação enantiosseletiva catalisada pela lipase CALB. Excelente enantiosseletividade (E >200) e altos excessos enantioméricos (até >99%) foram obtidos utilizando acetato de etila tanto como reagente doador de acila como solvente da reação. Adicionalmente, foi investigada a reação de biorredução de cetonas pró-quirais contendo boro. Os melhores resultados foram obtidos com as enzimas álcool desidrogenases (ADHs) purificadas dos microorganismos Rhodococcus ruber e Lactobacillus brevis. A ADH de R. ruber (ADH-A) promoveu a biorredução das cetonas aos respectivos álcoois de configuração-(R) com excelente enantiosseletividade (ee >99%), enquanto a ADH de L. brevis (ADH-LB) catalisou a redução de alguns exemplos de cetonas aos respectivos álcoois de configuração-(S), também com excelente enantiosseletividade. / In this work, was evaluated the reactivity of boron-containing organic compounds in enzyme-catalyzed reactions. Different examples of boron-containing chiral secondary alcohols were resolved by enantioselective acetylation mediated by lipase from Candida. antarctica (CALB). All reactions showed excellent enantioselectivities (E >200) and both remaining substrates and acetylated product were obtained in high enantiomeric excesses (up to >99%). Besides the enzymatic kinetic resolution, the chemoenzymatic dynamic kinetic resolution was also evaluated, using ruthenium complexes as racemization agents. After establishing the best conditions, the acetylated (R)-product was obtained with 83% conversion, maintaining the high enantioselectivity observed in the enzymatic kinetic resolution. As an expansion of work with boron-containing alcohols, the kinetic resolution of boron-containing chiral amines was studied. Several reaction conditions were studied to achieve the kinetic resolution of boron-containing amines via enantioselective acylation mediated by CAL-B. Excellent enantioselectivity (E >200) and high enantiomeric excess (up to >99%) of both the remaining amines and amides were obtained using ethyl acetate as both acyl donor and the reaction solvent. Additionally, the bioreduction of pro-chiral boron-containing ketones was investigated. The best results were obtained with purified alcohol dehydrogenases (ADH) from Rhodococcus ruber and Lactobacillus brevis. The ADH from R. ruber (ADH-A) mediated the bioreduction of ketones to their respective (R)-alcohols with excellent enantioselectivity (ee >99%), while the ADH from L. brevis (ADH-LB) catalyzed the reduction of some ketones to their respective (S)-alcohols, also with excellent enantioselectivity (ee >99%).

Abstract not available

The production of enantiopure compounds can be achieved by using dehydrogenases as biocatalysts catalyzing reduction reactions of prochiral compounds such as ketones, aldehydes and nitriles. These dehydrogenases are cofactor dependent enzyme where cofactor is Nicotinamide dinucleotite having some restrictions that limits usage of dehydrogenases in organic synthesis including instability of cofactor in water and high cost. Therefore suitable regeneration method is needed and developed which are enzymatic and electrochemical. We will use an electrochemical approach for the regeneration of reduced co-factors which has been shown in principal with mediators like pentamethylcyclopentadienyl rhodium bipyridine complexes or ferrocenes. This project is European Union project, whose name is Development of Electrochemical Reactors Using Dehydrogenases for Enantiopure Synthon Preparations. All active compounds / mediator, cofactor and enzyme, will be immobilized on the electrode surface of the constructed reactor surface. Therefore only educts and v products will exist in the reactor medium. A gas diffusion electrode will be employed as a counter electrode / which delivers clear protons to the system. Mediator will carry electrons to the cofactor for cofactor regeneration. Then enzyme will use the cofactor and convert substrates to the product in high stereoselectivity. Our part in this project is the synthesis of mediator and suitable linkers for enzyme, cofactor and mediator immobilization. In the first part of the study, Linkers which contain thiol group and disulfide linkage were synthesized because working electrode made of by gold nano particles and immobilization carried out by the help of these groups on gold nano surface. In the second part of the study, mediators were synthesized which are pentamethylcyclopentadienyl rhodium bipyridine complexes and ferrocene derivatives. Synthesized mediators were reacted with linkers by using Click Chemistry and by imine formation in order to convert mediator to the thiol functionalized form.

"December 2009" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Thesis advisor: David A. Wilson. Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

By applying the principles of Darwinian natural selection in the laboratory, directed evolution has become a powerful practical approach to study enzymes and optimize them to catalyze industrially relevant transformations. In this thesis, I applied this strategy to the engineering of amino acid dehydrogenases for biocatalysis of chiral amines, focusing on two crucial features for successful directed evolution experiments. A first key aspect is the development of technologies allowing the screening of large libraries of enzyme variants to explore sequence space efficiently. Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsions has recently led to the development of ultrahigh-throughput screening platforms that allow sorting of more than 106 variants per hour. So far, these microfluidic droplet sorters have relied exclusively on fluorescent readouts. To further extend the range of applications toward enzymes for which no fluorescent assays are available, I successfully developed a sorting module based on absorbance detection. Using this new module, microdroplets could be sorted based on an absorbance readout at rates of up to 1 million droplets per hour. To demonstrate the utility of this module for protein engineering, three rounds of directed evolution were performed to improve a poorly stable NAD+ dependent phenylalanine dehydrogenase (PheDH) toward its native substrate. Five hits showed increased activity (improved up to 10-fold in lysate; kcat increased >3.5-fold), soluble protein expression levels (>2.5-fold) and thermostability (Tm, 8 °C higher). To increase the sensitivity of the device (3–4 orders of magnitude lower than fluorescence assays) for detection of enzymes with limited stability and low turnovers, an extra step of growth in droplets from single cell encapsulation, followed by piconinection of substrates and lysis agents was implemented. As a result, a fivefold signal enhancement over background was achieved, for an amine dehydrogenase (AmDH) reaction shown to be undetectable in a droplet single cell assay. Second, I investigated how mutational robustness may correlate with protein stability and lead to successful hits after mutagenesis and screening. To examine this issue, I initially investigated various approaches (including ancestral resurrection and computational design) to identify stabilized PheDH variants. One such variant (dubbed Pross 4) showed increased expression levels (>3.3-fold) and thermostability (Tm, 13 °C higher) compared to the wild-type PheDH. I further compared the mutational tolerance and the hit rate between PheDH and Pross 4 by generating variant libraries focused on key active site residues and screening them for improved AmDH activity. The Pross 4 background generated 6.4 times more active variants than the PheDH background, the best hits displaying increased activity (up to 2.5-fold in lysate; kcat/KM increased up to 8-fold) compared to previously engineered AmDHs with the PheDH scaffold. In conclusion, this work highlights how directed evolution experiments could be designed for increased success rates, by combining reliable high-throughput screens with careful choice of evolutionary robust starting points.

In 2015, an estimated 46.8 million people were living with dementia, a number predicted to increase to 74.7 million by 2030 and 131.5 million by 2050. Whilst there are numerous causes for the development of dementia, Alzheimer's disease is by far the most common, accounting for approximately 50-70% of all cases. Current therapeutic agents against Alzheimer's disease are palliative in nature, managing symptoms without addressing the underlying cause and thus disease progression and patient death remain a certainty. Whilst the main underlying cause for the development of Alzheimer's disease was originally thought to be an abnormal deposition of insoluble amyloid-β peptide derived plaques within the brain, the failure of several high-profile therapeutic agents, which were shown to reduce the plaque burden without improving cognition, has recently prompted a shift in focus to soluble oligomeric forms of amyloid-β peptide. Such soluble oligomers have been shown to be toxic in their own right and to precede plaque deposition. Soluble amyloid-β oligomers have been identified in various subcellular compartments, including the mitochondria, where they form a complex with the 17β-HSD10 enzyme resulting in cytotoxicity. Interestingly, hallmarks of this toxicity have been shown to be dependent on the catalytic activity of the 17β-HSD10 enzyme, suggesting two therapeutic approaches may hold merit in treating Alzheimer's disease: disrupting the interaction between the 17β-HSD10 enzyme and amyloid-β peptide, or directly inhibiting the catalytic activity of the 17β-HSD10 enzyme. In 2006, Frentizole was identified as a small molecule capable of disrupting the 17β-HSD10/amyloid interaction. The work described herein details the generation of a robust screening assay allowing the catalytic activity of the 17β-HSD10 enzyme to be measured in vitro. This assay was subsequently employed for small molecule screening using two methodologies; first in a targeted approach using compounds derived from the Frentizole core scaffold, and second in an explorative manner using a diverse library of compounds supplied by the National Cancer Institute. As a result, a range of novel small molecule inhibitors of the 17β-HSD10 enzyme have been identified and the most promising characterised in terms of potency and mechanism of action. De-selection assays were developed to allow the efficient triage of hit compounds and work was begun on a cellular based assay which would allow the ability of compounds of interest to reverse a disease relevant phenotype to be assessed in a cellular environment. As such, we now have a number of hit compounds which will form the basis for the generation of subsequent series of derivatives with improved potency and specificity, as well as the robust assays required to measure such criteria, potentially leading to the generation of novel therapeutic agents against Alzheimer's disease.