Neste trabalho foi realizada a síntese de amidas, bem como de ácidos e ésteres borônicos, derivados dos aminoácidos L-cistina e L-cisteína, através de rota sintética simples, curta e de baixo custo, com o intuito de busca e a identificação de novo(s) inibidor(es) do proteassomo 20S. Esta classe de compostos possui estrutura que permite a inserção de diversos grupos funcionais, o que confere versatilidade e a construção de biblioteca de compostos que contém partes hidrofílicas e hidrofóbicas importantes para posterior avaliação inibitória. Para tanto, empregou-se rota sintética química convencional e rota biocatalisada para a formação da ligação amida. Os compostos derivados de L-cisteína foram obtidos via síntese clássica de peptídeos a qual forneceu os compostos desejados em rendimentos de até 85%. Por outro lado, tentativas de obtenção das amidas via biocatálise não se mostraram efetivas. Já amidas derivadas de L-cistina foram obtidas em rendimentos de até 79%, via síntese tradicional e até 100% de conversão através de rota biocatalítica. A inserção do átomo de boro nas estruturas se deu utilizando-se metodologias sintéticas já bem estabelecidas na literatura. Os ésteres borônicos derivados de L-cisteína foram obtidos em bons rendimentos (até 78%), enquanto que não foi possível obter-se compostos de boro derivados de L-cistina. Por sua vez, os compostos contendo ácido borônico na estrutura foram sintetizados via reação de hidrólise dos respectivos ésteres borônicos, em rendimentos moderados (até 34%). Após a obtenção dos compostos contendo grupamentos organoboro realizou-se avaliação inibitória dos mesmos frente ao proteassomo 20S. Valores de IC50 iguais a 52 µM foram obtidos para composto derivado de L-cisteína contendo grupamento éster borônico, que se mostraram inibidores moderados e reversíveis. Ácidos borônicos se mostraram sem capacidade de inibir o proteassomo 20S. Adicionalmente, realizaram-se estudos de modelagem molecular com a finalidade de elucidar os resultados obtidos experimentalmente. Inicialmente realizaram-se cálculos de modelagem molecular através da realização de docking de alguns compostos e após geração de modelo farmacofórico. De maneira geral observou-se que os inibidores derivados da L-cisteína não ocupam a mesma cavidade que o fármaco bortezomibe, o que pode explicar a diferença na atividade dos compostos frente à inibição do proteassomo 20S. Também se observou que, tendo-se a interação dos inibidores com a enzima, a vizinhança do átomo de boro tem grande influência na capacidade inibitória, uma vez que estes grupamentos determinam qual a região da cavidade do proteassomo 20S será ocupada pelo inibidor. / In our study, amides, boronic acids and esters derivatives from L-cysteine and L-cysteine were synthesized by simple, short and inexpensive synthetic route, in order to search for new inhibitor(s) of the 20S proteasome. This class of compounds has a structure that allows inclusion of various functional groups, giving it versatility and allowing the construction of library compounds containing hydrophilic and hydrophobic moieties, important for further evaluation. To this end, we used conventional chemical synthetic route and biocatalysis for peptide bond formation. The compounds derived from L-cysteine were obtained by classical synthesis of peptides which provided the desired compounds up to 85% yields. On the other hand, attempts to obtain the amides via biocatalysis were not effective. However, amides derived from L-cystine were obtained with up to 79% yields via chemical synthesis and conversion up to 100% using biocatalytic route. The insertion of the boron atom in the structures was possible using synthetic methodologies well established in literature. Boronic esters derived from L-cysteine were obtained in good yields (up to 78%), whereas it was not possible to obtain boron compounds derived from L-cystine. In turn, compounds containing boronic acids in the structure were synthesized by hydrolysis reaction of the respective boronic esters in moderate yields (up to 34%). With organoboron compounds in hand, we turned our attention to inhibitory assessment against the 20S proteasome. IC50 up to 52 µM were obtained when L-cysteine boronic ester derivatives were evaluated. These compounds are moderate and reversible inhibitors. L-cysteine boronic acids derivatives have shown not ability to inhibit the 20S proteasome. Additionally, molecular modeling studies were carried out in order to elucidate the results obtained experimentally. Initially molecular modeling calculations were carried out by performing docking experiments of some compounds. Generation of pharmacophoric model calculations was also executed. In general, it was observed that inhibitors derived from L-cysteine do not occupy the same cavity that drug bortezomib, which may explain the difference in the activity of compounds against the inhibition of 20S proteasome. We also observed that, with the interaction of inhibitors and enzyme, the side chains around boron atom has a great influence on inhibitory capacity, since these groups determine which region of the 20S proteasome cavity is occupied by the inhibitor.

Melanoma é o câncer mais agressivo e a mutação BRAF V600E é a mais frequente entre os pacientes. O vemurafenibe foi o primeiro inibidor específico desta mutação aprovado pela Food and Drug Administration. Entretanto, após cerca de seis meses há recidiva e superar os mecanismos de resistência responsáveis por este fenômeno ainda é um desafio. A curcumina é um tumérico com características antitumorais e anti-inflamatórias, entretanto sua baixa biodisponibilidade e estabilidade limitam seu uso e por isso impulsionaram a busca por análogos capazes de serem eficientes e comercializados. O DM-1, é um análogo monocetônico que apresentou efeitos antiumorais in vitro e in vivo em estudos anteriores. O objetivo deste trabalho foi avaliar os efeitos citotóxicos do DM-1 em células de melanoma sensíveis (naive) e resistentes ao vemurafenibe, bem como na modulação de metaloproteinases. As células de melanoma foram tratadas com diferentes concentrações de DM-1, e este composto foi citotóxico para linhagens sensíveis e resistentes ao vemurafenibe, além de induzir parada de ciclo celular em G1/G0 e diminuir o número de colônias, entretanto ele não foi seletivo em ensaios de citotoxicidade realizados com melanócitos e fibroblastos. O tratamento dessas células em doses subtóxicas resultou na modulação de metaloproteinases importantes no processo de invasão celular. O DM-1 reduziu as concentrações das metaloproteinases -1, -2 e -9 (MMP-1, -2 e -9) em um ensaio de quantificação de MMPs e a atividade das MMP-2 e -9 em um ensaio de zimografia de maneira célula dependente. As modulações negativas do inibidor de MMP TIMP-2 e MMP-14 para SKMEL-28 naive foram associadas a diminuição das atividades de MMP-2 e -9, enquanto que as modulações positivas para SKMEL-19 naive foram relacionadas ao aumento de MMP-2. Este composto ainda inibiu a migração das células e a formação de tubos por células endoteliais. / Malignant melanoma is the most aggressive cancer and the BRAF V600E mutation is the most frequent among patients. Vemurafenib was the first specific inhibitor for this mutation approved by Food and Drug Administration. Therefore around six months later there is relapse and overcoming it is still a challenge. Curcumin is a turmeric and it has been deeply researched because of its anti-inflammatory and antitumoral effects. However the low stability limits its use, therefore, encouraged the investigation of analogues capable to be efficient and commercialized. DM-1 is a monoketone curcumin analog and it showed antitumoral effects in vitro and in vivo in previous studies The aim of this project was to evaluate the cytotoxical effects of DM-1 for vemurafenib responsive (naïve) and resistant melanoma cells, as well as metalloproteinases modulation. Melanoma cells were treated with different DM-1 concentrations, and this compound was cytotoxic for responsive and resistant cell lines, besides inducing G1/G0 cell cycle arrest and reducing the number of colonies, nonetheless it was not selective in assays performed with melanocytes and fibroblasts. Subtoxic treatment of those cells modulated important MMPs in the cell invasion process. DM-1 reduced metalloproteinases -1, -2 and -9 (MMP-1,-2 and -9) in a quantification assay, and MMP-2 and -9 activities by zymography in a cell-dependent way. Negative modulations of MMP inhibitor TIMP-2 and MMP-14 for SKMEL-28 naïve were associated with MMP-2 and -9 reduced activities, whereas positive modulations for SKMEL-19 naïve were correlated to MMP-2 increase. Furthermore, this compound reduced migration of those cells and endothelial cell tube formation.

La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypes. / Dengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes.

No description available.

Le ribosome est un complexe cellulaire impliqué dans la catalyse et la coordination des différentes étapes de la traduction de l’information génétique, des ARN messagers aux protéines. Avec une masse moléculaire avoisinant 3.3 méga daltons, le ribosome eucaryote est 40% plus volumineux que son homologue bactérien. Le premier volet de la thèse présente la structure cristallographique du ribosome eucaryote 80S de levure à haute résolution. Cette structure constitue une base solide pour l’étude de la synthèse des protéines chez les eucaryotes ainsi que pour le développement de nouveaux composés thérapeutiques. Le deuxième volet est consacré aux structures cristallographiques de deux familles d’inhibiteurs spécifiques des eucaryotes, les glutarimides et les trichothecenes, en complexe avec le ribosome. Ces inhibiteurs constituent de nouveaux outils pour sonder les fonctions du ribosome et contribueront aux développements de composés par des approches de structure‐based drug design. / The ribosome is large cellular machinery that catalyzes and coordinates every step required to translate the genetic information encoded in messenger RNAs into proteins. With a molecular weight of 3.3 mega daltons, the eukaryotic ribosome is 40% larger compared to its bacterial counterpart. The fist axis of thesis presents the crystal structure of the complete eukaryotic 80S ribosome from yeast at high resolution. This structure constitutes a unique framework for future investigations of protein synthesis in eukaryotes and development of new therapeutic agents. The second axis of the thesis presents the crystal structures of two distinct families of eukaryote‐specific inhibitors, the glutarimides and the trichothecenes, bound the eukaryotic ribosome. These inhibitors constitute new tools to probe the ribosome functions and a starting point for future structure‐based drug design.

Le but des travaux est de caractériser l’influence de la signalisation VEGF, en particulier la signalisation autocrine VEGF, sur la biologie et la sensibilité/résistance aux médicaments anticancéreux de cellules de cancer colorectal. Nous avons souhaité caractériser l’impact de la signalisation autocrine VEGF dans des modèles de CRC avec une résistance naturelle au bevacizumab, un anticorps anti-VEGF. Bien que ce composé soit actif dans le CRC, une sous population de patients ne répondent pas au traitement. Nos résultats montrent une sur régulation de la voie autocrine HIF-VEGF-VEGFR en réponse à une exposition prolongée au bevacizumab dans les cellules bevacizumab résistantes. Si la résistance à cet anticorps est bien établie, d’autres inhibiteur de la voie VEGF restent actifs (comme la petite molécule ciblée nintedanib) et peuvent inhiber la voie mTOR. La signalisation VEGF autocrine joue un rôle dans la survie de cellules de CRC. Chez les sujets résistants au bevacizumab, il serait intéressant d’introduire le nintedanib seul ou en combinaison pour accentuer l’inhibition angiogénique. Une autre combinaison d’agents (anti VEGF(R) et anti EGFR) a montré une efficacité dans des modèles de CRC en préclinique. La combinaison du bevacizumab et d’une petite molécule ciblant EGFR (erlotinib) a montré une plus grande efficacité que le bevacizumab seul dans des modèles de CRC indépendamment du statut KRAS. Le bevacizumab induit une activation de la voie de survie EGFR dans les cellules tumorales et dans les cellules endothéliales associées à la tumeur. Cette activation se trouve diminuée avec l’introduction de l’erlotinib. Les résultats indiquent que la combinaison du bevacizumab et de l’erlotinib sont plus actif en thérapie de maintenance que le bevacizumab seul, même pour les patients mutés KRAS. Ces résultats ont mené à l’étude clinique positive GERCOR phase III DREAM dans le cancer du côlon métastatique. / The aim of the work is to characterize the influence of VEGF signaling , especially autocrine VEGF signaling , the biology and susceptibility / resistance to anticancer drugs of colorectal cancer cells. We wished to characterize the impact of the autocrine VEGF signaling in CRC models with natural resistance to bevacizumab , an anti -VEGF antibody. Although this compound is active in the CRC, a subpopulation of patients do not respond to treatment. Our results show an autocrine regulatory pathway HIF- VEGF- VEGFR in response to prolonged exposure to bevacizumab in bevacizumab resistant cells. If the resistance to the antibody is established, other inhibitos of VEGF pathway remain active (such as small molecule nintedanib ) and can inhibit the mTOR pathway. Autocrine VEGF signaling plays a role in CRC cell survival. In subjects resistant to bevacizumab, it would be interesting to introduce the nintedanib alone or in combination to enhance the angiogenic inhibition. Another combination of targeted agents ( anti-VEGF (R) and anti EGFR) has shown efficacy in preclinical models of CRC. The combination of bevacizumab and a small molecule targeting EGFR (erlotinib) showed greater efficacy than bevacizumab alone in CRC models regardless of KRAS status. Bevacizumab induces activation of the EGFR survival pathway in tumor cells and in endothelial cells associated with the tumor. This activation is decreased with the introduction of erlotinib. The results indicate that the combination of bevacizumab and erlotinib are more active in maintenance therapy than bevacizumab alone, even for patients mutated KRAS . These findings led to the positive Phase III clinical study GERCOR DREAM in metastatic colon cancer.

Abstract To investigate the turnover of type IV collagen in skeletal muscle in conditions where muscle function is impaired, type IV collagen and proteins regulating its degradation were studied during 1, 3 and 7 days of immobilization, 3- and 10-day glucocorticoid treatment and in neuromuscular diseases. In addition, fibrillar type I and III collagens were studied during immobilization and in neuromuscular diseases. The mRNA levels of type I, III and IV collagens were decreased during immobilization and during 10-day dexamethasone treatment. Gene expression and quantity of (pro)MMP-2 was increased during immobilization but decreased during dexamethasone treatment. The expression of TIMP-2 was decreased both during immobilization and dexamethasone treatment. Decreased gene expression and increased degradation caused decreased concentration of type IV collagen, suggesting net degradation of type IV collagen during immobilization. While the gene expression and degradation were decreased during dexamethasone treatment, the amount of type IV collagen was not changed. Dexamethasone thus seemed to slow down the turnover of type IV collagen. Decreased mRNA levels of collagens and prolyl 4-hydroxylase suggest decreased biosynthesis of collagens during immobilization. The mRNA levels of collagens I, III and IV were increased in polyneuropathy and polymyositis. The concentration and staining intensity of type IV collagen was increased in polyneuropathy, as was also the quantity and staining intensity of (pro)MMP-9. The results suggest accumulation of type IV collagen in the basement membranes of muscle cells and capillaries in polyneuropathy muscles. Lengthened position during immobilization partly prevented the atrophy and changes in collagen metabolism in plantarflexors. Endurance running was effective in preventing muscle atrophy during dexamethasone treatment, but exercise did however fail to prevent the changes observed in type IV collagen synthesis and degradation.

Dans cette étude, nous décrivons pour la première fois les propriétés anti-tumorales de composés de type thiomorpholine sulfonamide hydroxamate dans le cancer du sein. Nous avons montré que le TMI-1, un inhibiteur à spécificité double MMPs/ADAM-17, est un puissant inhibiteur de la croissance tumoral in vitro et in vivo. Cette activité anti-tumorale est indépendante de l’inhibition d’ADAM-17 et des MMPs. En effet, le TMI-1 inhibe la croissance tumorale in vitro de 89% des lignées tumorales mammaires à des IC50 de l’ordre du micromolaire, par sa double capacité d’induire l’arrêt du cycle cellulaire en G0/G1 et l’apoptose dépendante de la voie extrinsèque. TMI-1 n’a aucun effet sur les cellules normales. In vivo, le TMI-1 inhibe le développement tumoral et l’apparition de nouvelles tumeurs dans les souris transgéniques MMTV-ERBB2/neu. A la différence des drogues cytotoxiques conventionnelles, TMI-1 à une activité sélective sur les cellules tumorales en affectant le pool de cellules souches cancéreuses mammaires. De plus, le TMI-1 agit en synergie en association avec les drogues de chimiothérapie et de thérapies ciblées. TMI-1 a déjà été utilisé en clinique, est une molécule candidate au repositionnement clinique en cancérologie. D’une manière intéressante, une corrélation marquée a pu être mise en évidence entre TMI-1 et le statut mutationnel de P53. Finalement, TMI-1 représente une alternative thérapeutique prometteuse dans le traitement des cancers, d’autant que la sélectivité semble associée à un marqueur « compagnon » P53, muté dans environ 50% des cancers. Le lien existant entre ces composés et P53 passe par l’identification de la ou les cibles. Ce travail est actuellement en cours, utilisant des approches de protéomique inverse. / In this study, we focused on breast cancer and reported for the first time in vitro and in vivo antitumor properties of thiomorpholin sulfonamide hydroxamate compounds. We found that TMI-1, a dual ADAM-17 and MMP inhibitor is a strong anti-cancer agent. TMI-1 exerts its antitumor activity at micromolar range by induction of cell cycle arrest in G0/G1 and extrinsic apoptosis pathway. TMI-1 is not cytotoxic for normal cells. 89% of breast tumor cells are sensitive to TMI-1 in vitro. TMI-1 is also efficient in vivo, inhibits tumor growth and prevents the formation of additional tumors in MMTV-ERBB2/neu transgenic mice. Unlike conventional cytotoxic drugs, TMI-1 has a selective activity on tumor cells by affecting the pool of cancer stem cells. Interestingly TMI-1 is a potent synergistic drug for breast cancer therapy. TMI-1, already used to treat inflammatory disease, is a candidate for drug repositioning in oncology. Interestingly, a marked correlation was found between TMI-1 and the mutational status of P53. Finally, TMI-1 is of outstanding interest, as efficiency may be related to the surrogate marker P53 mutated in approximately 50% of cancers. The relationship between these compounds and P53 is currently in progress. This is done by a reverse proteomic approaches attempting to identify direct target(s).

Le déséquilibre HAT/HDAC aurait une influence sur le développement de certains cancers ainsi que dans l’addiction à l’alcool ou à la cocaïne. En inhibant les histones désacétylases, le taux d'acétylation de la chromatine augmente ce qui permet l’accès aux facteurs de transcription et l'expression des gènes. Aujourd'hui, il existe de nombreux inhibiteurs d'HDAC de structures diverses, mais ils ne sont pas spécifiques et présentent des effets secondaires importants. Les inhibiteurs d'HDAC comme le butyrate de sodium ou le MS-275 ont montré une modification de la dépendance à l'alcool chez le rat. MS-275 inhibe principalement la classe I de HDAC et conformément à ces observations nous nous intéressons aux inhibiteurs les plus sélectifs de la classe I tels que le Largazole thiol et le RedFK228. Notre but est de synthétiser de nouveaux cyclodepsipeptides analogues afin d'obtenir un inhibiteur sélectif de la classe I. Les HDAC de la classe I sont Zn-dépendants, ces analogues auront un groupement sulfonylhydrazide ayant une bonne affinité pour l’ion Zn2+ (ZBG). Il sera relié au cyclodepsipeptide par un bras espaceur dont la longueur sera adaptée (n = 2, 3). Une autre pharmacomodulation concerne l'incorporation d’hétérocycles différents (oxazole, thiazole et pyridine). Les inhibitions de ces composés ont pu être testées sur HDAC1, HDAC3 et HDAC6. Un composé a une spécificité pour HDAC3 et un autre a une spécificité pour HDAC1. Les tests sur des rats "binger" permettent de penser que HDAC1 est impliqué dans ce model de consommation et non HDAC3. / The imbalance HAT/HDAC would influence the development of cancers and alcohol or cocaine addiction. HDAC inhibition allows increase of both acetylation rate and gene expression. Today, there are many structurally diverse potent, but non-specific HDAC inhibitors displaying important side-effects. HDAC inhibitors such as sodium butyrate or MS-275 have been shown to alter the alcohol dependence in the rat. MS-275 inhibits mainly class I of HDAC and in line with these observations we are interested in more selective class I inhibitors such as Largazole thiol and RedFK228. Our purpose is to synthesize new cyclodepsipeptides analogues in order to obtain selective class I inhibitor. HDAC class I is a Zn-dependent enzyme and our target molecules have sulfonylhydrazide function as efficient Zinc binding group (ZBG). Additional pharmacomodulations concern the incorporation of different heterocycles (oxazole, thiazole, pyridine) and varying linker lengths (n = 2, 3). Inhibitions of these compounds have been tested on HDAC1, HDAC3 and HDAC6. A compound has specificity for HDAC3 and another has specificity for HDAC1. Tests on rats "binger" suggest that HDAC1 is involved in this model of consumption and not HDAC3.

Class of 2014 Abstract / Specific Aims: To identify reasons that an angiotensin converting enzyme inhibitor (ACEi) would not be indicated in diabetic patients with repeated clinical alerts to add ACEi therapy for preservation of renal function and/or hypertension. In addition, to identify if these repeated clinical alerts to add ACEi therapy are appropriate. Methods: Eligible patient charts were reviewed by researchers using a data dictionary to complete a standardized spreadsheet with patient demographic information (age, gender, and location), type of diabetes mellitus, evidence indicative of comorbid hypertension, action taken by pharmacist in response to clinical alert (letter sent to patient and letter sent to prescriber), and rationale of that action. This data, along with SOAP notes of patient interactions, was used by researchers to classify the repeated clinical alert as appropriate or inappropriate. Main Results: There were a total of 200 charts reviewed (male n = 61 (30.5%), female n = 139 (69.5%), mean age = 70 ± 11 years). Reasons for not contacting patients again include previous failure or adverse drug reaction (n = 62, 31.0%), patient did not meet call script requirements (n = 55, 27.5%), patient did not have diabetes or hypertension (n = 20, 10.0%), potential drug-disease interaction (n = 17, 8.5%), overlapping or previously addressed alerts (1.9%), or documentation was provided for “other” reasons (n = 43, 21.5%). The previous failure or adverse drug reaction rationale was appropriate in 32 of 62 repeated clinical alerts (52%; χ2= 10.15). The patient did not have diabetes or hypertension rationale was appropriate in 11 of 20 repeated clinical alerts (55%, χ2= 2.72). The potential drug-disease interaction rationale was appropriate in 3 of 17 repeated clinical alerts (8%, χ2= 9.89). The patient did not meet call script requirements rationale was appropriate in 31 of 55 repeated clinical alerts (56%, χ2= 6.91). The overlapping or previous alerts rationale was appropriate in 2 of 3 repeated clinical alerts (67%, χ2= 0.18). The “other” rationale were appropriate in 22 of 43 repeated clinical alerts (51%, χ2= 7.21) Overall, retrigger alerts were considered appropriate 50.5% of the time compared to the predicted value of 90% (χ2= 347 > critical value = 3.84 for p = 0.05 Conclusion: There are multiple reasons pharmacists do not recommend initiating ACEi therapy in patients with diabetes. Although the Medication Management Center (MMC) has rationale of these reasons documented after individual patient interactions, there are still several reasons why a retrigger alert would be appropriate despite that rationale. In addition, retrigger alerts were not considered appropriate as frequently as expected.