La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67. / Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

The dissertation describes a novel method for plant drug discovery based on mutation and selection of plant cells. Despite the industry focus on chemical synthesis, plants remain a source of potent and complex bioactive metabolites. Many of these have evolved as defensive compounds targeted on key proteins in the CNS of herbivorous insects, for example the insect dopamine transporter (DAT). Because of homology with the human DAT protein some of these metabolites have high abuse potential, but others may be valuable in treating drug dependence. This dissertation redirects the evolution of a native Lobelia species toward metabolites with greater activity at this therapeutic target, i.e. the human DAT. This was achieved by expressing the human DAT protein in transgenic plant cells and selecting gain-of-function mutants for survival on medium containing a neurotoxin that is accumulated by the human DAT. This created a sub-population of mutants with increased DAT inhibitory activity. Some of the active metabolites in these mutants are novel (i.e. not detectable in wild-type cells). Others are cytoprotective, and also protect DAergic neurons against the neurotoxin. This provides proof-of-concept for a novel plant drug discovery platform, which is applicable to many different therapeutic target proteins and plant species.

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques. / Fructose-1,6-bisphosphate aldolases (FBPA) are glycolytic enzymes (EC 4.1.2.13) that catalyze the reversible cleavage of fructose-1,6-bisphosphate (FBP) into the triose phosphates, glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). There are two classes of FBPAs that differ at the level of their mechanism. Class I FBPAs form a covalent iminium intermediate whereas class II FBPAs, being metalloenzymes, generally use a catalytic zinc in their reaction mechanism. In contrast to the mechanism of the class I FBPAs, which has been thoroughly studied, there are several unresolved inquiries as to the mechanism of class II FBPAs. We have therefore pursued a detailed analysis of the reaction mechanism using as a primary tool the elucidation of crystallographic structures. Several high resolution complexes have been resolved and have provided critical evidence to help us suggest the implication and role of several key residues in the active site. Consequently, we have correctly identified the residue which is responsible for the abstraction of the O4 proton from FBP, a vital step in the reaction mechanism. The residue responsible for this abstraction, which had incorrectly been assigned to Asp82 (in Helicobacter pylori), has been appropriately consigned to His180, a residue which is involved in coordinating the zinc molecule. Nevertheless, Asp82 remains an important residue as it orients, activates and stabilizes substrates. Finally, our study brings to evidence the dynamic character of our enzyme in which catalysis entails the relocalization of the catalytic zinc and several residues. The complexity of this reaction, notably one of the proton exchanges in the mechanism, could not be resolved solely by crystallographic means. In fact, the residue responsible for the stereospecific transfer of the pro(S) proton on carbon 3 (C3) of DHAP is situated on a loop that was not resolved in any of our structures. We therefore developed a molecular dynamics approach to study this intricate movement. After preliminary validation by inhibitor studies with class I FBPAs, the protocol was applied to class II FBPAs and several remarkable observations emerged: the residue responsible for this abstraction in Escherichia coli is Glu182 whereas a different residue, Glu149 (instead of Glu142) appears to assume this role in H. pylori. Our preliminary validations have confirmed this observation and shall be further consolidated in the future. Class II FBP aldolases, although absent from the human proteome, are prevalently found in several pathogens, and have further been found to be essential to a number of these organisms. As such, they are ideal targets for the development of novel anti-microbial agents. Developing new analogues of the cognate substrates of these enzymes is therefore not only advantageous for mechanistic studies, but has endless pharmacological potential. In the context of a collaborative effort involving a group of chemists, a compound that initially had an inhibition constant in the millimolar range was optimized and produced a series of compounds that inhibit in the nanomolar range.

La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67. / Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.

No description available.

La fructose-1,6-bisphosphate aldolase de classe I est une enzyme glycolytique (EC 4.1.2.13) qui catalyse le clivage réversible du fructose-1,6-bisphosphate (FBP) en dihydroxyacétone phosphate (DHAP) et glycéraldéhyde-3-phosphate (G3P). Des années de recherche sur FBP aldolase ont permis d’identifier les résidus impliqués dans son mécanisme réactionnel, ont tracé en grande partie les coordonnées de la réaction, ont révélé de nouvelles fonctions dites « moonlighting », et ont validé l’aldolase comme une cible attrayante pour des applications anti-glycolytiques tel que le cancer. Il existe néanmoins des questions en suspens relatives à ces activités que nous avons étudiées. Tout d'abord, la trajectoire détaillée de l'aldéhyde relatif à sa liaison au site actif allant jusqu’à la formation du lien carbone-carbone par condensation aldolique est indéfini. Pour élucider les détails moléculaires liés à ces événements, nous avons déterminé des structures cristallographiques à hautes résolution de l’aldolase de classe I chez Toxoplasma gondii, qui porte une identité de séquence élevée avec l’aldolase humaine (57%), en complexe avec l’intermédiaire ternaire de pré-condensation. Le complexe ternaire révèle un mode de liaison non-productive inhabituel pour G3P dans une configuration cis qui permet l’alignement de l'aldéhyde à proximité du nucléophile naissant. La configuration compétente pour la condensation aldolique provient d'une transposition cis-trans de l'aldéhyde qui produit une liaison hydrogène courte permettant la polarisation de l'aldéhyde et le transfert de proton au niveau de Glu-189. Nos résultats informent les chimistes synthétiques qui cherchent à développer l’aldolase comme biocatalyseur pour des réactions stéréo-contrôlées. Le rôle présumé de l’aldolase dans la production du méthyglyoxal (MGO), un métabolite dicarbonyle hautement réactif qui génère des « advanced glycation end products » (AGES) a également été étudié structurellement et enzymatiquement. Une enquête structurelle cristallographique de MGO générée par décomposition enzymatique chez l’aldolase de classe I a révélé que, contrairement aux indications préliminaires, l'apparition hypothétique de MGO et de phosphate inorganique (Pi) résultant de la décomposition enzymatique de DHAP dans le site actif de l’aldolase est mieux interprétée par une population mixte de DHAP et de molécules d'eau. Une étude enzymatique a révélé que la décomposition spontannée des trioses-phosphate est une source majeure de la production de MGO, alors qu’une production catalysée par l’aldolase est peu concluante. L’identification des sources de production de MGO continue d'être une priorité afin de développer des stratégies pour atténuer les manifestations cliniques de pathologies associées au MGO. La FBP aldolase est également reconnu pour ses activités « moonlighting » - du fait qu’elle effectue plus d'une activité sans rapport avec sa fonction glycolytique. Divers partenaires de l’aldolase sont rapportés dans la littérature, y compris les adhésines de surface cellulaire chez les parasites apicomplexes, dans lequel l’aldolase exécute une fonction d'échafaudage entre le complexe actomyosine et les adhésines - une interaction qui est décisive pour la motilité et l'invasion des cellules hôte. Le mode de liaison de cette interaction a été étudié et nos résultats sont compatibles avec une liaison au site actif. Les détails précis de cette interaction ont des implications thérapeutiques, étant donné que le ciblage de celui-ci réduit l'invasion des cellules hôte par les parasites. Enfin, l’aldolase de classe I est de plus en plus reconnu pour son potentiel comme cible anti-glycolytique dans les cellules qui sont fortement tributaires du flux glycolytique, comme les cellules cancéreuses et les parasites protozoaires. Le développement de nouveaux inhibiteurs de haute affinité est donc non seulement avantageux pour des études mécanistiques, mais représente un potentiel pharmacologique sans fin. Nous avons développé une nouvelle classe d’inhibiteurs de haute affinité de type inhibition lente et avons déterminé la base moléculaire de leur inhibition grâce à des structures cristallographiques à haute résolution et par un profilage enzymatique. Cette étude, qui combine plusieurs disciplines, y compris la cristallographie, enzymologie et chimie organique, souligne l'intérêt et l'importance d'une approche multidisciplinaire. / Class I Fructose-1,6-bisphosphate aldolases are glycolytic enzymes (EC 4.1.2.13) that catalyze the reversible cleavage of fructose-1,6-bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). Years of research on FBP aldolases has identified residues implicated in the reaction mechanism, mapped the greater part of the reaction coordinates, and revealed novel moonlighting functions. Further, FBP aldolase is recognized as an attractive target for anti-glycolytic applications such as cancer. There are nevertheless outstanding questions related to these activities that were investigated in this thesis. First, the detailed trajectory of the reaction mechanism from aldehyde binding in the active site to carbon-carbon bond formation by aldol condensation is undefined. To elucidate the molecular details related to these events, we solved high-resolution crystallographic structures of native class I aldolase from Toxoplasma gondii, which has a high sequence identity with human aldolase (57 %), in complex with the pre-condensation ternary intermediate. The ternary complex reveals a condensation-incompetent binding mode for G3P in a cis-configuration that aligns the aldehyde alongside the nascent nucleophile. The productive aldol-competent configuration arises from a cis-trans rearrangement of the aldehyde that produces a short hydrogen bond required for polarization of the aldehyde and coincident proton transfer at Glu-189. Our results inform synthetic chemists seeking to develop aldolases for stereo-controlled reactions in biosynthetic applications. The suspected role of aldolase in methylglyoxal (MGO) production, a highly reactive dicarbonyl metabolite that produces advanced glycation end-products (AGES) was also probed structurally and enzymatically. A crystallographic structural investigation of MGO generated by enzymatic decomposition in class I aldolase revealed that, contrary to preliminary indications, the appearance of MGO and inorganic phosphate (Pi) resulting from enzymatic decomposition of DHAP in the active site of aldolase is more appropriately modeled by a mixed population of DHAP and water molecules. Enzymatic investigation revealed triose-phosphate decomposition to be a major source of MGO production, whereas production by aldolase did not exceed assay background levels. Identifying the main sources of MGO production continues to be a priority for mitigating the clinical manifestations of MGO-derived pathologies. FBP aldolase is also recognized for its moonlighting properties – performing more than one activity unrelated to the glycolytic function. Diverse aldolase partners are reported, including cell surface adhesins in apicomplexan parasites, in which aldolase performs a bridging function between the actomyosin complex and the cytoplasmic domain of the adhesins – an interaction that is crucial for motility and host-cell invasion. The binding mode of this interaction was investigated and our results are consistent with active site binding. The precise details of aldolase-adhesin binding has therapeutic implications, since targeting of the latter reduces host-cell invasion by parasites. Finally, class I aldolase is gaining prominence as an anti-glycolytic target in cells that are highly dependent on glycolytic flux, such as cancer cells and protozoan parasites. Developing new high-affinity inhibitors for these enzymes is therefore not only advantageous for mechanistic studies, but has endless pharmacological potential. We developed a novel class of high-affinity aldolase inhibitors, bisphosphonates, and determined the molecular basis of their inhibition with high-resolution crystallographic structures and enzymatic profiling. This study, which combined several disciplines, including crystallography, enzymology, and organic chemistry, underscores the interest and significance of a multidisciplinary approach.

Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-E-vinylphosphonate and chemical stabilization of all nucleotides with 2'-O-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 μM concentration. Further concentration-response analysis revealed two compounds, and , as potent and selective CB2 ligands with sub-micromolar activities ( = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound were sought. Compound was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.