Synthesis and biological properties of oligonucleotides containing phosphoramidate internucleotide linkages

This thesis describes a series of studies on oligonucleotides containing cationic phosphoramidate modifications, with a focus on elucidating the changes in biological properties resulting from incorporation of cationic internucleotide linkages. In general, synthesis of phosphoramidate-modified linkages was achieved through incorporation of 3'-H-phosphonate monomers, followed by oxidative amination with primary and secondary amines.To facilitate our work on siRNAs and antisense oligonucleotides containing modified linkages, studies of the synthesis and stability of these linkages were undertaken. We subjected phosphoramidate-modified dimers to various conditions used in phosphoramidite chemistry and oligonucleotide deprotection. We demonstrated that, while phosphoramidate-modified linkages are unstable to treatment with triethylamine trihydrofluoride (a common desilylation reagent), desilylation of RNA containing phosphoramidate-modified linkages can be accomplished using either triethylamine trihydrofluoride/N-methylpyrrolidone/triethylamine or tetrabutylammonium fluoride. We also designed and developed an automated approach for the synthesis of phosphoramidate-modified oligonucleotides, allowing faster and more efficient production of this class of modified oligonucleotides; this process can be easily adapted for access to many other internucleotide linkage modifications. This work allowed further exploration of siRNA duplexes containing phosphoramidate-modified linkages targeting firefly luciferase mRNA. To this end, we synthesized a range of singly-modified and multiply-modified oligonucleotides containing various amine conjugates. Here, we showed that the terminal amine function of alkyldiamine phosphoramidate conjugates is beneficial with respect to binding affinity. We also demonstrated that phosphoramidate-modified linkages are mildly destabilizing in RNA:RNA duplexes, and that this destabilization can be mitigated through the use of 2'-modified residues, such as 2'-fluoro RNA and 2'-O-methyl RNA, in place of DNA. With respect to activity, DMEDA phosphoramidate modifications were well-tolerated in both the passenger and guide strands of an siRNA duplex. The synthesis and study of a series of antisense oligonucleotides modified with DMEDA phosphoramidates in a gapmer motif comprising cationic phosphoramidate flanks and phosphorothioated DNA/2'-F-ANA cores was explored using two targets: firefly luciferase mRNA and down-regulated in renal cell carcinoma mRNA. Although similar modification motifs were used for both sequences, the resulting AONs exhibited vastly different properties with respect to thermal stability. Phosphoramidate-modified AONs displayed moderate knockdown activity relative to analogous unmodified controls. Structural studies of phosphoramidate-modified siRNAs and AONs showed that the incorporation of phosphoramidate modifications has little effect on the global structure of the duplex. Finally, we demonstrated that, while cationic phosphoramidate modifications are known to impart high resistance to enzymes such as phosphodiesterase I and II and nuclease P1, phosphoramidate-modified siRNAs and AONs are less stable in serum than analogous unmodified controls.Le travail de la présente thèse s'articule autour d'une série d'études menées sur des oligonucléotides modifiés avec des fonctions phosphoramidates chargées positivement, en se concentrant notamment sur l'élucidation des changements de propriétés biologiques résultant de l'incorporation de ces liens cationiques internucléotidiques. De manière générale, la synthèse de liens modifiés par des fonctions phosphoramidates a été réalisée grâce à l'incorporation de monomères de type 3'-H-phosphonate, suivie par une amination oxydative par des amines primaires ou secondaires.Nous avons conduit une étude de synthèse et de stabilité de ces liens internucléotidiques visant à faciliter nos travaux sur les petits ARN interférents (pARNis) et les oligonucléotides antisens (OAs) contenant ces liens modifiés. Des dimères modifiés avec des fonctions phosphoramidates ont été soumis à des traitements divers couramment employés pendant la synthèse et la déprotection d'oligonucléotides selon la méthode phosphoramidite. Nous avons ainsi démontré que les fonctions phosphoramidates sont instables au traitement avec la triéthylamine-trihydrofluorure (un réactif communément utilisé pour la désilylation), alors que le clivage des groupements protecteurs silylés d'un ARN modifié avec des liens phosphoramidates peut être accompli à l'aide soit d'un mélange de triéthylamine-trihydrofluorure/N-méthylpyrrolidone/triéthylamine soit de fluorure de tétrabutylammonium. Nous avons aussi conçu et développé une approche automatisée pour la synthèse d'oligonucléotides modifiés avec des fonctions phosphoramidates, permettant de ce fait une production plus rapide et plus efficace de cette catégorie d'oligonucléotides et de nombreuses autres formes de modifications du lien internucléotidique. Ce travail a ouvert la voie à une exploration de pARNis modifiés avec des liens phosphoramidates et ciblant l'ARN messager de la luciférase de luciole. Pour ce faire, nous avons préparé un ensemble d'oligonucléotides contenant une ou plusieurs modifications conjuguées à des groupements amine de différentes natures. Nous avons montré que la fonction amine terminale de conjugués phosphoramidates-dialkylamine a un effet positif sur l'affinité de liaison du double brin. Nous avons aussi démontré que la présence de liens phosphoramidates s'accompagne d'une légère déstabilisation d'un double brin d'ARN et que cette déstabilisation peut être atténuée par l'utilisation de nucléotides modifiés en position 2', au lieu d'ADN. En ce qui concerne leur activité biologique, les modifications phosphoramidates diméthyléthylènediamine ont été bien tolérées à l'intérieur d'une double hélice de pARNi, que ce soit dans le brin passager ou le brin guide. Nous avons ensuite entrepris la synthèse et l'étude d'une série de OAs modifiés par des fonctions phosphoramidates selon un motif dit de <<gapmère>>, où les parties périphériques sont modifiées avec des fonctions phosphoramidates chargées positivement tandis que la partie centrale contient des nucléotides d'ADN ou de dérivé 2'-fluoré de l'arabinose (2'-F-ANA) aux liens internucléotidiques phosphorothioates. Nous avons choisi de nous concentrer sur deux cibles. Bien que la stratégie de modification des deux séquences ait été similaire, elle a très différemment affecté la stabilité thermique de ces OAs. Nos OAs phosphoramidates se sont révélés capable d'inactivation de l'expression génétique de manière modérée par rapport aux contrôles non-modifiés.Des études structurelles menées sur des pARNis et OAs modifiés avec des liens phosphoramidates ont montré que la structure globale du double brin n'est que peu affectée par la présence de modifications phosphoramidates. Enfin, nous avons démontré que, bien que les modifications phosphoramidates chargées positivement soient connues pour conférer une résistance aux nucléases, les pARNis et OAs modifiés avec des fonctions phosphoramidates sont apparus moins stables dans du sérum que les contrôles

Trichain cationic lipids: the potential of their lipoplexes for gene delivery

Lipoplexes (LDs) have been prepared from DNA and positively charged vesicles composed of the helper lipid, dioleoyl l-α-phosphatidylethanolamine (DOPE) and either a dichain (DC) oxyethylated cationic lipid or their corresponding novel trichain (TC) counterpart. This is the first study using the TC lipids for the preparation of LDs and their application. Here the results of biophysical experiments characterising the LDs have been correlated with the in vitro transfection activity of the complexes. Photon correlation spectroscopy, zeta potential measurements and transmission electron microscopy studies indicated that, regardless of the presence of a third chain, there were little differences between the size and charge of the TC and DC containing LDs. Small angle neutron scattering studies established however that there was a significant conformational re-arrangement of the lipid bilayer when in the form of a LD complex as opposed to the parent vesicles. This re-arrangement was particularly noticeable in LDs containing TC lipids possessing a third chain of C12 or a longer chain. These results suggested that the presence of a third hydrophobic chain had a significant effect on lipid packing in the presence of DNA. Picogreen fluorescence and gel electrophoresis studies showed that the TC lipids containing a third acyl chain of at least C12 were most effective at complexing DNA while the TC lipids containing an octanoyl chain and the DC lipids were least effective. The transfection efficacies of the TC lipids in the form of LDs were found to be higher than for the DC analogues, particularly when the third acyl chain was an octanoyl or oleoyl moeity. Little or no increase in transfection efficiency was observed when the third chain was a methyl, acetyl or dodecanoyl group. The large enhancement in transfection performance of the TC lipids can be attributed to their ability to complex their DNA payload. These studies indicate that presence of a medium or long third acyl chain was especially beneficial for transfection

Integration of Computational and Experimental Techniques for the Discovery of SARS-CoV-2 PLpro Covalent Inhibitors

Papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro or Mpro) are enzymes essential for the replication of SARS-CoV-2, the virus responsible for COVID-19. While 3CLpro has been the main target of many potential antivirals including nirmatrelvir (active ingredient of Paxlovid), PLpro has proven to be more difficult to target and only a handful of inhibitors have been disclosed. PLpro inhibitors would be highly valuable tools in the fight against COVID19 resistant strains and in future coronavirus pandemics. Combining our experience with 3CLpro covalent inhibitors with our expertise in structure-based covalent drug discovery, we rationally designed PLpro inhibitors achieving a maximum potency of 13 µM through fusion of GRL-0617 and VIR-251. In parallel, we launched an integrated large scale virtual screening/experimental approach, identifying four novel chemical series active at micromolar concentrations against PLpro. We report herein our investigations including rational design, virtual screening, synthesis of selected structures and in vitro assays leading to novel PLpro inhibitors

Design, Synthesis and Biological Evaluation of Novel SARS-CoV-2 3CLpro Covalent Inhibitors

Severe diseases such as the ongoing COVID-19 pandemic, as well as the previous SARS and MERS outbreaks, are the result of coronavirus infections and have demonstrated the urgent need for antiviral drugs to combat these deadly viruses. Due to its essential role in viral replication and function, 3CLpro> has been identified as a promising target for the development of antiviral drugs. Previously reported SARS-CoV 3CLpro non-covalent inhibitors were used as a starting point for the development of covalent inhibitors of SARS-CoV-2 3CLpro. We report herein our efforts in design and synthesis which led to submicromolar covalent inhibitors when the enzymatic activity of the viral protease was used as a screening platform