Triazole Substitution of a Labile Amide Bond Stabilizes Pantothenamides and Improves Their Antiplasmodial Potency

The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo. In this study, we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with 50% inhibitory concentrations (IC50s) of ∼50 nM, and three others have submicromolar IC50s. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA, and show that the compound inhibits the phosphorylation of [14C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings

Targeting antibiotic resistance through a versatile pantetheine scaffold: pantetheine derivatives as AAC(6')-Ii resistance inhibitors and novel pantothenamide antibacterial agents

Aminoglycosides are broad-spectrum antibiotics used in the treatment of serious infections. With the rapid emergence and spread of antibiotic resistance, their therapeutic use is becoming increasingly threatened. The most common mechanism of aminoglycoside resistance is the expression of aminoglycoside-modifying enzymes (AMEs). Aminoglycoside 6'-N-acetyltransferase-Ii (AAC(6')-Ii) is a chromosomally-encoded enzyme in Enterococcus faecium, which transfers an acetyl group from AcCoA to the 6'-NH2 of aminoglycosides. Many inhibitors of this enzyme have been reported by the Auclair group. Initially, bisubstrate inhibitors containing aminoglycoside and CoA moieties were generated, which were nanomolar inhibitors of AAC(6')-Ii, and served as useful structural and mechanistic probes. However, the presence of negatively charged phosphate groups precluded their membrane permeation and thus a second generation of truncated bisubstrate inhibitors was subsequently reported. It was demonstrated that the diphosphate group could effectively be mimicked by an acetoacetate moiety to afford a "lead" compound which was shown to be the first inhibitor active in cells, albeit at reduced activity over the bisubstrates. The work presented in this thesis encompasses a multi-faceted approach to targeting aminoglycoside resistance through 1) the inhibition of AAC(6')-Ii; and 2) the development of a novel class of antibacterial agents. A medicinal chemistry approach was used to improve the potency of the "lead" compound inhibitor through the insertion of alternative diphosphate mimics such as a squarate ester or an acetylsulfamate. As described in Chapter 2, we hoped this would improve upon the stability of the acetoacetate group, while maintaining key hydrogen-bonding interactions to the enzyme. The synthesis of these molecules however, proved more challenging than expected, and efforts were instead focused on the work described in subsequent chapters. In Chapter 3, a rigidification strategy was employed to improve the affinity of the lead molecule for AAC(6')-Ii by reducing the entropic cost of binding. Rigidified, triazole-containing derivatives were found to have a positive effect on the affinity of inhibitors for AAC(6')-Ii, and showed comparable in-cell activity to that of the lead. Next, Chapter 4 describes the use of a previously established prodrug strategy which capitalizes on the CoA biosynthetic pathway in order to extend aminoglycoside-pantetheine derivatives into potent bisubstrate inhibitors in cells. The effect of rigidification on the activity of the prodrugs was investigated. These compounds were not active in cells, which resulted from their poor in-cell extension. Finally, pantothenamide derivatives were synthesized and tested as reported in Chapter 5. Pantothenamides constitute a promising class of antibacterial agents, which has recently gained much attention. The molecules are extended by the CoA biosynthetic enzymes into inhibitors of downstream effector proteins. Chapter 5 reports the synthesis and biological activity of novel pantothenamides which were generated as part of a large-scale study. Although the molecules reported in this thesis were not the most active of the series, the results contribute to important SARs and provide a better understanding of the selectivity of PanK, the first, and rate-limiting step of CoA biosynthesis. Contributions and experimental methods comprise the final chapters of this thesis.Les aminoglycosides sont des antibiotiques à large-spectre utilisés pour les infections sérieuses. Avec l'émergence et la propagation de la résistance antibiotique, leur usage thérapeutique devient de plus en plus précaire. Le mécanisme de résistance le plus commun est l'expression d'enzymes qui modifient les aminoglycosides (AME). Les AMEs fonctionalizent les aminoglycosides de façon régiosélective, ce qui réduit leur affinité pour le ribosome bactérien. Aminoglycoside 6'-N-acetyltransférase-Ii (AAC(6')-Ii), qui est encodé sur le chromosome d'Enterococcus faecium, transfert un groupement acétyl de AcCoA à l'amine 6' des aminoglycosides. Plusieurs inhibiteurs de cet enzyme ont été découverts par le groupe Auclair. Initialement, des inhibiteurs bisubstrats contenant une moitié aminoglycoside et une moitié CoA ont été générés, qui avaient une activité inhibitrice nanomolaire sur AAC(6')-Ii et on servis comme sondes structurelles et mécanistiques. Cependant la présence de charges négatives sur les groupements phosphates prévenaient leur activité dans des cellules vivantes. Une deuxième génération de bisubstrats tronqués ont ensuite été développés. Ces derniers ont démontré que le groupement diphosphate pouvait effectivement être imités par un groupement acétoacétyl. Une de ces molécules était le premier inhibiteur actif dans des cellules ("lead"), quoiqu'ayant une activité réduite comparé au bisubstrat original. Le travail présenté dans cette thèse englobe une approche multiforme visant à cibler la résistance aux aminoglycosides par l'inhibition d'AAC(6')-Ii et à contribuer au developpement d'une nouvelle classe d'agents antibactériens. Une approche de chimie médicinale a été utilisée pour améliorer la puissance de la molécule "lead" par l'insertion d'un ester de squarate ou d'une acétylsulfamate afin d'imiter le diphosphate. Tel que décrit dans le chapitre 2, cet approche visait à améliorer la stabilité du groupement acétoacétyl tout en conservant les intéractions clés de pont-hydrogène avec l'enzyme. La synthèse ayant posé un plus élevé qu'attendu, et nos efforts se sont rapidement concentrés sur les projets décrits dans les chapitres suivants. Tel que décrit dans le chapitre 3, une stratégie de rigidification a aussi été utilisée pour améliorer l'affinité et l'activité inhibitrice du composé d'intérêt pour AAC(6')-Ii en réduisant l'entropie de liaison. Les derivés rigidifiés ont amelioré l'affinité des inhibiteurs envers AAC(6')-Ii, et ont demontré une activité cellulaire similaire à celle du "lead." Le chapitre 4 décrit l'usage d'une approche précédemment utilisée et basée sur une pro-drogue qui exploite la voie de biosynthèse de CoA pour allonger les dérivés aminoglycoside-pantothéine en bisubstrats inhibiteurs dans les cellules. L'effet de la rigidification de ces molecules sur leur activité a été investiguées. Ces composés ce sont cependant montrés inactifs dans les cellules, probablement en raison d'un faible taux d'activation. Finalement, des dérivés pantothénamides ont été synthétisés et évalués comme décrit dans le chapitre 5. Les pantothénamides sont une nouvelle classe d'agents antibactériens qui a récemment jouit de beaucoup d'attention. Ces molécules sont transformées par les enzymes de biosynthèse de CoA pour ensuite affecter les enzymes qui utilizent CoA. Le chapitre 5 explore des nouvelles pantothénamides qui ont été générés dans le cadre d'une étude à large spectre. Quoique les molécules rapportées dans cette thèse ne soient pas les plus actifs, les resultats obtenuent contribuent à révéler d'importantes relations structures-activité et offrent de la séléctivité de PanK, l'enzyme limitante dans la biosynthèse de CoA. Les contributions et les méthodes expérimentales composent les deux dernier chapitres de cette thèse

Probing the ligand preferences of the three types of bacterial pantothenate kinase

Pantothenate kinase (PanK) catalyzes the transformation of pantothenate to 4′-phosphopantothenate, the first committed step in coenzyme A biosynthesis. While numerous pantothenate antimetabolites and PanK inhibitors have been reported for bacterial type I and type II PanKs, only a few weak inhibitors are known for bacterial type III PanK enzymes. Here, a series of pantothenate analogues were synthesized using convenient synthetic methodology. The compounds were exploited as small organic probes to compare the ligand preferences of the three different types of bacterial PanK. Overall, several new inhibitors and substrates were identified for each type of PanK

Stereochemical modification of geminal dialkyl substituents on pantothenamides alters antimicrobial activity

Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial activity through interference with coenzyme A (CoA) biosynthesis or downstream CoA-utilizing proteins. A previous report has shown that replacement of the ProR methyl group of the benchmark N- pentylpantothenamide with an allyl group (R-anti configuration) yielded one of the most potent antibacterial pantothenamides reported so far (MIC of 3.2 μM for both sensitive and resistant Staphylococcus aureus). We describe herein a synthetic route for accessing the corresponding R-syn diastereomer using a key diastereoselective reduction with Baker's yeast, and report on the scope of this reaction for modified systems. Interestingly, whilst the R-anti diastereomer is the only one to show antibacterial activity, the R-syn isomer proved to be significantly more potent against the malaria parasite (IC50 of 2.4 ± 0.2 μM). Our research underlines the striking influence that stereochemistry has on the biological activity of pantothenamides, and may find utility in the study of various CoA-utilizing systems

Stereochemical modification of geminal dialkyl substituents on pantothenamides alters antimicrobial activity

Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial activity through interference with coenzyme A (CoA) biosynthesis or downstream CoA-utilizing proteins. A previous report has shown that replacement of the ProR methyl group of the benchmark N-pentylpantothenamide with an allyl group (R-anti configuration) yielded one of the most potent antibacterial pantothenamides reported so far (MIC of 3.2 μM for both sensitive and resistant Staphylococcus aureus). We describe herein a synthetic route for accessing the corresponding R-syn diastereomer using a key diastereoselective reduction with Baker’s yeast, and report on the scope of this reaction for modified systems. Interestingly, whilst the R-anti diastereomer is the only one to show antibacterial activity, the R-syn isomer proved to be significantly more potent against the malaria parasite (IC50 of 2.4 ± 0.2 μM). Our research underlines the striking influence that stereochemistry has on the biological activity of pantothenamides, and may find utility in the study of various CoA-utilizing systems

Optimizing the shipbuilding layout of Damex Shipbuilding and Engineering for cost efficiency

In this master thesis the shipyard layout of Damex Shipbuilding and Engineering is optimized for cost efficiency.Ship ProductionDPOMechanical, Maritime and Materials Engineerin

The Cardioprotective Effects of Semaglutide Exceed Those of Dietary Weight Loss in Mice With HFpEF

Obesity-related heart failure with preserved ejection fraction (HFpEF) has become a well-recognized HFpEF subphenotype. Targeting the unfavorable cardiometabolic profile may represent a rational treatment strategy. This study investigated semaglutide, a glucagon-like peptide-1 receptor agonist that induces significant weight loss in patients with obesity and/or type 2 diabetes mellitus and has been associated with improved cardiovascular outcomes. In a mouse model of HFpEF that was caused by advanced aging, female sex, obesity, and type 2 diabetes mellitus, semaglutide, compared with weight loss induced by pair feeding, improved the cardiometabolic profile, cardiac structure, and cardiac function. Mechanistically, transcriptomic, and proteomic analyses revealed that semaglutide improved left ventricular cytoskeleton function and endothelial function and restores protective immune responses in visceral adipose tissue. Strikingly, treatment with semaglutide induced a wide array of favorable cardiometabolic effects beyond the effect of weight loss by pair feeding. Glucagon-like peptide-1 receptor agonists may therefore represent an important novel therapeutic option for treatment of HFpEF, especially when obesity-related.</p

The Cardioprotective Effects of Semaglutide Exceed Those of Dietary Weight Loss in Mice With HFpEF

Obesity-related heart failure with preserved ejection fraction (HFpEF) has become a well-recognized HFpEF subphenotype. Targeting the unfavorable cardiometabolic profile may represent a rational treatment strategy. This study investigated semaglutide, a glucagon-like peptide-1 receptor agonist that induces significant weight loss in patients with obesity and/or type 2 diabetes mellitus and has been associated with improved cardiovascular outcomes. In a mouse model of HFpEF that was caused by advanced aging, female sex, obesity, and type 2 diabetes mellitus, semaglutide, compared with weight loss induced by pair feeding, improved the cardiometabolic profile, cardiac structure, and cardiac function. Mechanistically, transcriptomic, and proteomic analyses revealed that semaglutide improved left ventricular cytoskeleton function and endothelial function and restores protective immune responses in visceral adipose tissue. Strikingly, treatment with semaglutide induced a wide array of favorable cardiometabolic effects beyond the effect of weight loss by pair feeding. Glucagon-like peptide-1 receptor agonists may therefore represent an important novel therapeutic option for treatment of HFpEF, especially when obesity-related.</p