Design and synthesis of various nucleoside analogues as aminoacyl-tRNA synthetase inhibitors

Antibiotics are used to prevent and treat bacterial infections. They are essential components of modern medicine and have helped save millions of lives of people suffering from various common bacterial infections. However, due to excessive and irrational use of antibiotics, many of them are no longer effective against these resistant pathogenic microbes. This constitutes a severe risk for our ability to treat bacterial infections and for performing various medical procedures like organ transplants, chemotherapy, and surgeries. In comparison to the sharp rise in antibiotic resistance in microbes, the antibiotic discovery is moving at snail's pace. All the major antibiotic classes used commonly in current therapy were discovered 50 years ago. Therefore, it is crucial to find new antibacterial targets and simultaneously to develop new antibiotics against them. Aminoacyl-tRNA synthetases (aaRSs) have emerged as promising and clinically validated targets. This family of enzymes are responsible for the protein synthesis in a living organism. Because of their crucial role in protein synthesis, their selective inhibition would be detrimental to pathogens as exemplified by various naturally occurring antibiotics as well as synthesized compounds. Early efforts in the search for aaRS inhibitors yielded diverse sets of chemical structures, and among these mupirocin and tavaborole reached the market. The largest group of aaRS inhibitors are non-hydrolysable analogues of aminoacyl-adenylate (aa-AMP, Fig 1, structure a) where the acylated phosphate linkage has been replaced with a hydrolytically more stable acylated sulfamate linkage. The 5'-O-(N-aminoacyl)sulfamoyl adenosine (aaSAs) analogues (Fig 1, structure b) have shown to be the most potent inhibitors of the corresponding aaRSs. However, these compounds lack in vivo efficacy (due to limited uptake) and selectivity (due to structural similarity with aa-AMP). Therefore, the present chemical and biological studies have been carried out to overcome these issues. From literature, it has been observed that utilizing a non-adenine base could improve the selectivity of compounds. Inspired by these synthetic compounds and several natural antibiotics, we investigated the pharmacophoric importance of the adenine base regarding inhibitory activity and the development of resistance. Therefore, in chapter 2 and 3, we replaced the adenine base in 5'-O-(N-aminoacyl)sulfamoyl adenosine with three different bases viz. cytosine, uracil and N3-methyluracil and attached different amino acids to the sulfamoyl group (Fig 1, structure c). Three were targeting the class 1 aaRSs viz. leuRS, ileRS and tyrRS and another three belong to the class 2 aaRSs viz glyRS, serRS and aspRS. In collaboration with the bio-crystallography division, the affinity of the various compounds towards their corresponding enzymes was determined, a detailed structural analysis of the inhibitors in complex with the respective aaRS was performed, and their inhibitory activity was determined in cell extracts in the presence of N-acetyl transferases. In comparison to the parent aaSA analogues, all synthesized compounds showed a reduction in inhibitory activity. However, several analogues still displayed activity in the nanomolar range, and only slight differences in terms of interactions with the aaRSs were observed from the ligand bound X-ray crystallographic structures of the synthetases. Fig 1. The general structure of natural reaction intermediate aa-AMP, aaSA analogues, and various compounds synthesized. Next, the issue of in vivo efficacy was tackled using either a prodrug strategy or by including uptake enhancing moieties. In chapter 4 we addressed this issue with the intention to make these aaSA analogues more lipophilic by attaching an alkyl chain at N6 or by utilizing a general prodrug strategy in which we connected two different promoieties at the alpha amine of the amino acid part (Fig 1, structure d). The preliminary in vitro results for these compounds appeared to be promising and warrant further biological evaluation. Part of our work was focused on understanding the pharmacophoric importance of the aspartyl side chain for the inhibitory activity of aspartyl sulfamoyladenosine (DSA). All aaSAs are rapidly inactivated by acetylation of the alpha amine by transacetylases as found in resistant species. Likewise, the active warhead of the natural antibiotic Microcin C (McC) is an aspRS inhibitor, prone to acetylation and inactivation. Therefore, in chapter 5, we synthesized several compounds having different moieties replacing either the aspartyl side chain or the entire amino acid (Fig 1, structure e). We determined the inhibitory activity of the new derivatives with purified aspRS and performed detailed structural analysis of the synthesized compounds in complex with aspRS. It was observed that the inhibitory activity decreased already significantly only by replacement of the amine with a hydroxyl group and no inhibition was noted when simply removing the amine or substituting the amino acid for heterocyclic attachments. Finally, in chapter 6, we purified and synthesized various compounds targeting asparagine synthetase A (asnA), followed by detailed structural analysis of the synthesized compounds (Fig 1, structure f) in complex with asnA from Pyrococcus furiosus. In view of the high-resolution structures, we suggest that asnA could be used as a tool to understand the interaction of specific aspRS inhibitors, especially when having a modification in the base binding region, and provided the sulphonamide is acylated via the beta-carboxylic moiety of aspartate.status: publishe