Synthesis of Stable NAD⁺ Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC

Abstract

Stable NAD + analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD +-consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD + analogues, namely carbanicotinamide adenosine dinucleotide (c-NAD +), thionicotinamide adenosine dinucleotide (S-NAD +) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD + mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD + analogues in a scalable manner. We then compared the three NAD + mimics side-by-side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S-NAD + and BAD with IC 50 values of 28 and 39 μM, respectively.

Exploitation of the Host Ubiquitin System: Means by Legionella pneumophila

“…The SidE effector family has long known to be important for optimal virulence of L. pneumophila ( Song et al, 2021 ), and targeting the unique ubiquitin ligase activity with compounds is an anti-virulence strategy to control L. pneumophila infection. Several stable NAD + analogs capable of inhibiting the activity of SdeC at IC 50 of 28–39 μM have been developed ( Madern et al, 2020 ). It remains unclear whether these compounds can block intracellular replication of the pathogen.…”

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

“…The fluorescent derivative etheno-NAD + has been used to determine NAD + hydrolase activity of especially NADases and ADPribosylating toxins. [43,51] In this assay, the NAD + analog nicotinamide 1,N 6 -ethenoadenine dinucleotide is used as a substrate to produce a fluorescent product upon hydrolysis. The assay can be used for recombinant enzymes, cells, or tissue lysates.…”

C-Ribosylating Enzymes in the (Bio)Synthesis of C-Nucleosides and C-Glycosylated Natural Products

“…The C-nucleoside remdesivir (13) has drawn attention considerably for treating infections with SARS-CoV-2. 77,78 Related compounds like SRO-91 (14) and benzamide adenosine dinucleotide (15) are of interest for drug use against cancer 79−83 and bacterial, 84 as well as viral, infections. 84−86 Additionally, Ψ (2a) and its N1-methylated derivative (2b) are essential constituents of modern mRNA vaccines.…”

“…Although our focus here is on naturally occurring C -ribosylated molecules, we briefly consider synthetic C -β-riboside compounds. The C -nucleoside remdesivir ( 13 ) has drawn attention considerably for treating infections with SARS-CoV-2. , Related compounds like SRO-91 ( 14 ) and benzamide adenosine dinucleotide ( 15 ) are of interest for drug use against cancer − and bacterial, as well as viral, infections. − Additionally, Ψ ( 2a ) and its N1-methylated derivative ( 2b ) are essential constituents of modern mRNA vaccines. They also play a major role in the current therapeutic mRNA revolution. ,,,− Synthetic C -nucleosides, such as dZ ( 16 ) and NaM ( 17 ), are explored in synthetic biology as building blocks of xenobiotic nucleic acids. − Their enhanced stability compared with canonical N -nucleosides offers great advantage in synthetic biology applications. ,,, …”