Next generation Glucose-1-phosphate thymidylyltransferase (RmlA) inhibitors: An extended SAR study to direct future design

Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity

“…Inversion of stereochemistry at C4 in dTDP-β- l -6dTal ( 2 ) only showed inhibition against Pa RmlA. While thymidine analogues can inhibit RmlA, , these results highlight the importance of the sugar in forming contacts with RmlA, as seen in solved cocomplexes (Figure C, left). Variation of the nucleotide in UDP-β- l -Rha ( 12 ) and GDP-β- l -Rha ( 13 ) led to loss of inhibitory activity, except for weak inhibition of Pa RmlA by 12 .…”

Expanding the Substrate Scope of a Bacterial Nucleotidyltransferase via Allosteric Mutations

“…Solved crystal structures of RmlA proteins have revealed three functional subdomains: a core subdomain, a sugar binding subdomain, and a dimerization subdomain (Figure B, top left). ,, The core subdomain and sugar binding subdomain shape the main active site . The dimerization subdomain of RmlA assists in the assembly of RmlA homotetramers and helps to shape a distinct allosteric site that can bind to dTDP-β- l -Rha and other thymine-containing compounds. ,, We aimed to introduce mutations into the allosteric site of SALTY RmlA to abrogate ligand binding and to analyze the resulting effects on activity. While inhibition of SALTY RmlA by allosteric ligands is not well-characterized, there are several solved structures of P. aeruginosa RmlA with bound allosteric inhibitors. ,, SALTY RmlA and P. aeruginosa RmlA possess high sequence identities (Figure S2) (∼74%), and structural alignment (Figure S3) of SALTY RmlA and P. aeruginosa RmlA cocomplexed to dTDP-β- l -Rha indicated several conserved residues that make contacts with the allosteric ligand (Figure B, top right).…”

“…Past work has shown that derivatized thymine-based molecules can inhibit P. aeruginosa RmlA via allosteric site binding. ,, We synthesized the most potent small molecule inhibitor shown to complex RmlA from an initial report (Figure B) and tested its activity against wild-type and mutant RmlA constructs (Figure C). Surprisingly, D104N showed decreased sensitivity to 1 , even though the mutation is distant from the allosteric and active sites (Figure S1C).…”

“…44 The dimerization subdomain of RmlA assists in the assembly of RmlA homotetramers and helps to shape a distinct allosteric site that can bind to dTDP-β-L-Rha and other thymine-containing compounds. 43,44,51 We aimed to introduce mutations into the allosteric site of SALTY RmlA to abrogate ligand binding and to analyze the resulting effects on activity. While inhibition of SALTY RmlA by allosteric ligands is not well-characterized, there are several solved structures of P. aeruginosa RmlA with bound allosteric inhibitors.…”

“…While inhibition of SALTY RmlA by allosteric ligands is not well-characterized, there are several solved structures of P. aeruginosa RmlA with bound allosteric inhibitors. 43,44,51 SALTY RmlA and P. aeruginosa RmlA possess high sequence identities (Figure S2) (∼74%), and structural alignment (Figure S3) of SALTY RmlA and P. aeruginosa RmlA cocomplexed to dTDP-β-L-Rha indicated several conserved residues that make contacts with the allosteric ligand (Figure 1B, top right). Based on inspection of the overlaid structures, we made several mutations within the allosteric site of SALTY RmlA.…”