Emerging antibiotic resistance in pathogenic bacteria and reduction of compounds in the existing antibiotics discovery pipeline is the most critical concern for healthcare professionals. A potential solution aims to explore new or existing targets/compounds. Inhibition of bacterial aminoacyl-tRNA synthetase (aaRSs) could be one such target for the development of antibiotics. The aaRSs are a group of enzymes that catalyze the transfer of an amino acid to their cognate tRNA and therefore play a pivotal role in translation. Thus, selective inhibition of these enzymes could be detrimental to microbes. The 5′-O-(N-(L-aminoacyl)) sulfamoyladenosines (aaSAs) are potent inhibitors of the respective aaRSs, however due to their polarity and charged nature they cannot cross the bacterial membranes. In this work, we increased the lipophilicity of these existing aaSAs in an effort to promote their penetration through the bacterial membrane. Two strategies were followed, either attaching a (permanent) alkyl moiety at the adenine ring via alkylation of the N⁶-position or introducing a lipophilic biodegradable prodrug moiety at the alpha-terminal amine, totaling eight new aaSA analogues. All synthesized compounds were evaluated in vitro using either a purified Escherichia coli aaRS enzyme or in presence of total cellular extract obtained from E. coli. The prodrugs showed comparable inhibitory activity to the parent aaSA analogues, indicating metabolic activation in cellular extracts, but had little effect on bacteria. During evaluation of the N⁶-alkylated compounds against different microbes, the N⁶-octyl containing congener 6b showed minimum inhibitory concentration (MIC) of 12.5 µM against Sarcina lutea while the dodecyl analogue 6c displayed MIC of 6.25 µM against Candida albicans.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N¹-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N⁹-position, we obtained the N³-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N⁷-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these “base-flipped” analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N³-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N¹ and the N⁶-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

BK polyomavirus (BKPyV) reactivation poses a serious threat to both renal and hematopoietic transplant outcomes, with no approved antiviral therapies. Neutralizing monoclonal antibodies targeting the viral capsid protein VP1 have recently advanced into clinical development, driven by promising in vitro potency. Since naturally elicited neutralizing antibodies arise following infection, we examined whether host immune pressure may have shaped the VP1 surface, with implications for therapeutic application. To probe the impact of VP1 coevolution on therapeutic engagement, we evaluated two neutralizing antibody formats: the clinical-stage monoclonal antibody 319C07 and nanobodies VHH16 and VHH17. Our X-ray crystallographic analysis of the VP1 pentameric core in complex with antibody fragments showed a convergent receptor- mimetic engagement of the sialic acid-binding cleft, mapping to a shared epitope centered around the VP1 BC-loop. Analysis of over 900 BKPyV VP1 sequences from public databases, combined with longitudinal VP1 sequence data from transplant recipients, highlighted widespread pre-existing diversity at the BC-loop contact sites, underscoring the evolutionary adaptability of BKPyV at this antigenic surface that may compromise therapeutic recognition. Through integrated mutagenesis and binding analyses, we show that circulating BC-loop mutations, including single amino acid substitutions, are sufficient to abrogate 319C07 binding and neutralization. Nanobodies VHH16 and VHH17, selected for their compact size and potential to access intrarenal sites of BKPyV replication, also exhibited complete loss of binding across multiple clinically observed VP1 variants. Our findings may offer a mechanistic framework to interpret the translational gap between in vitro neutralization and clinical efficacy of these biologics. As part of ongoing efforts to target VP1, we demonstrate, for the first time, small molecules with nanomolar to sub-nanomolar affinities against BKPyV VP1 variants including potent binding to JCPyV VP1. Our work paves a path towards a new class of antiviral strategies that could block replication at intracellular replication sites, provide a higher genetic barrier to resistance and the potential to address both viral reactivation and persistent, high- burden viremia in transplant settings.