Fragment‐Linked Purine Derivatives as Inhibitors of (p)ppGpp Synthetases: Design, Synthesis, and Structure‐Activity Relationships

ABSTRACT

The bacterial stringent response, mediated by the alarmone nucleotides (p)ppGpp, plays a central role in persistence of infections, stress adaptation, and antimicrobial tolerance, making (p)ppGpp synthetases attractive targets for novel antibacterial strategies/therapies. In this study, we report the design, synthesis, and biological evaluation of fragment‐linked nucleobase derivatives targeting the RelA/SpoT homolog (RSH) enzymes. Guided by structural insights into adenine and guanine binding within small alarmone synthetase (SAS2/RelP), a series of purine‐based compounds was developed to simultaneously occupy conserved nucleotide‐binding regions. Initial linker‐bridged analogues showed limited activity, primarily due to poor solubility, prompting the introduction of ribose‐containing derivatives to improve physicochemical properties. Biochemical evaluation using HPLC‐based assays revealed selective inhibition of Staphylococcus aureus SAS1, with the most potent compound displaying micromolar activity (IC ₅₀  = 1.8 µM), while little or no inhibition was observed for related SAS enzymes. Structure‐activity relationship analysis highlights the importance of linker length and nucleobase positioning for enzyme selectivity. These findings establish fragment‐linked nucleobase scaffolds as a promising starting point for the development of inhibitors targeting (p)ppGpp biosynthesis and the bacterial stringent response.