Structure-Based Design and Development of Functionalized Mercaptoguanine Derivatives as Inhibitors of the Folate Biosynthesis Pathway Enzyme 6‑Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from <i>Staphylococcus aureus</i>

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), an enzyme from the folate biosynthesis pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin and is a yet-to-be-drugged antimicrobial target. Building on our previous discovery that 8-mercaptoguanine (8MG) is an inhibitor of Staphylococcus aureus HPPK (SaHPPK), we have identified and characterized the binding of an S8-functionalized derivative (<b>3</b>). X-ray structures of both the SaHPPK/<b>3</b>/cofactor analogue ternary and the SaHPPK/cofactor analogue binary complexes have provided insight into cofactor recognition and key residues that move over 30 Å upon binding of <b>3</b>, whereas NMR measurements reveal a partially plastic ternary complex active site. Synthesis and binding analysis of a set of analogues of <b>3</b> have identified an advanced new lead compound (<b>11</b>) displaying >20-fold higher affinity for SaHPPK than 8MG. A number of these exhibited low micromolar affinity for dihydropteroate synthase (DHPS), the adjacent, downstream enzyme to HPPK, and may thus represent promising new leads to bienzyme inhibitors