The identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

Abstract

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.

The Structural and Functional Basis for Recurring Sulfa Drug Resistance Mutations in Staphylococcus aureus Dihydropteroate Synthase

“…Our studies reveal the same basic mechanism of resistance in DHPS but also show the importance of secondary mutations that can partially offset the negative impact of primary resistance mutations on enzyme function. Developing inhibitors that only occupy the volume assumed by native substrates will continue to be a key strategy in our drug discovery efforts on DHPS and other key enzymes in the folate pathway (Yun et al, 2014 ). An important conclusion from our studies is that continued development of the sulfonamide scaffold focused on the ring extending beyond the native substrate binding pocket is fundamentally flawed.…”

Structural Basis for the Selective Binding of Inhibitors to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Staphylococcus aureus and Escherichia coli

“…In particular, compounds 13 – 15 were noted to bind to SaHPPK ( K D = 0.57, 0.60, 0.69 μM, respectively) and EcHPPK ( K D = 0.70, 0.82, 0.92 μM, respectively) with remarkably similar affinities. The apparent 13-fold weaker EcHPPK vs SaHPPK binding reported previously for 14 , is attributable instead to the different SPR conditions employed in the two separate studies. Specifically, a subsaturation concentration of the cofactor analogue AMPCPP was employed in the EcHPPK SPR experiments of Yun et al compared to saturated levels of ATP in our studies …”

“…Owing to differences in instrumentation and experimental setup, including the method of surface coupling and enzyme capture, we measured the affinity of some previously reported 8MG derivatives , alongside our new series of derivatives for direct reference (Tables , , and ). Notably, the cysteine-conjugated biotinylated SaHPPK used in this study returned affinities approximately 2-fold tighter than those previously obtained with SaHPPK immobilized directly via one or more lysine residues …”

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase–dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance

“…It is noteworthy that the PvHPPK-DHPS residues involved in recognition of pterin moiety in both domains are highly conserved. This presents yet another opportunity to target conserved motifs within PvHPPK-DHPS now that the crystal structure is available (37). The presented PvHPPK-DHPS structure indicates that sulfa-drug resistance mutations emanate from a structural compromise in the mutant drug resistance enzyme that enables rejection of the drug while minimally altering affinity for its substrate pABA.…”

“…The presented PvHPPK-DHPS structure indicates that sulfa-drug resistance mutations emanate from a structural compromise in the mutant drug resistance enzyme that enables rejection of the drug while minimally altering affinity for its substrate pABA. Although our pABA-bound PvHPPK-DHPS crystal structure can explain SDX resistance for most mutations, we feel that SDX-bound crystal structures of mutant and WT PvDHPSs are required for a deeper understanding of this enzyme/drug system (37). From our structural analysis of PvHPPK-DHPS and its mutations in context of sulfadoxine resistance, we have generated several insights including (a) the presented structure should be exploited to identify nonsulfa drugs that do not mimic pABA and thus inhibit the enzyme irreversibly, (b) the Plasmodium HPPK domain can now be utilized for focusing on pterin-based inhibitors (38), (c) designing drugs that target the triple mutant in PvHPPK-DHPS will be valuable because they can be selectively administered in regions of prevalent SDX resistance, and (d) twin targeting of Plasmodium HPPK and the DHPS subdomains within PvHPPK-DHPS may provide more potent inhibition of the enzyme.…”