The structure of Plasmodium falciparum hydroxymethyldihydropterin pyrophosphokinase‐dihydropteroate synthase reveals the basis of sulfa resistance

Abstract

The clinical efficacy of sulfa drugs as antimalarials has declined owing to the evolution of resistance in Plasmodium falciparum (Pf) malaria parasites. In order to understand the basis of this resistance and to design more effective antimalarials, we have solved 13 structures of the bifunctional enzyme 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (HPPK)–dihydropteroate synthase (DHPS) from wild‐type (WT) P. falciparum and sulfa‐resistant mutants, both as apoenzyme and as complexes with pteroate (PTA) and sulfa derivatives. The structures of these complexes show that PTA, which effectively inhibits both the WT and mutants, stays in active sites without steric constraint. In contrast, parts of the sulfa compounds situated outside of the substrate envelope are in the vicinity of the resistance mutations. Steric conflict between compound and mutant residue along with increased flexibility of loop D2 in the mutants can account for the reduced compound binding affinity to the mutants. Kinetic data show that the mutants have enhanced enzyme activity compared with the WT. These PfDHPS structural insights are critical for the design of novel, substrate envelope–compliant DHPS inhibitors that are less vulnerable to resistance mutations.DatabasesThe data reported in this paper have been deposited in the Protein Data Bank, www.wwpdb.org. PDB ID codes: 6JWQ for apoWT; 6JWR, 6JWS, and 6JWT for PTA complexes of WT, A437G (3D7), and V1/S; 6JWU, 6JWV, and 6JWW for STZ‐DHP complexes of WT, 3D7, and V1/S; 6JWX, 6JWY, and 6JWZ for SDX‐DHP complexes of WT, 3D7, and W2; 6KCK, 6KCL, and 6KCM for Pterin/pHBA complexes of WT, TN1, and W2.

Plasmodium falciparum DHFR and DHPS Mutations Are Associated With HIV-1 Co-Infection and a Novel DHPS Mutation I504T Is Identified in Western Kenya

“…The minor frequency clone (1%) included the I504T mutation along with A437G and K540E while the major clone included only A437G and K540E. While A437G and K540E are well known sulfa drug resistance mutations ( Chitnumsub et al, 2019 ), the role of I504T is unknown, so we developed a homology model to predict substrate and drug binding interactions. The newly published P. falciparum DHPS model ( Chitnumsub et al, 2019 ) included 13 crystal structures of different mutants in complex with sulfa drugs.…”

“…While A437G and K540E are well known sulfa drug resistance mutations ( Chitnumsub et al, 2019 ), the role of I504T is unknown, so we developed a homology model to predict substrate and drug binding interactions. The newly published P. falciparum DHPS model ( Chitnumsub et al, 2019 ) included 13 crystal structures of different mutants in complex with sulfa drugs. Supplemental Figure 2A shows the superposition of our homology modeled structure of P. falciparum DHPS (violet) on the crystal structure (yellow) from Chitnumsub et al (2019) .…”

“…The newly published P. falciparum DHPS model ( Chitnumsub et al, 2019 ) included 13 crystal structures of different mutants in complex with sulfa drugs. Supplemental Figure 2A shows the superposition of our homology modeled structure of P. falciparum DHPS (violet) on the crystal structure (yellow) from Chitnumsub et al (2019) . The structural similarity is very high with an RMSD of 0.8 Å.…”

“…The 3D structure of P. falciparum DHPS was not available at the time this study was initiated but has recently become available ( Chitnumsub et al, 2019 ). Accordingly, we used a crystal structure of P. vivax DHPS in complex with substrates 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and 4-aminobenzoic acid ( p ABA) ( Yogavel et al, 2018 ) to build a homology model of P. falciparum DHPS, which we subsequently compared to the published structure ( Chitnumsub et al, 2019 ).…”

“…The 3D structure of P. falciparum DHPS was not available at the time this study was initiated but has recently become available ( Chitnumsub et al, 2019 ). Accordingly, we used a crystal structure of P. vivax DHPS in complex with substrates 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and 4-aminobenzoic acid ( p ABA) ( Yogavel et al, 2018 ) to build a homology model of P. falciparum DHPS, which we subsequently compared to the published structure ( Chitnumsub et al, 2019 ). The experimental structure of P. vivax DHPS in complex with DHPPP and p ABA substrates was downloaded from the Protein Data Bank (PDB) server (PDB ID: 5Z79) ( Manickam et al, 2018 ) and the structure prediction wizard from the PRIME module of Schrodinger suite was used for homology modeling ( Jacobson et al, 2002 ; Jacobson et al, 2004 ).…”