Crystal Structures of Candida albicans Dihydrofolate Reductase Bound to Propargyl‐Linked Antifolates Reveal the Flexibility of Active Site Loop Residues Critical for Ligand Potency and Selectivity

Abstract

Candida albicans and Candida glabrata cause fungal bloodstream infections that are associated with significant mortality. As part of an effort to develop potent and selective antifolates that target dihydrofolate reductase (DHFR) from Candida species, we report three ternary crystal structures of Candida albicans DHFR (CaDHFR) bound to novel propargyl-linked analogs. Consistent with earlier modeling results, these structures show that hydrophobic pockets in the binding site may be exploited to increase ligand potency. The crystal structures also confirm that loop residues Thr 58- Phe 66, which flank the active site and influence ligand potency and selectivity, adopt multiple conformations. To aid the development of a dual Candida spp. inhibitor, three new crystal structures of C. glabrata DHFR (CgDHFR) bound to similar ligands as those bound in the ternary structures of CaDHFR are also reported here. Loop residues 58-66 in CgDHFR and human DHFR are 1 Å and 3 Å closer to the folate binding site, respectively, than loop residues in CaDHFR, suggesting that a properly size ligand could be a potent and selective dual inhibitor of CaDHFR and CgDHFR.

Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities

“…B. anthracis (PDB ID: 3E0B), C. albicans (PDB ID: 3QLW), C. glabrata (PDB ID: 3CSE) and S. aureus (PDB ID: 3FQ0), with cocrystallized N22. The IC 50 values of N22 for these enzymes were 890 nM [47], 100 nM [48], 8.2 nM [49] and 68 nM [50], respectively. Table 6A provides the list of significant stabilizing and destabilizing contacts made by N22 in these DHFRs.…”

Acetylenic Linkers in Lead Compounds: A Study of the Stability of the Propargyl-Linked Antifolates

“…The crystal structures of C. glabrata DHFR and S. aureus DHFR bound to biphenyl-propargyl-linked antifolates (Frey et al, 2009(Frey et al, , 2010aPaulsen et al, 2011) guided the placement of solubilityenhancing functionality. In particular, these structures reveal that although substitutions at the meta and para positions of the aryl ring interact productively with the enzyme, the region is largely solvent exposed ( Fig.…”

“…1) (Pelphrey et al, 2007;Bolstad et al, 2008;Liu et al, 2008Liu et al, , 2009Paulsen et al, 2009). Crystal structures of DHFR from Staphylococcus aureus (Frey et al, 2009(Frey et al, , 2010bViswanathan et al, 2012), Candida glabrata, Candida albicans (Liu et al, 2008Paulsen et al, 2011), and Bacillus anthracis (Beierlein et al, 2008) reveal that the propargyl linker occupies a narrow space bridging two critical pockets in the enzyme, one of which binds the diaminopyrimidine group and the other that is primarily hydrophobic. Using these crystal structures, several compounds with this generalized scaffold and a biphenyl moiety (example compound 1 in Fig.…”

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

“…The value of K m and V max for FH 2 was determined to be 0.14 mM and 0.053 mM min characterized DHFR proteins revealed the presence of certain conserved motifs and residues (Fig. S2), including a motif Ile-Val-Ala(7-9) involved in contact with the 6-methylpteridine moiety of MTX; residue Trp24 that interacts with the carboxamide of NADPH; two residues (Ala31 and Arg81) involved in the MTX binary complex; residue Asp29 involved in the hydride transfer of NADPH; residue Leu26 in regions that form hydrogen bonds between Met20 and the F-G or G-H loops; three residues (Ile16, Ile22 and Ile145) that would overlap partially with atoms of the nicotinamide rings of NADPH (Liu et al, 2008;Schnell et al, 2004); a NADPH binding residue Asp175 that is involved in a critical hydrogen bonding interaction with the Met20 loop that stabilizes the closed active-site conformation (Schnell et al, 2004); four residues (Ile6, Met30, Phe33 and Tyr151) interacting with the PYR ring of the inhibitor (Liu et al, 2008;Paulsen et al, 2011); a dimethoxyphenyl ring fits nicely in a hydrophobic pocket composed of Met30, Thr67, Ile71, Pro72, Phe75 and Leu78 (Liu et al, 2008); and loop residues Thr67-Phe75 critical for ligand potency and selectivity (Paulsen et al, 2011). Previous evolutionary analysis of DHFRs from 233 species identified three prominent phylogenetically coherent events (PCEs), with strong implications regarding their importance in the preservation and divergence of enzyme functions through time (Liu et al, 2013).…”