Crystal Structures of Klebsiella pneumoniae Dihydrofolate Reductase Bound to Propargyl-Linked Antifolates Reveal Features for Potency and Selectivity

Abstract

Resistance to the antibacterial antifolate trimethoprim (TMP) is increasing in members of the family Enterobacteriaceae, driving the design of next-generation antifolates effective against these Gram-negative pathogens. The propargyl-linked antifolates are potent inhibitors of dihydrofolate reductases (DHFR) from several TMP-sensitive and -resistant species, including Klebsiella pneumoniae. Recently, we have determined that these antifolates inhibit the growth of strains of K. pneumoniae, some with MIC values of 1 g/ml. In order to further the design of potent and selective antifolates against members of the Enterobacteriaceae, we determined the first crystal structures of K. pneumoniae DHFR bound to two of the propargyl-linked antifolates. These structures highlight that interactions with Leu 28, Ile 50, Ile 94, and Leu 54 are necessary for potency; comparison with structures of human DHFR bound to the same inhibitors reveal differences in residues (N64E, P61G, F31L, and V115I) and loop conformations (residues 49 to 53) that may be exploited for selectivity.

Crystal Structures of Trimethoprim-Resistant DfrA1 Rationalize Potent Inhibition by Propargyl-Linked Antifolates

“…Enzyme inhibition assays were performed as previously described by monitoring the rate of NADPH oxidation by DHFR at an absorbance of 340 nm(19). Assays were carried out in a buffer containing 20 mM TES pH 7.0, 50 mM KCl, 10 mM 2-mercaptoethanol, 0.5 mM EDTA and 1 mg/mL BSA.…”

“…Structural alignment of the sequences of the wild-type DHFR from E. coli (PDB 1DRE) (20), K. pneumoniae (PDB ID 4OR7) (19), and Staphylococcus aureus (PDB ID 3SGY) (18). The sequences of DfrA1 from E. coli (GenBank ADH82150.1) and DfrA1 from K. pneumoniae (GenBank ADH82140.1) are aligned to the DHFR enzymes with structures.…”

“…Recently, we reported the activity of several of these compounds against K. pneumoniae and we reported the crystal structure of K. pneumoniae DHFR (KpDHFR) bound to two PLA compounds (19). Here, we expand on that work to report that several of the PLAs not only inhibit the wild-type DHFR enzymes from Enterobacteriaceae but also significantly inhibit the DfrA1 enzyme as well.…”

Antibacterial Antifolates: From Development through Resistance to the Next Generation

“…2) is currently under development to inhibit both Gram-positive and Gram-negative bacteria; they are specifically designed to target both trimethoprim-sensitive and trimethoprim-resistant enzymes. Using a structure-based approach founded on the determination of several tens of structures of DHFR from many species, including S. aureus (Frey et al 2009), mutant forms of S. aureus (Frey et al 2009, 2010; Keshipeddy et al 2015), E. coli , K. pneumoniae (Lamb et al 2014), and plasmid-encoded trimethoprim-resistant DHFR such as DfrA1 (Lombardo et al 2015), new DHFR inhibitors were designed to overcome mutations that cause TMP resistance as well as whole enzymes that are horizontally transferred for resistance. The development of these compounds has led to very potent inhibitors of wild-type and mutant forms of S. aureus DHFR as well as the growth of the S. aureus (wild-type and mutant) bacteria.…”

MRSA Isolates from United States Hospitals Carry dfrG and dfrK Resistance Genes and Succumb to Propargyl-Linked Antifolates

“…Over the past decade, we have focused on the development of next generation propargyl-linked antifolates (PLAs) that maintain activity against many of the important pathogenic bacterial DHFR enzymes while expanding coverage to include both mutant and naturally TMP-insensitive DHFR enzymes that give rise to TMP resistance within S. aureus (Frey et al, 2009, 2010a, 2012; Keshipeddy et al, 2015; Lamb et al, 2014; Lombardo et al, 2016). The occurrence of a common F98Y mutation shared by resistant S. aureus mutants and plasmid-encoded DHFRs provided rationale that new antifolates that are effective against this mutant could expand coverage for these resistant enzymes.…”