Ligand-Induced Conformational Changes in the Crystal Structures ofPneumocystis cariniiDihydrofolate Reductase Complexes with Folate and NADP+ ,

dPneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with K i values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased K m values for dihydrofolic acid (DHFA). The catalytic rate constant (k cat ) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of k cat , as well as a K m for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

Section: Resultsmentioning

confidence: 99%

“…The deduced structure of P. jirovecii DHFR was calculated by homology modeling to the crystal structure of the P. carinii DHFR ternary complex with methotrexate and NADPH (15)(16)(17). The homology data for the pjDHFR complex with OAAG324 ( Fig.…”

Section: Fig 1 Schematics Of the Pjdhfr Inhibitors Trimethoprim And Omentioning

confidence: 99%

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

Queener

Cody

Pace

et al. 2013

“…The loop on one side of the site extends from residues 20 to 33, partially encompassing helix A, and is believed to contain regulatory elements for binding in some bacterial species (19,44). The largest differences are seen at residues 21 to 25, with shifts in the main chain conformation of Ͼ1 Å relative to the MTX complex (see Fig.…”

Section: Structure Of B Anthracis Dhfr-rab1 Complexmentioning

confidence: 99%

Crystal Structure of Bacillus anthracis Dihydrofolate Reductase with the Dihydrophthalazine-Based Trimethoprim Derivative RAB1 Provides a Structural Explanation of Potency and Selectivity

Bunce

Bourne²,

Berlin

et al. 2009

Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target. In the present work, we have synthesized one compound (RAB1) displaying favorable 50% inhibitory concentration (54 nM) and MIC (<12.8 g/ml) values. RAB1 was cocrystallized with the B. anthracis DHFR in the space group P2 1 2 1 2 1 , and X-ray diffraction data were collected to a 2.3-Å resolution. Binding of RAB1 causes a conformational change of the side chain of Arg58 and Met37 to accommodate the dihydrophthalazine moiety. Unlike the natural substrate or trimethoprim, the dihydrophthalazine group provides a large hydrophobic anchor that embeds within the DHFR active site and accounts for its selective inhibitory activity against B. anthracis.

“…Since immunosuppressed patients and those with AIDS are severly affected by these pathogens, efforts have been focused on the design of antifolates that are selective against P. carinii DHFR (pc DHFR) and T. gondii (or tgDHFR) (12, 18, 22-24, 27, 28, 33, 42, 44, 48). In most of these studies, antifolate selectivity reported as a ratio of 50% inhibitory concentrations (IC 50 s) from two DHFR species was measured against crude DHFR preparations from rat liver (44,47,48).Evidence from sequence analysis and three-dimensional crystal structures of DHFR from many species shows that there is a high degree of conservation at the primary sequence and structural level among DHFRs (8,[10][11][12][13][14]17,19,29,34,36,37,41,43,50,53,55). However, kinetic and biochemical characterization data reveal differences in the mechanism of action that result in significant species specificity by selected inhibitors (15-17, 21, 25, 26, 31, 36, 39, 43).…”

mentioning

confidence: 99%

“…Evidence from sequence analysis and three-dimensional crystal structures of DHFR from many species shows that there is a high degree of conservation at the primary sequence and structural level among DHFRs (8,[10][11][12][13][14]17,19,29,34,36,37,41,43,50,53,55). However, kinetic and biochemical characterization data reveal differences in the mechanism of action that result in significant species specificity by selected inhibitors (15-17, 21, 25, 26, 31, 36, 39, 43).…”

mentioning

confidence: 99%

Isolation of Rat Dihydrofolate Reductase Gene and Characterization of Recombinant Enzyme

Wang

Bruenn

Queener

et al. 2001

Self Cite

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.Dihydrofolate reductase (DHFR) catalyzes the NADPHdependent reduction of dihydrofolate to tetrahydrofolate, which serves as a substrate for a number of one-carbon transfer reactions in purine and pyrimidine synthesis, including that of thymidylate. DHFR, along with other enzymes in the folate metabolic pathway, is critical for the biosynthesis of DNA, RNA, and certain amino acids (2-4, 51). Consequently, inhibition of DHFR enzymatic activity depletes the tetrahydrofolate pool inside the cell and inhibits DNA synthesis, subsequently leading to cell death. For this reason, DHFR has been studied extensively and many antifolates have been synthesized and tested as potential candidates for drugs (5, 19-24, 28, 30, 35, 44-48, 52). More recently, antifolates have been shown effective against such opportunistic infectious agents as Pneumocystis carinii and Toxoplasma gondii (1, 6, 9, 10). Since immunosuppressed patients and those with AIDS are severly affected by these pathogens, efforts have been focused on the design of antifolates that are selective against P. carinii DHFR (pc DHFR) and T. gondii (or tgDHFR) (12, 18, 22-24, 27, 28, 33, 42, 44, 48). In most of these studies, antifolate selectivity reported as a ratio of 50% inhibitory concentrations (IC 50 s) from two DHFR species was measured against crude DHFR preparations from rat liver (44,47,48).Evidence from sequence analysis and three-dimensional crystal structures of DHFR from many species shows that there is a high degree of conservation at the primary sequence and structural level among DHFRs (8,[10][11][12][13][14]17,19,29,34,36,37,41,43,50,53,55). However, kinetic and biochemical characterization data reveal differences in the mechanism of action that result in significant species specificity by selected inhibitors (15-17, 21, 25, 26, 31, 36, 39, 43). For example, trimethoprim (TMP) [2,4-diamino-5...