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Literature summary for 1.5.1.3 extracted from

  • Lamb, K.M.; Lombardo, M.N.; Alverson, J.; Priestley, N.D.; Wright, D.L.; Anderson, A.C.
    Crystal structures of Klebsiella pneumoniae dihydrofolate reductase bound to propargyl-linked antifolates reveal features for potency and selectivity (2014), Antimicrob. Agents Chemother., 58, 7484-7491 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Klebsiella pneumoniae

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant His-tagged enzyme in complex with NADPH and inhibitor 3 or 4, sitting drop vapour diffusion method, mixing of 15 mg/ml protein with 1 mM NADPH, and 1 mM inhibitor 4 and concentration to 24 mg/ml protein, addition of reservoir solution containing 100 mM cacodylate, pH 7.4, 150 mM sodium acetate, 25% w/v PEG 8000 in a 1:2 ratio to 0.01 ml sitting drops, equilibration against 1 ml reservoir solution with additional 1 mM (final concentration) L-glutathione, or mixing of 15 mg/ml protein with 1 mM NADPH, and 1 mM inhibitor 3 and concentration to 24 mg/ml protein, addition of reservoir solution, containing 100 mM cacodylate, pH 7.4, 125 mM sodium acetate, 25% w/v PEG 8000, and 11 mM calcium chloride, in a 1:2 ratio to 0.01 ml sitting drops, equilibration against 1 ml reservoir solution with additional 11 mM (final concentration) betaine hydrochloride, both 1-5 days at 4°C, X-ray diffraction structure determination and analysis at 1.76 A resolution, molecular replacement Klebsiella pneumoniae

Inhibitors

Inhibitors Comment Organism Structure
5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
Homo sapiens
5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
Klebsiella pneumoniae
5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
Homo sapiens
5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
Klebsiella pneumoniae
6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
-
Homo sapiens
6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
-
Klebsiella pneumoniae
6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine
-
Homo sapiens
6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine active site binding structure, overview Klebsiella pneumoniae
6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
-
Homo sapiens
6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine active site binding structure, overview Klebsiella pneumoniae
additional information development of a series of propargyl-linked antifolates that are potent inhibitors of DHFR from multiple species. One of the strengths of this series is the flexibility and versatility to target TMP-resistant species, regardless of whether that resistance occurs natively or via acquired mutations. Determination and comparison of inhibition kinetics and binding structures of human and Klebsiella enzymes with propargyl-linked antifolate inhibitors, overview Homo sapiens
additional information development of a series of propargyl-linked antifolates that are potent inhibitors of DHFR from multiple species. One of the strengths of this series is the flexibility and versatility to target TMP-resistant species, regardless of whether that resistance occurs natively or via acquired mutations. Determination and comparison of inhibition kinetics and binding structures of human and Klebsiella enzymes with propargyl-linked antifolate inhibitors, overview Klebsiella pneumoniae
trimethoprim resistance of enterobacteria generally occurs through the acquisition of a TMP-insensitive DHFR Klebsiella pneumoniae
trimethoprim-sulfamethoxazole resistance of enterobacteria generally occurs through the acquisition of a TMP-insensitive DHFR Klebsiella pneumoniae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
7,8-dihydrofolate + NADPH + H+ Homo sapiens
-
5,6,7,8-tetrahydrofolate + NADP+
-
r
7,8-dihydrofolate + NADPH + H+ Klebsiella pneumoniae
-
5,6,7,8-tetrahydrofolate + NADP+
-
r

Organism

Organism UniProt Comment Textmining
Homo sapiens P00374
-
-
Klebsiella pneumoniae Q7BPB0
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Klebsiella pneumoniae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7,8-dihydrofolate + NADPH + H+
-
Homo sapiens 5,6,7,8-tetrahydrofolate + NADP+
-
r
7,8-dihydrofolate + NADPH + H+
-
Klebsiella pneumoniae 5,6,7,8-tetrahydrofolate + NADP+
-
r

Synonyms

Synonyms Comment Organism
DHFR
-
Homo sapiens
DHFR
-
Klebsiella pneumoniae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
assay at room temperature Homo sapiens
22
-
assay at room temperature Klebsiella pneumoniae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Homo sapiens
7
-
assay at Klebsiella pneumoniae

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Homo sapiens
NADP+
-
Klebsiella pneumoniae
NADPH
-
Homo sapiens
NADPH
-
Klebsiella pneumoniae

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.000015
-
pH 7.0, 22°C Klebsiella pneumoniae 5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.000016
-
pH 7.0, 22°C Klebsiella pneumoniae 6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine
0.000023
-
pH 7.0, 22°C Klebsiella pneumoniae 6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
0.000058
-
pH 7.0, 22°C Klebsiella pneumoniae 5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.000108
-
pH 7.0, 22°C Klebsiella pneumoniae 6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
0.00016
-
pH 7.0, 22°C Homo sapiens 6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
0.00033
-
pH 7.0, 22°C Homo sapiens 6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
0.00075
-
pH 7.0, 22°C Homo sapiens 5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.0013
-
pH 7.0, 22°C Homo sapiens 6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine
0.0017
-
pH 7.0, 22°C Homo sapiens 5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine