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Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

Walsh HA, O'Shea KC, Botting NP - BMC Biochem. (2003)

Bottom Line: The potency of the various inhibitors was found to be species specific.The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase.The modified purification described is relatively quick, simple and cost effective.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemistry, University of St, Andrews, St Andrews, Fife, KY16 9ST UK. haw2@st-andrews.ac.uk

ABSTRACT

Background: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim.

Results: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed.

Conclusion: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective.

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Synthesis of inhibitors a) (CH3)3COCl, pyridine, 0°C (79%); b) CuBr, ethyl acetate, CHCl3, reflux, 4 hr (63%); c) NaH, AcNHCH(CO2Et)2, DMF, 0°C (57%); d) HCl, diethyl ether, reflux, 6 hr (76%)
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Figure 6: Synthesis of inhibitors a) (CH3)3COCl, pyridine, 0°C (79%); b) CuBr, ethyl acetate, CHCl3, reflux, 4 hr (63%); c) NaH, AcNHCH(CO2Et)2, DMF, 0°C (57%); d) HCl, diethyl ether, reflux, 6 hr (76%)

Mentions: The two inhibitors were synthesised using adaptations of previously reported methods [11]. The 3-hydroxydesaminokynurenine (4) was prepared from 3-hydroxyacetophenone (6) (Figure 6). The hydroxy group was first protected as the pivalate ester derivative and then the methyl group brominated to give (7). The bromide (7) was then coupled to anion of diethyl acetamidomalonate in DMF to give the fully protected amino acid (8). Deprotection under acidic conditions provided the target compound. In the case of the 3-methoxydesaminokynurenine (5), the starting material was 3-methoxyacetophenone which could be used without necessity for an initial protection step. The spectral data for the two final products are as follows; 3-Hydroxydesaminokynurenine (4): m.p. 188°C (dec.); (Found: C, 48.61; H, 5.00; N, 5.63 C10H12ClNO4 requires C, 48.89; H, 4.92; N, 5.69%); νmax (nujol)/cm-1 3383 (NH), 1739 (CO, acid), 1660 (CO); δH (300 MHz, 2H2O) 3.73 (2H, d, J3,2 5.0 Hz, 3-C H2), 4.39 (1H, t, J2,3 5.0 Hz, 2-CH), 7.06 (1H, dd, J 2.4, 1.0 Hz, 4'-H), 7.26 (1H, m, 5'-H), 7.30 (1H, m, 2'-H), 7.41 (1H, dd, J 7.8, 1.0 Hz, 6'-H); δC(75.4 MHz, 2H2O) 38.21 (s, 3-CH2), 48.88 (s, 2-CH), 114.47 120.78 (s, 4'-C), 121.90 (s, 2'-C), 121.90 (s, 6'-C), 130.51 (s, 5'-C), 136.56 (1'-C), 156.10 (s, 3'-C), 171.77 (s, OCOH), 199.07 (s, 4-CO); m/z (CI) 210 ([M + H]+, 13), 195 (100, ([M+H-NH]+), 177 (18, [C10H8O3 +H]+), 164 (17, [M-(CO2H)+]), 149 (35, [C9H9O2]+), 43 (95, [CH2CNH2]+). 3-Methoxydesaminokynurenine (5): m.p. 164°C (dec.); νmax(nujol)/cm-1 3378 (NH), 1738 (CO, acid), 1681 (CO); δH (200 MHz, 2H2O) 3.77 (3H, s, OCH3), 3.80 (2H, d, J3,2 5 Hz, 3-CH2), 4.47 (1H, t, J2,3 5 Hz, 2-CH), 7.17 (1H, dt, J 8, 2.8 Hz, 4'-H), 7.39 (2H, m, 2', 5'-H), 7.53 (1H, m, 6'-H); δC (50.31 MHz, 2H2O), 41.26 (s, 3-CH2), 51.82 (s, 2-CH), 58.48 (s, OCH3), 115.72 (s, 2'-C), 123.64 (s, 4'-C), 124.35 (s, 6'-C), 133.22 (s, 5'-C), 139.06 (s, 1'-C), 162.06 (s, 3'-C), 174.46 (s, CO2H), 201.66 (s, 4-CO); m/z (CI) 224.0913 (MH+) C11H14NO4requires 224.0922, 209 (100, ([M+H-CH3]+), 191 (49, [HO-C6H4COCH2CHCO2HNH2-H2O]+), 178 (73, [M+H-C2H2O2]+), 135 (18, [CH3OC6H4CO]+). Analysis by HPLC (C18, reverse phase silica, 3 μ, flow rate 3 mL/min, 1% acetic acid, 20% methanol) confirmed the purity of both inhibitors.


Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

Walsh HA, O'Shea KC, Botting NP - BMC Biochem. (2003)

Synthesis of inhibitors a) (CH3)3COCl, pyridine, 0°C (79%); b) CuBr, ethyl acetate, CHCl3, reflux, 4 hr (63%); c) NaH, AcNHCH(CO2Et)2, DMF, 0°C (57%); d) HCl, diethyl ether, reflux, 6 hr (76%)
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Related In: Results  -  Collection

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Figure 6: Synthesis of inhibitors a) (CH3)3COCl, pyridine, 0°C (79%); b) CuBr, ethyl acetate, CHCl3, reflux, 4 hr (63%); c) NaH, AcNHCH(CO2Et)2, DMF, 0°C (57%); d) HCl, diethyl ether, reflux, 6 hr (76%)
Mentions: The two inhibitors were synthesised using adaptations of previously reported methods [11]. The 3-hydroxydesaminokynurenine (4) was prepared from 3-hydroxyacetophenone (6) (Figure 6). The hydroxy group was first protected as the pivalate ester derivative and then the methyl group brominated to give (7). The bromide (7) was then coupled to anion of diethyl acetamidomalonate in DMF to give the fully protected amino acid (8). Deprotection under acidic conditions provided the target compound. In the case of the 3-methoxydesaminokynurenine (5), the starting material was 3-methoxyacetophenone which could be used without necessity for an initial protection step. The spectral data for the two final products are as follows; 3-Hydroxydesaminokynurenine (4): m.p. 188°C (dec.); (Found: C, 48.61; H, 5.00; N, 5.63 C10H12ClNO4 requires C, 48.89; H, 4.92; N, 5.69%); νmax (nujol)/cm-1 3383 (NH), 1739 (CO, acid), 1660 (CO); δH (300 MHz, 2H2O) 3.73 (2H, d, J3,2 5.0 Hz, 3-C H2), 4.39 (1H, t, J2,3 5.0 Hz, 2-CH), 7.06 (1H, dd, J 2.4, 1.0 Hz, 4'-H), 7.26 (1H, m, 5'-H), 7.30 (1H, m, 2'-H), 7.41 (1H, dd, J 7.8, 1.0 Hz, 6'-H); δC(75.4 MHz, 2H2O) 38.21 (s, 3-CH2), 48.88 (s, 2-CH), 114.47 120.78 (s, 4'-C), 121.90 (s, 2'-C), 121.90 (s, 6'-C), 130.51 (s, 5'-C), 136.56 (1'-C), 156.10 (s, 3'-C), 171.77 (s, OCOH), 199.07 (s, 4-CO); m/z (CI) 210 ([M + H]+, 13), 195 (100, ([M+H-NH]+), 177 (18, [C10H8O3 +H]+), 164 (17, [M-(CO2H)+]), 149 (35, [C9H9O2]+), 43 (95, [CH2CNH2]+). 3-Methoxydesaminokynurenine (5): m.p. 164°C (dec.); νmax(nujol)/cm-1 3378 (NH), 1738 (CO, acid), 1681 (CO); δH (200 MHz, 2H2O) 3.77 (3H, s, OCH3), 3.80 (2H, d, J3,2 5 Hz, 3-CH2), 4.47 (1H, t, J2,3 5 Hz, 2-CH), 7.17 (1H, dt, J 8, 2.8 Hz, 4'-H), 7.39 (2H, m, 2', 5'-H), 7.53 (1H, m, 6'-H); δC (50.31 MHz, 2H2O), 41.26 (s, 3-CH2), 51.82 (s, 2-CH), 58.48 (s, OCH3), 115.72 (s, 2'-C), 123.64 (s, 4'-C), 124.35 (s, 6'-C), 133.22 (s, 5'-C), 139.06 (s, 1'-C), 162.06 (s, 3'-C), 174.46 (s, CO2H), 201.66 (s, 4-CO); m/z (CI) 224.0913 (MH+) C11H14NO4requires 224.0922, 209 (100, ([M+H-CH3]+), 191 (49, [HO-C6H4COCH2CHCO2HNH2-H2O]+), 178 (73, [M+H-C2H2O2]+), 135 (18, [CH3OC6H4CO]+). Analysis by HPLC (C18, reverse phase silica, 3 μ, flow rate 3 mL/min, 1% acetic acid, 20% methanol) confirmed the purity of both inhibitors.

Bottom Line: The potency of the various inhibitors was found to be species specific.The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase.The modified purification described is relatively quick, simple and cost effective.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemistry, University of St, Andrews, St Andrews, Fife, KY16 9ST UK. haw2@st-andrews.ac.uk

ABSTRACT

Background: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim.

Results: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed.

Conclusion: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective.

Show MeSH
Related in: MedlinePlus