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

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.

Figure 1

Reaction catalysed by kynureninase

Figure 2

Discontinuous gel electrophoresis of human kynureninase. 10% SDS-Page gel image of purified recombinant kynureninase (20 μg) at 52.4 kDa in the presence of PLP. This NuPAGE Novex Bis-Tris Gel (prepacked) was produced using the Xcell SureLock Mini-Cell from Invitrogen. The pertained Mark 12 standards were also acquired from Invitrogen. Run conditions were 200 V (constant)/35 minute in MES buffer with expected current of 100–125 mA/gel at start going to 60–80 ma/gel at the end.

Figure 3

Structures of new inhibitors

Figure 4

Inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) Primary Lineweaver Burk (L/B) plot of kinetic data for inhibition of rat hepatic kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (■); I = 50 nM (▲) I = 100 nM (▼); I = 200 nM (◀ ▶); I = 400 nM (●); I = 600 nM (□)) depicting mixed inhibition. s = substrate (3-hydroxykynurenine) and v = specific activity. The inset is a secondary plot of slope against [I] to determine the K_i (40 nM). The slopes were calculated from a L/B plot (n = 3).

Figure 5

Inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) Dixon plot illustrating inhibition of bacterial kynureninase by 3-hydroxydesaminokynurenine (4) (I = 0 (◆); I = 200 nM (▼); I = 400 nM (▲); I = 600 nM (■). The inhibition is mixed and gives a K_I = 100 nM and the graph is a replot of the data (n = 3) used to construct a Michael's–Menten plot. The inset is a secondary plot of slope (from L/B) against inhibitor concentration for recombinant human kynureninase (n= 3 and r² = 0.99) to calculate K_I (5 nM). The concentration of substrate was varied between 2.5 μM – 20 μM. Lines were fitted to a straight line equation for linear regression.

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%)