Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects

Syntheses of polypyrrolic macrocycles

Pentaphyrins are pentapyrrolic macrocycles containing five bridging methine groups between the five pyrrolic subunits. The syntheses of decamethylpentaphyrin 71, 2-ethoxycarbonyl-3,7,8,12,13,17,18,22,23-nonamethylpentaphyrin 74, 2-ethoxycarbonyl-3,7,8,22,23-pentamethyl-12,13,17,18-tetraethylpentaphyrin 75, 3,12-dimethoxycarbonylethyl-2,7,8,-13,17,18,22,23-octamethylpentaphyrins 29 and the zinc complexes of 71 and 74 are described. The physical properties of the pentaphyrins show them to be aromatic like sapphyrins, porphyrins and corroles. This aromaticity is reflected in the large shielding of the NH protons and the deshielding of the methine protons in the nmr spectra and by their optical spectra, which exhibit Soret and visible bands similar to sapphyrins and porphyrins.Science, Faculty ofChemistry, Department ofGraduat

Synthesis of substrates and analogs for the enzymes of vitamin B₁₂ biosynthesis

Typescript (photocopy).The synthesis of some of the postulated intermediates in the pathway of vitamin B12 biosynthesis, precorrins-4A, -4B and -5, were initiated. The different ring precursors necessary for the precorrins were prepared as racemic mixtures apart from ring C precursors of precorrin-5 and -4A and -4B, which were obtained optically pure from (+)-camphor and pyrrole respectively. A new method was developed to synthesize a meso-substituted symmetrical dipyrromethane which served as a key intermediate in the synthesis of 20-methyluroporphyrin III and its derivative, 12-decarboxylated-20-methyl-uroporphyrin III. Unlike the 20-H analogs, these uro'gens were found to be unstable. From initial experiments, incubation of 20-methyl-uro'gen III with methylase-1 (M-1) enzyme resulted in the production of a chlorins and isobacteriochlorins as detected by high pressure liquid chromatography coupled with UV, while the action of M-1 on 12- decarboxylated-20-methyl uro'gen III did not show production of any methylated porphyrin. A number of substrate analogs for two enzymes, PBG deaminase and ALA dehydratase, involved in the vitamin B12 biosynthesis were synthesized. Three novel oxa analogs of PBG : the oxa analog, 2-aminomethyl-4-(2-carboxyethyl)-3- carboxymethyl furan, the oxa analog of iso PBG, 2-aminomethyl-3-(2-carboxyethyl)- 4-carboxymethyl furan and its hydroxy derivative, 2-hydroxymethyl-3-(2- carboxyethyl)-4-carboxymethyl furan were prepared. These products have been tested with PBG deaminase and found not to be substrates of the enzyme. In parallel with this study, 5-hydroxylevulinic acid (the hydroxy analog of ALA) was synthesized and found not to be a substrate of ALA dehydratase when tested with the enzyme. Another novel analog of PBG, 2-(hydroxy-1-propene)-4-(2-carboxyethyl)- 3-carboxymethyl pyrrole, was synthesized as the Z isomer. The intermediate, 2- (3-phthalimido-1-propene)-4-(2-methoxycarbonylethyl)-3-methoxycarbonylmethyl pyrrole, was found to be unstable and in the presence of traces of water gave 2 - (hydroxy-1-propene)-4-(2-methoxycarbonylethyl)-3-methoxycarbonylm ethyl pyrrole. Interestingly, the action of PBG deaminase on this PBG analog showed the formation of an ES1 enzyme-substrate complex

Design, Synthesis, and Antisickling Investigation of a Nitric Oxide-Releasing Prodrug of 5HMF for the Treatment of Sickle Cell Disease

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies

Refinement parameters for the human PL kinase structure with bound inhibitors.

a<p>Numbers in parenthesis refer to the outermost resolution bin.</p>b<p>Rmerge  =  Σ<i>_hkl</i>Σ<i>_i</i>|<i>I_hkli</i> - 〈<i>I_hkli</i>〉|/Σ<i>_hkl</i>Σ<i>_i</i>〈<i>I_hkli</i>〉.</p>c<p>5% of the reflection were excluded from the refinement and used for the calculation of Rfree.</p

Figure 5

<p>(A) Interactions between ginkgotoxin (yellow sticks) and the active site residues (green stick). Water molecules are red sphere. (B) Superimposed binding of ginkgotoxin (yellow stick, from hPL kinase) and pyridoxamine (cyan stick from sheep PL kinase). Protein residues are green and cyan sticks for the hPL kinase and sheep PL kinase, respectively. Water molecules are green and red spheres for the hPL kinase and sheep PL kinase, respectively. (C) Schematic diagram showing interactions between active side residues, water molecules and ginkgotoxin. Dotted and heavy lines are hydrogen-bond and hydrophobic interactions, respectively. (D) Interactions between active site residues (green stick), ginkgotoxin phosphate (cyan stick), ATP (green and brown sticks), Mg ions (brown sphere), Na ions (blue sphere) and water molecules (red sphere). (E) Schematic diagram showing interactions between ginkgotoxin phosphate, ATP, water molecules, Mg ions and the protein residues. Dotted and heavy lines are hydrogen-bond and hydrophobic interactions, respectively. Only potential hydrogen-bond interactions less than 3.6 Å are shown with dotted lines. For brevity, ginkgotoxin and its phosphorylated analog are denoted as GI and GIP in the figure.</p

Structures of potential PL kinase inhibitors.

<p>Structures of potential PL kinase inhibitors.</p