Colin H. MacKinnon

A mechanistic rationalisation for the substrate specificity of recombinant mammalian 4-hydroxyphenylpyruvate dioxygenase (4-HPPD)

Abstract The isolation and purification of α-ketoisocaproate dioxygenase [α-KICD] from rat liver is described. Sequence determination of the purified protein revealed it to have complete homology to rat liver 4-hydroxyphenyl-pyruvate dioxygenase [4-HPPD] which was confirmed by the cloning and expression of the gene encoding 4-HPPD in E. coli. Examination of the substrate specificity of the resulting soluble recombinant protein revealed it to be capable of the oxidative decarboxylation of a range of ketoacids derived from proteinogenic amino acids. The significance of the turnover of these different ketoacids is discussed in relation to the mechanism of this fascinating enzyme.

The C-terminal of rat 4-hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity.

We have cloned and overexpressed rat 4‐hydroxyphenylpyruvate dioxygenase (4HPPD) in Escherichia coli. The soluble, active recombinant enzyme was shown to contain both 4HPPD and α‐ketoisocaproate dioxygenase (αKICD) activity. However, upon truncation of the 14 amino acids at the C‐terminus by site‐directed mutagenesis, the resulting mutant enzyme (rat F antigen) exhibited complete loss of 4HPPD and αKICD activities. This finding suggests that the C‐terminal extension domain plays an essential role in the catalytic activity of the enzyme.

4-Hydroxyphenylpyruvate dioxygenase appears to display α-ketoisocaproate dioxygenase activity in rat liver

Abstract Isolation and sequence determination of a protein exhibiting α-ketoisocaproate dioxygenase activity from rat liver revealed that the protein was identical to the liver-specific rat F antigen which is believed to be a species variant of 4-hydroxyphenylpyruvate dioxygenase. Isolation and sequence determination of a protein exhibiting α-ketoisocaproate dioxygenase activity from rat liver revealed that the protein was identical to the liver-specific rat F antigen which is believed to be a species variant of 4-hydroxyphenylpyruvate dioxygenase.

Property- and structure-guided discovery of a tetrahydroindazole series of interleukin-2 inducible T-cell kinase inhibitors.

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.

Enzymatic synthesis of monocyclic β-lactams

Abstract An Mg2+ and ATP dependent β-lactam synthetase (BLS) catalyses formation of a β-lactam ring during the biosynthesis of clavulanic acid, an important β-lactamase inhibitor. An epimeric mixture of a 2-methylated derivative of the natural BLS substrate N2-(2-carboxyethyl)- l -arginine was synthesised and found to be a substrate for the enzyme. The epimeric products were characterised by 1H NMR and mass spectrometric analyses. The results suggest that a modified version of BLS might be used to catalyse the preparation of intermediates useful for the synthesis of β-lactam antibiotics.

INITIAL STUDIES ON THE SUBSTRATE SPECIFICITY OF SOLUBLE RECOMBINANT 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE FROM RAT LIVER

The molecular cloning and expression of the cDNA encoding 4-hydroxyphenylpyruvate dioxygenase (rat F antigen) into E.coli and initial studies concerning the substrate specificity of the recombinant protein are described.

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK).

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.

Stereochemical course of the conversion of α-ketoisocaproate to β-hydroxyisovalerate by soluble, recombinant mammalian 4-hydroxyphenylpyruvate dioxygenase☆

Abstract The stereochemical course of the conversion of α-ketoisocaproate to β-hydroxyisovalerate catalysed by the enzyme 4-hydroxyphenylpyruvate dioxygenase is described.

IDENTIFICATION AND STEREOCHEMISTRY OF THE PRODUCT OF 4-HPPD CATALYZED OXIDATION OF THE KETOACID OF METHIONINE

Abstract 4-Hydroxyphenylpyruvate dioxygenase catalyzes the conversion of 2-oxo-5-thiahexanoic acid 1 , the ketoacid from methionine (Scheme 1) to an unequal mixture of 4-thiapentanoic acid-4-oxide enantiomers 2a .

Design, synthesis and structure-activity relationships of a novel class of sulfonylpyridine inhibitors of Interleukin-2 inducible T-cell kinase (ITK).

Starting from benzylpyrimidine 2, molecular modeling and X-ray crystallography were used to design highly potent inhibitors of Interleukin-2 inducible T-cell kinase (ITK). Sulfonylpyridine 4i showed sub-nanomolar affinity against ITK, was selective versus Lck and its activity in the Jurkat cell-based assay was greatly improved over 2.