A study of the binding requirements of calyculin A and dephosphonocalyculin A with PP1, development of a molecular recognition model for the binding interactions of the okadaic acid class of compounds with PP1.

The interactions of the okadaic acid class of compounds, with special emphasis on the solution structures of calyculin A and dephosphonocalyculin A with PP1 are reported. After examination of the interactions of all docked structures, a receptor based pharmacophore model for the interactions of the protein phosphatase inhibitors has been developed. Calyculin A or dephosphonocalyculin A can interact with the enzyme in either a manner similar to the reported crystal structure, or in an extended form. The inhibitors require two essential regions interacting with the hydrophobic region and the central metal binding regions of the enzyme. This simplified model is consistent with previously published models of the okadaic acid class of compounds with PP1.

Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library.

The inability to generate soluble, correctly folded recombinant protein is often a barrier to successful structural and functional studies. Access to affordable synthetic genes has, however, made it possible to design, make and test many more variants of a target protein to identify suitable constructs. We have used rational design and gene synthesis to create a controlled randomised library of the EphB4 receptor tyrosine kinase, with the aim of obtaining soluble, purifiable and active catalytic domain material at multi-milligram levels in Escherichia coli. Three main parameters were tested in designing the library--construct length, functional mutations and stability grafting. These variables were combined to generate a total of 9720 possible variants. The screening of 480 clones generated a 3% hit rate, with a purifiable solubility of up to 15 mg/L for some EphB4 constructs that was largely independent of construct length. Sequencing of the positive clones revealed a pair of hydrophobic core mutations that were key to obtaining soluble material. A minimal kinase domain construct containing these two mutations exhibited a +4.5°C increase in thermal stability over the wild-type protein. These approaches will be broadly applicable for solubility engineering of many different protein target classes. Atomic coordinates and structural factors have been deposited in PDB under the accession 2yn8 (EphB4 HP + staurosporine).

Sequence-specific interaction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the adenine-tract DNA duplex d(CTTTTGCAAAAG)2 has been studied in both D2O and H2O solutions by 1D and 2D 1H NMR spectroscopy. Thirty-one nuclear Overhauser effects between drug and nucleotide protons within the minor groove of the duplex, together with ring-current induced perturbations to the chemical shifts of basepair and deoxyribose protons, define the position and orientation of the bound dye molecules. Two drug molecules bind cooperatively and in symmetry related orientations at the centre of the 5'-TTTT and 5'-AAAA sequences with the binding interactions spanning only the four A-T basepairs. The positively charged N-methylpiperazine moieties point towards the centre of the duplex while the phenol groups are disposed towards the 3'-ends of the sequence. Resonance averaging is apparent for both the D2/D6 and D3/D5 phenol protons and D2"'/D6"' and D3"'/D5"' of the N-methylpiperazine ring and is consistent with these groups being involved in rapid rotation or ring-flipping motions in the bound state. Interstrand NOEs between adenine H2s and deoxyribose H1' are consistent with a high degree of propeller twisting of the A-T basepairs at the binding site of the aromatic benzimidazole and phenol rings of Hoechst. The data imply that the minor groove is particularly narrow with many contacts between the complementary curved surfaces of the drug and DNA indicating that strong van der Waals interactions, involving the floor and the walls of the minor groove, stabilize the complex. In our model the NH groups of the benzimidazole rings are positioned to make a pair of bifurcated hydrogen bonds with the adenine N3 and thymine O2 on the floor of the minor groove.

Structural analysis of the decanucleotide duplex d(GGTAATTACC)2 using 1H NMR spectroscopy.

The conformation of the decanucleotide duplex d(GGTAATTACC)2 has been investigated in solution by one- and two-dimensional proton NMR spectroscopy. Intra- and inter-nucleotide two-dimensional nuclear Overhauser enhancement data, recorded at mixing times between 15 and 250 ms, reveal a right-handed B-DNA structure. The data also show that the A-T basepairs of the TAATTA tract are highly propeller twisted and the minor groove is particularly narrow.

Interaction of Hoechst 33258 with the minor groove of the A + T-rich DNA duplex d(GGTAATTACC)2 studied in solution by NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the A + T-rich DNA duplex d(GGTAATTACC)2 has been investigated in solution by two-dimensional 1H-NMR spectroscopy. Many intermolecular NOEs define the position and orientation of the Hoechst molecule at the centre of the duplex, binding to a single site located within the minor groove of the AATT sequence. This is despite the opportunity for Hoechst 33258 to bind to as many as five unique sites containing the minimum requirement of three consecutive A.T base pairs. All intermolecular NOEs are detected between protons on the concave edge of Hoechst, many with the adenine H2s on the floor of the minor groove. The NOEs are consistent with interproton distances measured within a partially restrained energy-minimised structure of the complex. The AATT sequence provides the key recognition features for tight binding, including a particularly narrow minor groove, with no evidence for the partial G.C specificity previously proposed to be necessary to accommodate the bulky N-methylpiperazine ring. While chemical exchange cross-peaks between symmetry-related adenine H2s on opposite strands of the duplex identify a very slow rate of dissociation of the drug from the complex at 298 K (exchange rate approximately 1.2 s-1). Averaging of chemical shifts in the phenyl ring of Hoechst 33258 are indicative of rapid ring-flipping motions in the bound state.

Use of transferred nuclear-Overhauser-effect spectroscopy to measure the bound conformation of a disulphide-replaced analogue of glutathione disulphide as an inhibitor of yeast glutathione reductase.

The analogue of glutathione disulphide (GSSG) in which the disulphide bridge of GSSG is replaced by -CH2-S- was synthesised from L-cystathionine using t-butoxycarbonyl and t-butyl ester protection with triethylsilane-promoted deprotection. This analogue (GCSG) was found to be a linear, competitive inhibitor of yeast glutathione reductase (Ki value 981 microM at pH 7.0), a very poor substrate and not to act as an irreversible inhibitor of glutathione reductase. The weak binding of GCSG to glutathione reductase permitted the use of transferred nuclear Overhauser effect spectroscopy (TRNOESY) to investigate the bound conformation of GCSG in its complex with glutathione reductase. The solution structure of free GCSG was investigated by NMR spectroscopy using a range of NMR techniques. The TRNOESY experiment allowed a range of conformations to be determined for the central bridge region (containing the -CH2-S- replacement) of GCSG bound to yeast glutathione reductase. Using the nuclear Overhauser effect constraints thus derived, in combination with molecular graphics and energy minimisation based on the known crystal coordinates of glutathione disulphide (GSSG) bound to human erythrocyte glutathione reductase, allowed an explanation of the lack of substrate activity of GCSG, its inactivity as a suicide inactivator and its relatively weak binding in terms of the enforced mislocation of the -CH2-S- bridge with respect to the catalytic residues (relative to GSSG). Thus, the simple replacement of -S- by -CH2-, common in medicinal chemistry, can lead to poor receptor binding if the replacement occurs in a central, rather than peripheral, part of the ligand under modification.

Rational inhibitor design, synthesis and NMR spectroscopic study by transferred nuclear overhauser spectroscopy of novel inhibitors of cinnamyl alcohol dehydrogenase, a critical enzyme in lignification.

Cinnamyl alcohol dehydrogenase is one of the enzymes controlling the first two committed steps of lignification. Using a 3-dimensional similarity model of this enzyme, a series of novel phosphonates (1-5) was designed as potential inhibitors. Phosphonates 1-5 were synthesized in good yield by reaction of the corresponding cinnamaldehydes with tetraethylmethylene diphosphonate. Monophosphonic acids 6 and 7 were obtained by basic hydrolysis of the corresponding phosphonates while phosphonamidate 8 was synthesized by reacting benzylamine with the iminium salt intermediate of the monophosphonic acid. Using recombinant cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) the inhibitory activity of these compounds was evaluated and compared with that of the carbonyl analogues. Inhibition kinetic studies showed compounds 2 and 3 to be mixed type linear inhibitors while compound 4 was uncompetitive. 1H NMR studies of inhibitor 2, for which Ki and Ki' were 20 and 86 microM, respectively, in the presence of CAD based on selective line-broadening showed an increased interaction of the 3-OMe group of the aromatic ring of the inhibitor with the active site of the CAD. A transferred nuclear overhauser effect spectroscopy (TRNOESY) experiment for inhibitor 2 with CAD was used to determine the conformation of this compound bound to CAD. These results were found to be consistent with the 3-dimensional structural model of the enzyme.