Carboxyl Methyltransferase Catalysed Formation of Mono- and Dimethyl Esters under Aqueous Conditions: Application in Cascade Biocatalysis.

Carboxyl methyltransferase (CMT) enzymes catalyse the biomethylation of carboxylic acids under aqueous conditions and have potential for use in synthetic enzyme cascades. Herein we report that the enzyme FtpM from Aspergillus fumigatus can methylate a broad range of aromatic mono- and dicarboxylic acids in good to excellent conversions. The enzyme shows high regioselectivity on its natural substrate fumaryl-l-tyrosine, trans, trans-muconic acid and a number of the dicarboxylic acids tested. Dicarboxylic acids are generally better substrates than monocarboxylic acids, although some substituents are able to compensate for the absence of a second acid group. For dicarboxylic acids, the second methylation shows strong pH dependency with an optimum at pH 5.5-6. Potential for application in industrial biotechnology was demonstrated in a cascade for the production of a bioplastics precursor (FDME) from bioderived 5-hydroxymethylfurfural (HMF).

Structure-based design of nucleoside-derived analogues as sulfotransferase inhibitors.

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group ('sulfation') from the enzyme co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer's disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

Small Molecule Inhibitors of Cyclophilin D To Protect Mitochondrial Function as a Potential Treatment for Acute Pancreatitis.

Opening of the mitochondrial permeability transition pore (MPTP) causes mitochondrial dysfunction and necrosis in acute pancreatitis (AP), a condition without specific drug treatment. Cyclophilin D (CypD) is a mitochondrial matrix peptidyl-prolyl isomerase that regulates the MPTP and is a drug target for AP. We have synthesized urea-based small molecule inhibitors of cyclophilins and tested them against CypD using binding and isomerase activity assays. Thermodynamic profiles of the CypD/inhibitor interactions were determined by isothermal titration calorimetry. Seven new high-resolution crystal structures of CypD-inhibitor complexes were obtained to guide compound optimization. Compounds 4, 13, 14, and 19 were tested in freshly isolated murine pancreatic acinar cells (PACs) to determine inhibition of toxin-induced loss of mitochondrial membrane potential (ΔΨm) and necrotic cell death pathway activation. Compound 19 was found to have a Kd of 410 nM and a favorable thermodynamic profile, and it showed significant protection of ΔΨm and reduced necrosis of murine as well as human PACs. Compound 19 holds significant promise for future lead optimization.

Ligand binding and aggregation of pathogenic SOD1.

Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, ß-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

Alkali metal reductions of organic molecules: why mediated electron transfer from lithium is faster than direct reduction.

Lithium metal reductions are widely employed in organic synthesis, where it is common to employ a "mediator" to speed up the electron transfer kinetics. We present experimental data for the electrode kinetics of the reduction of the most common mediator, 4,4'-di-tert-butyl-1,1'-biphenyl (DBB) in tetrahydrofuran (THF) over a range of temperatures. Using corresponding data for the oxidation of lithium we present quantitative estimates of the kinetic advantage for the use of DBB as a mediator in lithium reductions, over, in particular, direct reduction using lithium metal.

Synthetic analogues of the manzamenones and plakoridines which inhibit DNA polymerase.

An array of novel analogues of the marine oxylipins, the manzamenones and plakoridines, have been prepared in divergent fashion using an approach modelled on a biogenetic theory. Many of the target compounds show potent inhibition of DNA polymerases alpha and beta and human terminal deoxynucleotidyl transferase (TdT).