Steroidomimetic Tetrahydroisoquinolines for the Design of New Microtubule Disruptors.

Structure-activity relationship translation offers an expeditious means for discovery of new active series. This approach was applied to discover tetrahydroisoquinoline (THIQ)-based steroidomimetic microtubule disruptors. The two A-ring elements of a three-point steroidal pharmacophore were incorporated into a THIQ-based A,B-ring mimic to which an H-bond acceptor was attached as the third motif. Optimization of the representative 6c through conformational biasing delivered a 10-fold gain in activity and a new series of microtubule disruptors (e.g., 9c) with antiproliferative activity in the nanomolar range. The THIQ derivatives match, or surpass, the activities of the steroidal series and exhibit improved physicochemical properties.

Chimeric microtubule disruptors.

A chimeric approach is used to discover microtubule disruptors with excellent in vitro activity and oral bioavailability; a ligand-protein interaction with carbonic anhydrase that enhances bioavailability is characterised by protein X-ray crystallography. Dosing of a representative chimera in a tumour xenograft model confirms the excellent therapeutic potential of the class.

Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase.

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.

Remarkably different structures and reaction mechanisms of ketoreductases for the opposite stereochemical control in the biosynthesis of BIQ antibiotics.

Two ketoreductases, RED1 and RED2, are involved in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) and dihydrogranaticin in S. violaceoruber Tu22, respectively. They are responsible for the stereospecific reductions of the bicyclic intermediate to give (S)- or (R)-DNPA, although there is no similarity between their amino acid sequences. Biotransformation using synthetic analogous substrates revealed that the substrate specificities are quite different. Homology modelling studies and site directed mutagenesis showed remarkable differences in three-dimensional structures and catalytic mechanisms between RED1 and RED2.