Dimerization and DNA recognition rules of mithramycin and its analogues.

The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me(2+))-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM=MTM SDK>MTM SA-Trp>MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.

Arginine-rhamnosylation as new strategy to activate translation elongation factor P.

Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-ß-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development.

Structural insight into MtmC, a bifunctional ketoreductase-methyltransferase involved in the assembly of the mithramycin trisaccharide chain.

More and more post-PKS tailoring enzymes are recognized as being multifunctional and codependent on other tailoring enzymes. One of the recently discovered intriguing examples is MtmC, a bifunctional TDP-4-keto-d-olivose ketoreductase-methyltransferase, which-in codependence with glycosyltransferase MtmGIV-is a key contributor to the biosynthesis of the critical trisaccharide chain of the antitumor antibiotic mithramycin (MTM), produced by Streptomyces argillaceus. We report crystal structures of three binary complexes of MtmC with its methylation cosubstrate SAM, its coproduct SAH, and a nucleotide TDP as well as crystal structures of two ternary complexes, MtmC-SAH-TDP-4-keto-d-olivose and MtmC-SAM-TDP, in the range of 2.2-2.7 Å resolution. The structures reveal general and sugar-specific recognition and catalytic structural features of MtmC. Depending on the catalytic function that is conducted by MtmC, it must bind either NADPH or SAM in the same cofactor binding pocket. A tyrosine residue (Tyr79) appears as a lid covering the sugar moiety of the substrate during the methyl transfer reaction. This residue swings out of the active site by ~180° in the absence of the substrate. This unique conformational change likely serves to release the methylated product and, possibly, to open the active site for binding the bulkier cosubstrate NADPH prior to the reduction reaction.

Enzymatic methylation and structure-activity-relationship studies on polycarcin V, a gilvocarcin-type antitumor agent.

Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure-activity relationships of the gilvocarcin group of antitumor antibiotics due to a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary O-methyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2'-OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important hydrogen bond donor for the interaction with histone H3, and converting 3'-OH into an OCH3 group improved the bioactivity. Bis-methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two hydrogen bond donors in the sugar are necessary for optimal binding.

Semi-synthetic mithramycin SA derivatives with improved anticancer activity.

Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved mithramycin analogues mithramycin SK and mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to mithramycin SK. This work shows the ability to transform the previously useless mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.