Thursday, October 22, 2015

The Sodium-Potassium Pump, “Neoglycosylation” and The Death of Emil Fischer

            The effects of cardenolides and related cardiotonic steroids on cardiac contractility is caused by a specific interaction with the sodium-potassium pump (Na+,K+-ATPase), which maintains normal gradients of sodium and potassium across the plasma membrane of eukaryotic cells. Partial inhibition of the ion-pumping function of the enzyme leads to increased strength of myocardial muscle contraction and this so-called positive inotropic pharmacological action is the basis of digoxin’s clinical utility in the treatment of congestive heart failure. More recently, the sodium-potassium pump has been shown to participate in protein-protein interactions that stimulate growth-related signal transduction pathways that are also essential to increased myocardial contractility. This Na+,K+-ATPase-induced second messenger signaling is thought to have different downstream consequences in various cell types (e.g. cancer versus non-malignant). Indeed, up-regulation of the sodium-potassium pump has been observed in a variety of cancers including ovarian, pancreatic and melanoma. This has led to the notion that cardiotonic steroids, as inhibitors of the Na+,K+-ATPase catalytic alpha subunit, represent viable lead compounds for the chemotherapeutic treatment of cancer. In a small cohort of breast cancer patients, it was reported that women who were taking a cardiotonic steroid at the time of their breast cancer diagnosis had tumors with less aggressive phenotypes than the breast tumors of women not taking a steroid such as digoxin. The same authors later reported a higher recurrence rate of cancer among women not taking a cardiotonic steroid drug. However, subsequent epidemiological studies of the association between cardenolide use and breast cancer incidence gave conflicting results. 
            Drug discovery research targeting the Na+,K+-ATPase is now facilitated by the recently reported crystallographic studies depicting the enzyme (from pig kidney) in complex with ouabain and digoxin, extended to 3.4 angstrom-resolution. The crystal structures clearly illustrate the extracellular region of the sodium-potassium pump to which cardiotonic steroids bind. A relatively small set of amino acids in the steroid-binding pocket serve as the primary contributors to binding the sugar substructure of the ligand. Interestingly, a handful of mutations near the binding site confer protection from poisons such as ouabain to a diverse range of insects, amphibians, reptiles and mammals, illustrating that similar selection pressures have resulted in convergent evolution across the animal kingdom.
            Based on its in vitro anticancer properties, and in conjunction with selected patient profiling data suggesting that the survival rate of cancer patients taking digitalis-derived drugs is statistically enhanced, digitoxin has been identified as a lead compound for oncology treatment applications. Jon Thorson’s research group at the University of Kentucky has used digitoxin as a model system for chemical derivatization by a technique that he refers to as ‘neoglycorandomization.’ The method applies a chemoselective glycosylation reaction developed in the late 1990’s to derivatized terpenoid aglycone substrates to produce ‘glycodiverse’ libraries via a one-step divergent process. The protocol circumvents the need for protecting group strategies, selective anomeric activation and stereochemical control over carbohydrate coupling steps. The neoglycosylation reacton, itself, is a chemoselective glycosylation between an N,O-dialkylhydroxyamine (a nucleophilic alkoxyamine) and an unprotected reducing aldose (hemiacetal). The adduct that is formed between the reacting partners, a ‘neoglycoside,’ exists as a cyclized saccharide containing an intact N-O bond. Recently, Thorson and co-workers used the neoglycorandomization technique to probe the structure-activity relationships associated with the sugar/amine regiochemistry of a set of digitoxigenin neoglycosides. They quickly identified a 3-amino-substitution on the sugar to be most advantageous, affording a digitoxigenin monosaccaride derivative (structure depicted above) that is equipotent to digitoxin in an in vitro assay against the non-small lung cancer cell line A549. The authors rationalize the cytotoxicity data using a docking model of the sodium-potassium pump derived from human Na+,K+-ATPase ligand-bound crystal structures. In this case, molecular modeling revealed a correlation between the determined anticancer activity with ligand-binding site occupancies wherein the polar sugar/amine moiety is fully solvent-exposed.
            It must be stated that when a chemist conducts drug discovery research for a living, it is indoctrinated at a relatively early stage of one’s career that molecules containing two consecutive heteroatoms linked by a sigma bond should not show up in screening libraries. So, compounds that contain a heteroatom-heteroatom bond that is not part of a heteroaryl ring system, for example hydrazides and oximes, are strongly discouraged as lead generation starting points in spite of their synthetic accessibility. When you talk to career medicinal chemists about why this should be the case, they usually say something like, “Well that’s what killed Emil Fischer.” For the record, Fischer’s death was actually self-inflicted; but it probably didn’t help that he was suffering from an excruciatingly painful case of intestinal carcinoma that was likely caused by exposure to a molecule that he discovered, phenylhydrazine (see structure above). More anecdotal evidence along these lines lies in the mushroom-derived toxin, gyromitrin, a carcinogen present in several members of the distinctive fungal genus Gyromitra. Gyromitrin is basically a pro-drug delivery system for methylhydrazine. In the body, methylhydrazine reacts with pyridoxal 5-phosphate, the active form of vitamin B6, to form a hydrazone, resulting in reduced production of GABA which leads to neurological symptoms. Gyromitrin is also metabolized to reactive nitrosamide intermediates that decompose to methyl radicals causing liver necrosis. Because of examples like these, I’m not a great supporter of ‘neoglycorandomization’ as an approach to drug discovery in spite of its conciseness. The neoglycoside molecules that comprise a ‘glycodiverse’ library all contain R1R2N-OMe functionality that seems like it might not be an ideal starting point for drug discovery. Personally, I would rather invest time and resources towards the additional effort required to synthesize a smaller, more focused set of well-designed screening targets. Moreover, the bigger problem with digitoxin and related cardiotonic steroids as chemotherapeutic lead compounds is their notoriously low cytotoxic selectivity for human cancer cells versus human non-malignant cells, which is typically not higher than ten-fold. To this point, Thorson, in his recent MedChem manuscript, notes that the aminosugars described in his study present opportunities for conjugation to cancer-targeting antibodies as a strategy to improve their therapeutic window of efficacy and enable their application in the chemotherapeutic treatment of cancer.

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