Structural Requirements for Bisphosphonate Binding on Hydroxyapatite: NMR Study of Bisphosphonate Partial Esters

Eighteen different bisphosphonates, including four clinically used bisphosphonate acids and their phosphoesters, were studied to evaluate how the bisphosphonate structure affects binding to bone. Bisphosphonates with weak bone affinity, such as clodronate, could not bind to hydroxyapatite after the addition of one ester group. Medronate retained its ability to bind after the addition of one ester group, and hydroxy-bisphosphonates could bind even after the addition of two ester groups. Thus, several bisphosphonate esters are clearly bone binding compounds. The following conclusions about bisphosphonate binding emerge: (1) a hydroxyl group in the geminal carbon takes part in the binding process and increases the bisphosphonate’s ability to bind to bone; (2) the bisphosphonate’s ability to bind decreases when the amount of ester groups increases; and (3) the location of the ester groups affects the bisphosphonate’s binding ability

<i>In Silico</i> Prediction of the Site of Oxidation by Cytochrome P450 3A4 That Leads to the Formation of the Toxic Metabolites of Pyrrolizidine Alkaloids

In humans, the metabolic bioactivation of pyrrolizidine alkaloids (PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation of their necine bases at C3 or C8 of heliotridine- and retronecine-type PAs or at the N atom of the methyl substituent of otonecine-type PAs. However, no attempts have been made to identify which C atom is the most favorable site for hydroxylation <i>in silico</i>. Here, in order to determine the site of hydroxylation that eventually leads to the formation of the toxic metabolites produced from lasiocarpine, retrorsine, and senkirkin, we utilized the ligand-based electrophilic Fukui function <i>f</i>^–(<b>r</b>) and hydrogen-bond dissociation energies (BDEs) as well as structure-based molecular docking. The ligand-based computations revealed that the C3 and C8 atoms of lasiocarpine and retrorsine and the C26 atom of senkirkin were chemically the most susceptible locations for electrophilic oxidizing reactions. Similarly, according to the predicted binding orientation in the active site of the crystal structure of human CYP3A4 (PDB code: 4I4G), the alkaloids were positioned in such a way that the C3 atom of lasiocarpine and retrorsine and the C26 of senkirkin were closest to the catalytic heme Fe. Thus, it is concluded that the C3 atom of lasiocarpine and retrorsine and C26 of senkirkin are the most favored sites of hydroxylation that lead to the production of their toxic metabolites

Identification of a New Reactive Metabolite of Pyrrolizidine Alkaloid Retrorsine: (3<i>H</i>‑Pyrrolizin-7-yl)methanol

Pyrrolizidine alkaloids (PAs) such as retrorsine are common food contaminants that are known to be bioactivated by cytochrome P450 enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites known as dehydropyrrolizidine alkaloids (DHPs). We compared how both electrochemical (EC) and human liver microsomal (HLM) oxidation of retrorsine could produce short-lived intermediate metabolites; we also characterized a toxicologically important metabolite, (3<i>H</i>-pyrrolizin-7-yl)­methanol. The EC cell was coupled online or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas the HLM oxidation was performed in 100 mM potassium phosphate (pH 7.4) in the presence of NADPH at 37 °C. The EC cell oxidation of retrorsine produced 12 metabolites, including dehydroretrorsine (<i>m</i>/<i>z</i> 350, [M + H⁺]), which was degraded to a new reactive metabolite at <i>m</i>/<i>z</i> 136 ([M + H⁺]). The molecular structure of this small metabolite was determined using high-resolution mass spectrometry and NMR spectroscopy followed by chemical synthesis. In addition, we also identified another minor but reactive metabolite at <i>m</i>/<i>z</i> 136, an isomer of (3<i>H</i>-pyrrolizin-7-yl)­methanol. Both (3<i>H</i>-pyrrolizin-7-yl)­methanol and its minor isomer were also observed after HLM oxidation of retrorsine and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the presence of reduced glutathione (GSH), each isomer formed identical GSH conjugates at <i>m</i>/<i>z</i> 441 and <i>m</i>/<i>z</i> 730 in the negative ESI-MS. Because (3<i>H</i>-pyrrolizine-7-yl)­methanol) and its minor isomer subsequently reacted with GSH, it is concluded that (3<i>H</i>-pyrrolizin-7-yl)­methanol may be a common toxic metabolite arising from PAs

Enantiomers of 3‑Methylspermidine Selectively Modulate Deoxyhypusine Synthesis and Reveal Important Determinants for Spermidine Transport

Eukaryotic translation initiation factor 5A (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [<i>N</i>^ε-(4-amino-2-hydroxybutyl)­lysine]. Deoxyhypusine synthase (DHS) is the rate-limiting enzyme of this two-step process, and the polyamine spermidine is the only natural donor of the butylamine group for this reaction, which is very conservedhypusine biosynthesis suffers last when the intracellular spermidine pool is depleted. DHS has a very strict substrate specificity, and only a few spermidine analogs are substrates of the enzyme and can support long-term growth of spermidine-depleted cells. Herein, we compared the biological properties of earlier unknown enantiomers of 3-methylspermidine (3-MeSpd) in deoxyhypusine synthesis, in supporting cell growth and in polyamine transport. Long-term treatment of DU145 cells with α-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (<i>R</i>)-3-MeSpd did not cause depletion of hypusinated eIF5A, and the cells were still able to grow, whereas the combination of α-difluoromethylornithine with a racemate or (<i>S</i>)-3-MeSpd caused cessation of cell growth. Noticeably, DHS preferred the (<i>R</i>)- over the (<i>S</i>)-enantiomer as a substrate. (<i>R</i>)-3-MeSpd competed with [¹⁴<i>C</i>]-labeled spermidine for cellular uptake less efficiently than the (<i>S</i>)-3-MeSpd (<i>K</i>_i = 141 μM vs 19 μM, respectively). The cells treated with racemic 3-MeSpd accumulated intracellularly mainly (<i>S</i>)-3-MeSpd, but not DHS substrate (<i>R</i>)-3-MeSpd, explaining the inability of the racemate to support long-term growth. The distinct properties of 3-MeSpd enantiomers can be exploited in designing polyamine uptake inhibitors, facilitating drug delivery and modulating deoxyhypusine synthesis