Robert J. Kempton

Molecular determinants of sarco/endoplasmic reticulum calcium ATPase inhibition by hydroquinone‐based compounds

The ion transport activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA) is specifically and potently inhibited by the small molecule 2,5‐di‐tert‐butylhydroquinone (BHQ). In this study, we investigated the relative importance of the nature and position of BHQs four substituents for enzyme inhibition by employing a combination of experimental and computational techniques. The inhibitory potencies of 21 commercially available or synthesized BHQ derivatives were determined in ATPase activity assays, and 11 compounds were found to be active. Maximum inhibitory potency was observed in compounds with two para hydroxyl groups, whereas BHQ analogues with only one hydroxyl group were still active, albeit with a reduced potency. The results also demonstrated that two alkyl groups were an absolute requirement for activity, with the most potent compounds having 2,5‐substituents with four or five carbon atoms at each position. Using the program GOLD in conjunction with the ChemScore scoring function, the structures of the BHQ analogues were docked into the crystal structure of SERCA mimicking the enzymes E2 conformation. Analysis of the docking results indicated that inhibitor binding to SERCA was primarily mediated by a hydrogen bond between a hydroxyl group and Asp‐59 and by hydrophobic interactions involving the bulky inhibitor alkyl groups. Attempts to dock BHQ into crystal structures corresponding to the E1 conformation of the enzyme failed, because the conformational changes accompanying the E2/E1 transition severely restricted the size of the binding site, suggesting that BHQ stabilizes the enzyme in its E2 form. The potential role of Glu309 in enzyme inhibition is discussed in the context of the computational results. The docking scores correlated reasonably well with the measured inhibitory potencies and allowed the distinction between active and inactive compounds, which is a key requirement for future virtual screening of large compound databases for novel SERCA inhibitors. Proteins 2008.

High-performance liquid chromatography of methotrexate analogs containing terminal lysine or ornithine and their dansyl derivatives

A procedure utilizing a reverse-phase semipreparative high-performance liquid chromatography column and a binary solvent system consisting of trifluoroacetic acid and 1-propanol has been developed for the semipreparative scale purification and analytical identification of four newly synthesized analogs of methotrexate. The methotrexate analogs containing a lysine or an ornithine residue in place of a terminal glutamate residue together with their respective dansyl derivatives were purified in milligram quantities by the procedures described.

Design, synthesis, and biological evaluation of hydroquinone derivatives as novel inhibitors of the sarco/endoplasmic reticulum calcium ATPase.

Analogues of the compound 2,5-di-tert-butylhydroquinone (BHQ) are capable of inhibiting the enzyme sarco/endoplasmic reticulum ATPase (SERCA) in the low micromolar and submicromolar concentration ranges. Not only are SERCA inhibitors valuable research tools, but they also have potential medicinal value as agents against prostate cancer. This study describes the synthesis of 13 compounds representing several classes of BHQ analogues, such as hydroquinones with a single aromatic substituent, symmetrically and unsymmetrically disubstituted hydroquinones, and hydroquinones with omega-amino acid tethers attached to their hydroxyl groups. Structure-activity relationships were established by measuring the inhibitory potencies of all synthesized compounds in bioassays. The assays were complemented by computational ligand docking for an analysis of the relevant ligand/receptor interactions.

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-L-lysine analogue of methotrexate, N alpha-(4-amino-4-deoxy-10- methylpteroyl )-N epsilon-(5-[N,N-dimethylamino]-1-naphthalenesulfonyl)-L-lysine, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [3H]methotrexate transport with a Ki of 7.02 microM, a value virtually identical to the Kt for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-L-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.