Paul W. Erhardt

Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis

Purpose: We have identified the phytoalexin compounds glyceollins I, II, and III, which exhibit marked antiestrogenic effects on estrogen receptor function and estrogen-dependent tumor growth in vivo. The purpose of this study was to investigate the interactions among the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in ovariectomized athymic mice. Experimental Design: Four treatment groups for each cell line were used: vehicle control, 20 mg/kg/mouse/d glyceollin mixture injection, 0.72 mg estradiol (E2) implant, and E2 implant + 20 mg/kg/mouse/d glyceollin injection. Results: Treatment with glyceollin suppressed E2-stimulated tumor growth of MCF-7 cells (−53.4%) and BG-1 cells (−73.1%) in ovariectomized athymic mice. These tumor-inhibiting effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumors. In contrast to tamoxifen, the glyceollins had no estrogen-agonist effects on uterine morphology and partially antagonized the uterotropic effects of estrogen. Conclusions: These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma.

Synthesis and Evaluation of Indole-Based Chalcones as Inducers of Methuosis, a Novel Type of Nonapoptotic Cell Death

Methuosis is a novel caspase-independent form of cell death in which massive accumulation of vacuoles derived from macropinosomes ultimately causes cells to detach from the substratum and rupture. We recently described a chalcone-like compound, 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MIPP), which can induce methuosis in glioblastoma and other types of cancer cells. Herein, we describe the synthesis and structure-activity relationships of a directed library of related compounds, providing insights into the contributions of the two aryl ring systems and highlighting a potent derivative, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MOMIPP) that can induce methuosis at low micromolar concentrations. We have also generated biologically active azide derivatives that may be useful for future studies aimed at identifying the protein targets of MOMIPP by photoaffinity labeling techniques. The potential significance of these studies is underscored by the finding that MOMIPP effectively reduces the growth and viability of Temozolomide-resistant glioblastoma and doxorubicin-resistant breast cancer cells. Thus, it may serve as a prototype for drugs that could be used to trigger death by methuosis in cancers that are resistant to conventional forms of cell death (e.g., apoptosis).

Ultra-short acting beta-blockers: A proposal for the treatment of the critically ill patient

Beta-blockade is of proven value in the therapy of acute myocardial infarction but, unfortunately, may produce cardiac failure by removal of needed sympathetic support. The long duration of action of available blockers (hours) makes reversal of failure a complicated problem and precludes rapid modification of therapy to match changing autonomic conditions. To improve the safety and efficacy of beta-blockade in this setting we have developed the concept of ultra-short beta-blockade and have identified a novel beta-blocker (ASL-8052) which possesses a duration of action less than 15 minutes. This compound is cardioselective and possesses efficacy in an animal model of acute myocardial infarction. It, therefore, appears to be suitable for rapid attainment of controlled levels of beta-blockade via intravenous infusion and rapid recovery from beta-blockade if required by the clinical situation. The compound should, therefore, be useful for safe therapy in critically ill cardiac patients.

Medicinal Chemistry in the New Millennium: A Glance into the Future

The future of medicinal chemistry as both a pure and an applied science has been considered relative to trends that are already having a significant impact upon drug discovery and development. After quickly reviewing how medicinal chemistry has been practiced to date, topics considered into the future include: pursuing therapeutic efficacy, addressing 3D structure within database settings, assuring absorption, directing distribution, controlling metabolism, optimizing elimination, and avoiding toxicity. It is suggested that as the exploration of these topics proceeds into the new millennium by deploying combinatorial chemistry coupled to high-throughput screening, medicinal chemistry will play a key role as a central interpreter of the underlying structureactivity relationships such that the overall process of drug discovery and development will be knowledge-generating. As fundamental knowledge accumulates across all of these areas, virtual approaches will eventually become firmly anchored to experimental and theoretical databases having validated clinical predictability. The potential impact of some of the recent trends in process chemistry, and in analytical chemistry using X-ray diffraction as an exemplary method, are additionally highlighted before reiterating the articles major points in a summary section. From this purview, the summary also considers the education of future medicinal chemists, notes potential issues related to the future of pharmaceutical-related intellectual property, and concludes by alluding to a brewing paradox between enhanced knowledge and enhanced molecular diversity relative to the future discovery of new drugs.

Total Syntheses of Racemic, Natural (−) and Unnatural (+) Glyceollin I

The first total syntheses of racemic glyceollin I and its enantiomers are described. A Wittig approach was utilized as an entry to the appropriately substituted isoflav-3-ene so that an osmium tetroxide mediated asymmetric dihydroxylation could be deployed for stereospecific introduction of the 6a-hydroxy group. While using triphenylphosphine hydrobromide, a novel method was found for gently removing MOM from protected phenolic hydroxyl groups present within sensitive systems.

Microplate Screening of the Differential Effects of Test Agents on Hoechst 33342, Rhodamine 123, and Rhodamine 6G Accumulation in Breast Cancer Cells that Overexpress P-Glycoprotein:

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 μM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.

Glyceollins, Soy Isoflavone Phytoalexins, Improve Oral Glucose Disposal by Stimulating Glucose Uptake

Soy glyceollins, induced during stress, have been shown to inhibit cancer cell growth in vitro and in vivo. In the present study, we used prediabetic rats to examine the glyceollins effect on blood glucose. During an oral glucose tolerance test (OGTT), the blood glucose excursion was significantly decreased in the rats treated with oral administration of either 30 or 90 mg/kg glyceollins. Plasma analysis demonstrated that glyceollins are absorbed after oral administration, and duration of exposure extends from 20 min to at least 4 h postadministration. Exposure of 3T3-L1 adipocytes to glyceollins significantly increased both insulin-stimulated and basal glucose uptake. Basal glucose uptake was increased 1.5-fold by exposure to 5 μM glyceollin in a dose-response manner. Coincubation with insulin significantly stimulated maximal glucose uptake above basal uptake levels and tended to increase glucose uptake beyond the levels of either stimulus alone. On a molecular level, polymerase chain reaction showed significantly increased levels of glucose transporter GLUT4 mRNA in 3T3-L1 adipocytes, especially when the cells were exposed to 5 μM glyceollins for 3 h in vitro. It correlated with elevated protein levels of GLUT4 detected in the 5 μM glyceollin-treated cells. Thus, the simulative effect of the glyceollins on adipocyte glucose uptake was attributed to up-regulation of glucose transporters. These findings indicate potential benefits of the glyceollins as an intervention in prediabetic conditions as well as a treatment for type 1 and type 2 diabetes by increasing both the insulin-mediated and the basal, insulin-independent, glucose uptake by adipocytes.

Theoretical Studies of Salt-Bridge Formation by Amino Acid Side Chains in Low and Medium Polarity Environments

Salt-bridge formation between Asp/Glu···Lys and Asp/Glu···Arg side chains has been studied by model systems including formic and acetic acids as proton donors and methylamine, guanidine, and methylguanidine as proton acceptors. Calculations have been performed up to the CCSD(T)(CBS)//MP2/aug-cc-pvtz level with formic acid proton donors. Complexes formed with acetic acid were studied at the CCSD(T)/aug-cc-pvdz//MP2/aug-cc-pvdz level. Protein environments of low and moderate polarity were mimicked by a continuum solvent with dielectric constants (ε) set to 5 and 15, respectively. Free energy differences, ΔG(tot), were calculated for the neutral, hydrogen-bonded form and for the tautomeric ion pair. These values predict that a salt bridge is not favored for the Asp/Glu···Lys pair, even in an environment with ε as large as 15. In contrast, the Asp/Glu···Arg salt bridge is feasible even in an environment with ε = 5. Charge transfers for the complexes were calculated on the basis of CHELPG and AIM charges.

On the Interaction of Aliphatic Amines and Ammonium Ions with Carboxylic Acids in Solution and in Receptor Pockets

Association energies of the acetate ion with cationic amines bearing one to three methyl groups were calculated in the range of -14 to -17 kcal/mol in aqueous solution by means of the IEF-PCM method at the CCSD(T)/CBS//MP2/aug-cc-pvdz and DFT/B97D/CBS//B97D/aug-cc-pvtz levels. The main stabilization factor for the association is the possibility for the formation of an ionic intermolecular hydrogen bond between the elements of the complex. For a quaternary ammonium ion, the favorable electrostatic interaction energy is the only driving force, and the stabilization energy for the complex is reduced to -4 kcal/mol. The internal free energies of the ion-pair tautomers of the studied species are higher by 10-15 kcal/mol in water than those for the neutral, hydrogen-bonded forms. Monte Carlo free energy perturbation calculations at T = 298 K and p = 1 atm predict -11 to -16 kcal/mol relative solvation free energy in favor of the corresponding ionic form. As a result, the ion-pair tautomer is the prevailing form in aqueous solution and on the extracellular surface of a receptor. Modeling the complex of a protonated ligand interacting with an Asp/Glu carboxylate side-chain in the binding cavity of a receptor, two strongly bound water molecules were considered so as to form hydrogen-bonded water bridges between the elements of the ion-pair. Nonetheless, the low polarity environment mimicked by a chloroform solvent cannot stabilize the ionic tautomer. A proton jump was predicted, which suggests that acetylcholine, an inherent cation by structure, might have evolved as the natural agonist for muscarinic receptors because a quaternary ammonium system assures the maintenance of the ion-pair form with a carboxylate side-chain in a protein cavity, the latter perhaps then being needed for receptor activation.

Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity.

Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.