Mechanism and Kinetics of Microtubule Perturbing Agents

Microtubules are cellular cytoskeletal components that play an integral part in many cell functions. Compounds that bind to microtubules and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. A series of analogues of the microtubule stabilizing dictyostatin were examined to probe biological and biochemical structure-activity relationships. The results were consistent with previous reports showing that 16-normethyldictyostatin and 15Z,16-normethyldictyostatin lose potency in pacitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of â-tubulin. 6-epi-Dictyostatin and 7-epi-dictyostatin were potent analogues of dictyostatin, and 6-epi-dictyostatin was chosen for milligram scale for pre-clinical studies. The thalidomide analogue 5HPP-33 was identified as an easily synthesized small microtubule perturbing agent, and experiments with isolated tubulin were performed to determine its mechanism of action. Tubulin polymerization was used to determine the effect of 5HPP-33 on normal microtubule formation. In experiments utilizing microtubule associated proteins (MAPs) to induce polymer formation, 5HPP-33 inhibited tubulin polymerization, but under a different set of conditions appeared to form and stabilize microtubules. The polymer was imaged using electron microscopy, which showed that 5HPP-33 caused the formation of spirals and rings. Due to 5HPP-33 failing to compete with known radiolabeled microtubule perturbing agents for their respective binding sites, a tritiated version of 5HPP-33 was synthesized. The binding experiments performed showed that [3H]5HPP-33 had a slight affinity for isolated MAPs, and this was the reason for the discrepancy between the tubulin polymerization experiments. A binding site for 5HPP-33 could not be determined, making it a possible novel microtubule perturbing agent. (-)-Pironetin is a microtubule inhibitor that appears to form a covalent linkage to the tubulin heterodimer. Although immunoflourescent images showed (-)-pironetin to work in the same manner as vinblastine, cellular and biochemical experiments proved that (-)-pironetin is mechanistically different from vinblastine. The tubulysins are known microtubule destabilizers and bind to the vinca domain on â-tubulin. Three analogues of the tubulysins were synthesized and their effects on cell growth and microtubule perturbation experiments were determined. WZY-111-63C (N14-desacetoxytubulysin H) was found to be 50 times more cytotoxic than paclitaxel and vincristine

Quantitation of zoledronic acid in murine bone by liquid chromatography coupled with tandem mass spectrometry

a b s t r a c t An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250-50.0 g/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06 ± 0.52 vs. 0.76 ± 0.09 ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment

Associations between Dietary Intake of Fruits and Vegetables in Relation to Urinary Estrogen DNA Adduct Ratio

BACKGROUND: Estrogen exposure plays a role in breast cancer (BC) development. A novel estrogen biomarker, the estrogen DNA adduct (EDA) ratio, was shown to be elevated in women at high-risk of BC and among BC cases. Modifiable factors may impact the EDA ratio, with studies demonstrating that resveratrol reduces EDA ratio in vitro. We sought to examine the hypothesis that dietary intake of fruits and vegetables is inversely associated with EDA ratio. METHODS: This analysis was conducted in 53 pre-menopausal, healthy women aged 40–45 years from a cross-sectional study in which participants provided first-void urine samples and 3-day food records. Urine samples were analyzed using ultraperformance liquid chromatography/tandem mass spectrometry. The EDA ratio was calculated as the estrogen-DNA adducts divided by estrogen metabolites and conjugates. A trend test was used to assess associations between tertiles of dietary intake using linear regression. RESULTS: After adjustment for age, total energy, percent adiposity, serum estradiol and estrone-sulfate, we observed inverse associations of EDA ratio with carbohydrate consumption (P=0.01) and vegetable intake (P =0.01). EDA ratio was inversely associated with 5 botanical groups (Chenopodiaceae: P=0.02; Umbelliferae: P=0.03; Compositae: P=0.01; Ericaceae: P=0.01; Musaceae: P=0.03) but not fruit intake overall. CONCLUSION: Although these data require replication before conclusions are drawn, this report suggests an inverse association between vegetable and carbohydrate consumption and EDA ratio. IMPACT: While more information is still needed, these findings suggest a link between dietary intake and a biomarker that is both associated with high-risk BC status and associated with modifiable factors

Recipient Pretransplant Inosine Monophosphate Dehydrogenase Activity in Nonmyeloablative Hematopoietic Cell Transplantation

AbstractMycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5′-monophosphate to xanthosine 5′-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients

Microtubule Binding and Disruption and Induction of Premature Senescence by Disorazole C1S⃞

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization, with an IC50 of 11.8 ± 0.4 μM, and inhibited [3H]vinblastine binding noncompetitively, with a Ki of 4.5 ± 0.6 μM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C1, with a Ki of 10.6 ± 1.5 μM, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence