Mikell Paige

Asymmetric Synthesis of 2,3-Dihydro-2-arylquinazolin-4-ones: Methodology and Application to a Potent Fluorescent Tubulin Inhibitor with Anticancer Activity

For several decades the 2,3-dihydroquinazolinone (DHQZ) heterocycle has been known to possess a variety of important biological and medicinal properties. Despite the many interesting facets of these molecules, synthetic access to nonracemic DHQZ analogues has remained elusive. Herein, we disclose a synthetic route that allows access to either enantiomer of a variety of DHQZ derivatives. We illustrate the utility of this chemistry with the asymmetric preparation and biological evaluation of a new chiral fluorescent tubulin binding agent with extremely potent antiproliferative properties against human cancer cells. A computational rationale for the increased potency of the (S)-enantiomer over the (R)-enantiomer is given, based on the crystal structure of alpha,beta-tubulin complexed with colchicine. Taking advantage of the inherent fluorescence of these molecules, confocal images of GMC-5-193 (compound 7) in the cytoplasm of human melanoma cells (MDA-MB-435) cells are presented.

Sazetidine-A, a Selective α4β2 Nicotinic Receptor Desensitizing Agent and Partial Agonist, Reduces Nicotine Self-Administration in Rats

Adequate treatment of tobacco addiction remains problematic. Part of the problem with treatment is a poor understanding of the pharmacologic aspects of nicotine contributing to addiction. In addition to activating nicotinic acetylcholine receptors, nicotine also desensitizes them. It is currently not known how much of each of nicotines actions contribute to its particular behavioral effects. Sazetidine-A (saz-A) is a novel nicotinic receptor-desensitizing agent and partial agonist with high selectivity for α4β2 receptors. The current experiments were conducted to determine whether saz-A would reduce nicotine self-administration in rats and to characterize its ancillary effects. Adult male Sprague-Dawley rats were allowed to self-administer nicotine. After initial food pellet training followed by 10 sessions of nicotine self-administration training, the rats were administered saz-A (0.1–3 mg/kg s.c.) or the saline vehicle in a repeated-measures counterbalanced design. Saz-A at the 3 mg/kg dose significantly decreased nicotine self-administration relative to performance of the same rats after saline injections. In a second study, long-term administration of this dose of sazetidine-A over the course of 10 sessions significantly reduced nicotine self-administration with no apparent diminution of effect. Saz-A in this dose range had only modest effects on locomotor activity, without any overall decrease in activity over a 1-h-long session. Saz-A significantly reduced food self-administration, but this effect was smaller than its effect on nicotine self-administration. Saz-A, which is a selective α4β2-desensitizing agent and partial agonist, effectively reduces nicotine self-administration. This type of treatment holds promise for a new therapy to aid smoking cessation.

A boronic acid chalcone analog of combretastatin A-4 as a potent anti-proliferation agent

Chalcones represent a class of natural products that inhibits tubulin assembly. In this study we designed and synthesized boronic acid analogs of chalcones in an effort to compare biological activities with combretastatin A-4, a potent inhibitor of tubulin polymerization. Systematic evaluation of the positional effects of the carbonyl moiety towards inhibition of tubulin polymerization, cancer cell proliferation and angiogenesis revealed that placement of the carbonyl adjacent to the trimethoxybenzene A-ring resulted in more active compounds than when the carbonyl group was placed adjacent to the C-ring. Our study identified a boronic acid chalcone with inhibition towards 16 human cancer cell lines in the 10-200nM range, and another three cell lines with GI(50)-values below 10nM. Furthermore, this drug has significant anti-angiogenesis effects demonstrated by HUVEC tube formation and aortic ring assay.

Fluorescent epigenetic small molecule induces expression of the tumor suppressor ras-association domain family 1A and inhibits human prostate xenograft.

Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance. Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.

Pilot Analysis of the Plasma Metabolite Profiles Associated with Emphysematous Chronic Obstructive Pulmonary Disease Phenotype

The current pilot study examined the hypothesis that cigarette smokers who developed an emphysematous phenotype of Chronic Obstructive Pulmonary Disease (COPD) were associated with distinctive patterns in their corresponding metabolomics profile as compared to those who did not. Peripheral blood plasma samples were collected from 38 subjects with different phenotypes of COPD. They were categorized into three groups: healthy non-smokers (n=16), smokers without emphysema (n=8), and smokers with emphysema (n=14). Ultra High Performance Liquid Chromatography/quadrupole-Time-of-Flight Mass Spectrometry techniques were used to identify a large number of metabolite markers (3534). Unsupervised clustering analysis accurately separated the smokers with emphysema from others without emphysema and demonstrated potentials of this metabolomics data. Subsequently predictive models were created with a supervised learning set, and these predictive models were found to be highly accurate in identifying the subjects with the emphysematous phenotype of COPD with excellent sensitivity and specificity. Our methodology provides a preliminary model that differentiates an emphysematous COPD phenotype from other COPD phenotypes on the basis of the metabolomics profiles. These results also suggest that the metabolomics profiling could potentially guide the characterization of relevant metabolites that leads to an emphysematous COPD phenotype.

Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells.

Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers. CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhibitory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors (VMY-1-101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undiminished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, combining therapeutic and diagnostic properties in the same molecule.

Role of LTB4 in the pathogenesis of elastase-induced murine pulmonary emphysema

Exaggerated levels of the leukotriene B₄ (LTB₄) frequently coexist at sites of inflammation and tissue remodeling. Therefore, we hypothesize that the LTB₄ pathway plays an important role in the pathogenesis of neutrophilic inflammation that contributes to pulmonary emphysema. In this study, significant levels of LTB₄ were detected in human lung tissues with emphysema compared with lungs without emphysema (9,497 ± 2,839 vs. 4,142 ± 1,173 pg/ml, n = 9 vs. 10, P = 0.04). To further determine the biological role of LTB₄ in the pathogenesis of emphysema, we compared the lungs of wild-type (WT) and LTA₄ hydrolase-/- mice (LTB₄ deficient, LTA₄H-/-) exposed to intranasal elastase or vehicle control. We found that intranasal elastase induced accumulation of LTB₄ in the lungs and caused progressively worsening emphysema between 14 and 28 days after elastase exposure in WT mice but not in LTA₄H-/- mice. Premortem physiology documented increased lung compliance in elastase-exposed WT mice compared with elastase-exposed LTA₄H-/- mice as measured by Flexivent (0.058 ± 0.005 vs. 0.041 ± 0.002 ml/cmH₂O pressure). Postmortem morphometry documented increased total lung volume and alveolar sizes in elastase-exposed WT mice compared with elastase-exposed LTA₄H-/- mice as measured by volume displacement and alveolar chord length assessment. Furthermore, elastase-exposed LTA₄H-/- mice were found to have significantly delayed influx of the CD45(high)CD11b(high)Ly6G(high) leukocytes compatible with neutrophils compared with elastase-exposed WT mice. Mechanistic insights to these phenotypes were provided by demonstrating protection from elastase-induced murine emphysema with neutrophil depletion in the elastase-exposed WT mice and by demonstrating time-dependent modulation of cysteinyl leukotriene biosynthesis in the elastase-exposed LTA₄H-/- mice compared with elastase-exposed WT mice. Together, these findings demonstrated that LTB₄ played an important role in promoting the pathogenesis of pulmonary emphysema associated with neutrophilic pulmonary inflammation.

Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema.

The leukotriene A(4) hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A(4) to leukotriene B(4) and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro-inflammatory properties of its metabolite, leukotriene B(4). Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A(4) hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models.

ApoE mimetic peptide decreases Aβ production in vitro and in vivo

BackgroundApolipoprotein E (apoE) is postulated to affect brain Aβ levels through multiple mechanisms--by altering amyloid precursor protein (APP) processing, Aβ degradation, and Aβ clearance. We previously showed that an apoE-derived peptide containing a double repeat of the receptor-binding region was similarly effective in increasing APP processing in vivo. Here, we further examined whether peptides containing tandem repeats of the apoE receptor-binding region (amino acids 141-149) affected APP trafficking, APP processing, and Aβ production.ResultsWe found that peptides containing a double or triple tandem repeat of the apoE receptor-binding region, LRKLRKRLL, increased cell surface APP and decreased Aβ levels in PS1-overexpressing PS70 cells and in primary neurons. This effect was potentiated by a sequential increase in the number of apoE receptor-binding domain repeats (trimer > dimer > monomer). We previously showed that the apoE dimer increased APP CTF in vivo; to determine whether the dimer also affected secreted APP or Aβ levels, we performed a single hippocampal injection of the apoE dimer in wild-type mice and analyzed its effect on APP processing. We found increased sAPPα and decreased Aβ levels at 24 hrs after treatment, suggesting that the apoE dimer may increase α-secretase cleavage.ConclusionsThese data suggest that small peptides consisting of tandem repeats of the apoE receptor-binding region are sufficient to alter APP trafficking and processing. The potency of these peptides increased with increasing repeats of the receptor binding domain of apoE. In addition, in vivo administration of the apoE peptide (dimer) increased sAPPα and decreased Aβ levels in wild-type mice. Overall, these findings contribute to our understanding of the effects of apoE on APP processing and Aβ production both in vitro and in vivo.

Expansion of microvascular networks in vivo by phthalimide neovascular factor 1 (PNF1)

Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2(-/-) (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.