Venkata Mahidhar Yenugonda

Tumor-targeting nanodelivery enhances the anticancer activity of a novel quinazolinone analogue

GMC-5-193 (GMC) is a novel anticancer small-molecule quinazolinone analogue with properties that include antimicrotubule activity and inherent fluorescence. The aim of this study was to produce and optimize a systemically administered liposomal formulation for tumor-targeting delivery of GMC to enhance the anticancer effect of this compound and evaluate its bioefficacy. GMC was encapsulated within a cationic liposome, which was decorated on the surface with an anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the tumor-targeting moiety to form a nanoscale complex (scL/GMC). Confocal imaging of fluorescent GMC uptake in a human melanoma cell line, MDA-MB-435, showed higher cellular uptake of GMC when delivered via the liposome complex compared with free GMC. Delivery of GMC by the tumor-targeting liposome nanoimmunocomplex also resulted in a 3- to 4-fold decrease in IC50 values in human cancer cells [DU145 (prostate) and MDA-MB-435] compared with the effects of GMC administered as free GMC. In addition, the GMC nanoimmunocomplex increased the sensitivity of cancer cells to doxorubicin, docetaxel, or mitoxantrone by ∼3- to 30-fold. In the MDA435/LCC6 athymic nude mice xenograft lung metastases model, GMC was specifically delivered to tumors by the nanoimmunocomplex. These data show that incorporation of small-molecule therapeutic GMC within the tumor-targeting liposome nanocomplex enhances its anticancer effect. [Mol Cancer Ther 2008;7(3):559–68]

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.

VMY-1-103, a dansylated analog of purvalanol B, induces caspase-3-dependent apoptosis in LNCaP prostate cancer cells

The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy. ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 µM, VMY-1-103 increased both the proportion of cells in G1 and p21CIP1 protein levels. At higher concentrations (5 µM or 10 µM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 µM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo.

Quantifying the CDK inhibitor VMY-1-103’s activity and tissue levels in an in vivo tumor model by LC-MS/MS and by MRI

The development of new small molecule-based therapeutic drugs requires accurate quantification of drug bioavailability, biological activity and treatment efficacy. Rapidly measuring these endpoints is often hampered by the lack of efficient assay platforms with high sensitivity and specificity. Using an in vivo model system, we report a simple and sensitive liquid chromatography-tandem mass spectrometry assay to quantify the bioavailability of a recently developed novel cyclin-dependent kinase inhibitor VMY-1-103, a purvalanol B-based analog whose biological activity is enhanced via dansylation. We developed a rapid organic phase extraction technique and validated wide and functional VMY-1-103 distribution in various mouse tissues, consistent with its enhanced potency previously observed in a variety of human cancer cell lines. More importantly, in vivo MRI and single voxel proton MR-Spectroscopy further established that VMY-1-103 inhibited disease progression and affected key metabolites in a mouse model of hedgehog-driven medulloblastoma.

Chemistry and pharmacological studies of 3-alkoxy-2,5-disubstituted-pyridinyl compounds as novel selective α4β2 nicotinic acetylcholine receptor ligands that reduce alcohol intake in rats.

Neuronal acetylcholine receptors mediate the addictive effects of nicotine and may also be involved in alcohol addiction. Varenicline, an approved smoking cessation medication, showed clear efficacy in reducing alcohol consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors, was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder, we designed and synthesized novel sazetidine-A analogues containing a methyl group at the 2-position of the pyridine ring. In vitro pharmacological studies revealed that some of the novel compounds showed overall pharmacological property profiles similar to that of sazetidine-A but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In rat studies, compound (S)-9 significantly reduced alcohol uptake. More importantly, preliminary results from studies in a ferret model indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.

Design, synthesis and discovery of picomolar selective α4β2 nicotinic acetylcholine receptor ligands.

Developing novel and selective compounds that desensitize α4β2 nicotinic acetylcholine receptors (nAChRs) could provide new effective treatments for nicotine addiction, as well as other disorders. Here we report a new class of nAChR ligands that display high selectivity and picomolar binding affinity for α4β2 nicotinic receptors. The novel compounds have Ki values in the range of 0.031-0.26 nM and properties that should make them good candidates as drugs acting in the CNS. The selected lead compound 1 (VMY-2-95) binds with high affinity and potently desensitizes α4β2 nAChRs. At a dose of 3 mg/kg, compound 1 significantly reduced rat nicotine self-administration. The overall results support further characterizations of compound 1 and its analogues in preclinical models of nicotine addiction and perhaps other disorders involving nAChRs.

VMY-1-103 is a novel CDK inhibitor that disrupts chromosome organization and delays metaphase progression in medulloblastoma cells

Medulloblastoma is the most prevalent of childhood brain malignancies, constituting 25% of childhood brain tumors. Craniospinal radiotherapy is a standard of care, followed by a 12-month regimen of multi-agent chemotherapy. For children less than 3 years of age, irradiation is avoided due to its destructive effects on the developing nervous system. Long-term prognosis is worst for these youngest children and more effective treatment strategies with a better therapeutic index are needed. VMY-1-103, a novel dansylated analog of purvalanol B, was previously shown to inhibit cell cycle progression and proliferation in prostate and breast cancer cells more effectively than purvalanol B. In the current study, we have identified new mechanisms of action by which VMY-1-103 affected cellular proliferation in medulloblastoma cells. VMY-1-103, but not purvalanol B, significantly decreased the proportion of cells in S phase and increased the proportion of cells in G2/M. VMY-1-103 increased the sub G1 fraction of apoptotic cells, induced PARP and caspase-3 cleavage and increased the levels of the Death Receptors DR4 and DR5, Bax and Bad while decreasing the number of viable cells, all supporting apoptosis as a mechanism of cell death. p21CIP1/WAF1 levels were greatly suppressed. Importantly, we found that while both VMY and flavopiridol inhibited intracellular CDK1 catalytic activity, VMY-1-103 was unique in its ability to severely disrupt the mitotic spindle apparatus significantly delaying metaphase and disrupting mitosis. Our data suggest that VMY-1-103 possesses unique antiproliferative capabilities and that this compound may form the basis of a new candidate drug to treat medulloblastoma.

Preclinical studies of the potent and selective nicotinic α4β2 receptor ligand VMY-2-95.

The discovery and development of small molecules that antagonize neuronal nicotinic acetylcholine receptors may provide new ligands for evaluation in models of depression or addiction. We discovered a small molecule, VMY-2-95, a nAChR ligand with picomolar affinity and high selectivity for α4β2 receptors. In this study, we investigated its preclinical profile in regards to solubility, lipophilicity, metabolic stability, intestinal permeability, bioavailability, and drug delivery to the rat brain. Metabolic stability of VMY-2-95·2HCl was monitored on human liver microsomes, and specific activity of VMY-2-95·2HCl on substrate metabolism by CYP1A2, 2C9, 2C19, 2D6, and 3A4 was tested in a high-throughput manner. The intestinal transport of VMY-2-95·2HCl was studied through Caco-2 cell monolayer permeability. VMY-2-95·2HCl was soluble in water and chemically stable, and the apparent partition coefficient was 0.682. VMY-2-95·2HCl showed significant inhibition of CYP2C9 and 2C19, but weak or no effect on 1A2, 2D6, and 3A4. The Caco-2 cell model studies revealed that VMY-2-95·2HCl was highly permeable with efflux ratio of 1.11. VMY-2-95·2HCl achieved a maximum serum concentration of 0.56 mg/mL at 0.9 h and was orally available with a half-life of ∼9 h. Furthermore, VMY-2-95·2HCl was detected in the rat brain after 3 mg/kg oral administration and achieved a maximal brain tissue concentration of 2.3 μg/g within 60 min. Overall, the results demonstrate that VMY-2-95·2HCl has good drug like properties and can penetrate the blood–brain barrier with oral administration.

Trans-resveratrol boronic acid exhibits enhanced anti-proliferative activity on estrogen-dependent MCF-7 breast cancer cells

Resveratrol (RSV), a natural compound present in the skin and seeds of red grapes, is considered a phytoestrogen and has structural similarity to the synthetic estrogen diethylstilbestrol. RSV inhibits tumor cell growth in estrogen receptor-positive (ER+) and negative (ER-) breast cancer cell lines resulting in cell specific regulation of the G1/S and G2/M stages of the cell cycle. However apoptotic cell death was only observed in ER+ MCF-7 cells. In this study, we designed and synthesized boronic acid derivative of RSV and evaluated their biological effects on ER+ MCF-7 breast cancer cells. The trans-4 analog inhibited the growth of MCF-7 cells and is not a substrate for p-glycoprotein. The trans-4 analog induces G1 cell cycle arrest, which coincides with marked inhibition of G1 cell cycle proteins and a greater pro-apoptotic effect. Finally, the trans-4 analog had no effect on the estrogen-stimulated growth of MCF-7 cells. Our results demonstrate that the trans-4 analog inhibits MCF-7 breast cancer cells by a different mechanism of action than that of RSV (S-phase arrest), and provides a new class of novel boronic acids of RSV that inhibit breast cancer cell growth.

Effects of the sazetidine-a family of compounds on the body temperature in wildtype, nicotinic receptor β2−/− and α7−/− mice

Nicotine elicits hypothermic responses in rodents. This effect appears to be related to nicotinic receptor desensitization because sazetidine-A, an α4β2 nicotinic receptor desensitizing agent, produces marked hypothermia and potentiates nicotine-induced hypothermia in mice. To determine the specificity of sazetidine-A induced hypothermia to β2 subunit-containing nicotinic receptors, we tested its efficacy in β2 knockout (β2(-/-)) mice. These effects were compared with wildtype (WT) and α7 knockout (α7(-/-)) mice. Confirming our earlier results, sazetidine-A elicited a pronounced and long-lasting hypothermia in WT mice. In comparison, sazetidine-A induced a much attenuated and shorter hypothermic response in β2(-/-) mice. This indicates that the greater proportion of sazetidine-A induced hypothermia is mediated via actions on β2-containing nicotinic receptors, while a smaller component of hypothermia induced by sazetidine-A is mediated by non-β2 receptors. Similar to WT mice, α7(-/-) mice showed the full extent of the sazetidine-A effect, suggesting that the hypothermia produced by sazetidine-A did not depend on actions on α7 nicotinic receptor subtype. Three other novel nicotinic receptor desensitizing agents derived from sazetidine-A, triazetidine-O, VMY-2-95 and YL-1-127 also produced hypothermia in WT and α7(-/-) mice. Furthermore, unlike sazetidine-A, triazetidine-O and YL-1-127 did not show any hint of a hypothermic effect in β2(-/-) mice. VMY-2-95 like sazetidine-A did show a residual hypothermic effect in the β2(-/-) mice. These studies show that the hypothermic effects of sazetidine-A and the related compound VMY-2-95 are mainly mediated by nicotinic receptors containing β2 subunit, but that a small component of the effect is apparently mediated by non-β2 containing receptors.