Sterically induced conformational restriction: Discovery and preclinical evaluation of novel pyrrolo[3,2-d]pyrimidines as microtubule targeting agents.

The discovery, synthesis and biological evaluations of a series of nine N5-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds with microtubule depolymerizing activity were identified. Some of these compounds also circumvent clinically relevant drug resistance mechanisms (expression of P-glycoprotein and ßIII tubulin). Compounds 4, 5, and 8-13 were one to two-digit nanomolar (IC50) inhibitors of cancer cells in culture. Contrary to recent reports (Banerjee et al. J. Med. Chem.2018, 61, 1704-1718), the conformation of the most active compounds determined by 1H NMR and molecular modeling are similar to that reported previously and in keeping with recently reported X-ray crystal structures. Compound 11, freely water soluble as the HCl salt, afforded statistically significant inhibition of tumor growth in three xenograft models [MDA-MB-435, MDA-MB-231 and NCI/ADR-RES] compared with controls. Compound 11 did not display indications of animal toxicity and is currently slated for further preclinical development.

Janus Compounds, 5-Chloro-N4-methyl-N4-aryl-9H-pyrimido[4,5-b]indole-2,4-diamines, Cause Both Microtubule Depolymerizing and Stabilizing Effects.

While evaluating a large library of compounds designed to inhibit microtubule polymerization, we identified four compounds that have unique effects on microtubules. These compounds cause mixed effects reminiscent of both microtubule depolymerizers and stabilizers. Immunofluorescence evaluations showed that each compound initially caused microtubule depolymerization and, surprisingly, with higher concentrations, microtubule bundles were also observed. There were subtle differences in the propensity to cause these competing effects among the compounds with a continuum of stabilizing and destabilizing effects. Tubulin polymerization experiments confirmed the differential effects and, while each of the compounds increased the initial rate of tubulin polymerization at high concentrations, total tubulin polymer was not enhanced at equilibrium, likely because of the dueling depolymerization effects. Modeling studies predict that the compounds bind to tubulin within the colchicine site and confirm that there are differences in their potential interactions that might underlie their distinct effects on microtubules. Due to their dual properties of microtubule stabilization and destabilization, we propose the name Janus for these compounds after the two-faced Roman god. The identification of synthetically tractable, small molecules that elicit microtubule stabilizing effects is a significant finding with the potential to identify new mechanisms of microtubule stabilization.

The design and discovery of water soluble 4-substituted-2,6-dimethylfuro[2,3-d]pyrimidines as multitargeted receptor tyrosine kinase inhibitors and microtubule targeting antitumor agents.

The design, synthesis and biological evaluations of fourteen 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines are reported. Four compounds (11-13, 15) inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor ß (PDGFR-ß), and target tubulin leading to cytotoxicity. Compound 11 has nanomolar potency, comparable to sunitinib and semaxinib, against tumor cell lines overexpressing VEGFR-2 and PDGFR-ß. Further, 11 binds at the colchicine site on tubulin, depolymerizes cellular microtubules and inhibits purified tubulin assembly and overcomes both ßIII-tubulin and P-glycoprotein-mediated drug resistance, and initiates mitotic arrest leading to apoptosis. In vivo, its HCl salt, 21, reduced tumor size and vascularity in xenograft and allograft murine models and was superior to docetaxel and sunitinib, without overt toxicity. Thus 21 affords potential combination chemotherapy in a single agent.

Synthesis of N(4)-(substituted phenyl)-N(4)-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells.

A series of fourteen N(4)-(substituted phenyl)-N(4)-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlorination and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nanomolar GI(50) values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6 inhibited tubulin polymerization and [(3)H]colchicine binding to tubulin. The most potent compounds were all effective in cells expressing P-glycoprotein or the ßIII isotype of tubulin, which have been associated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15 within the colchicine site.

Oral and Craniofacial Stem Cells: An Untapped Source for Neural Tissue Regeneration.

Nerve tissue regeneration continues to represent an intractable obstacle to realizing the promise of tissue engineering. Although neurobiology works to shed light on the mechanisms governing neuronal growth and repair, considerable technical gaps remain that hinder progress. Chief among these is the absence of an appropriate culture environment to faithfully reproduce the neuronal niche ex vivo. We propose that the various multipotent cells found in the oral cavity may represent an important yet underutilized resource for preparing such neurogenic microenvironments. Similar to those of nerve tissue, these cell populations are of ectodermal origin and have clinically demonstrated neurogenic potential. Although there is a lack of consensus on whether putative types of oral and craniofacial stem cells constitute distinct populations, their contribution to neural tissue engineering may be twofold: as a cellular feedstock for neoneurogenesis and for the production of specialized in vitro environments for neurogenic differentiation, phenotype maintenance, and use in therapeutic applications. Impact statement We propose that addressing gaps in understanding the neurogenic role of dental stem cells and their microenvironment may yield efficient and reliable strategies for long-term neuronal cell culture and open new avenues for neural regeneration in both dental, nerve, and other tissues.

Correction: Regulation of E-cadherin localization by microtubule targeting agents: rapid promotion of cortical E-cadherin through p130CAS/Src inhibition by eribulin.

[This corrects the article DOI: 10.18632/oncotarget.23798.].

Regulation of E-cadherin localization by microtubule targeting agents: rapid promotion of cortical E-cadherin through p130Cas/Src inhibition by eribulin.

Microtubule targeting agents (MTAs) are some of the most effective anticancer drugs used to treat a wide variety of adult and pediatric cancers. Building evidence suggests that these drugs inhibit interphase signaling events and that this contributes to their anticancer actions. The effects of diverse MTAs were evaluated following a 2 hour incubation with clinically relevant concentrations to test the hypothesis that these drugs rapidly and differentially disrupt epithelial-to-mesenchymal transition (EMT)-related signaling. The MTAs rapidly promoted the cortical localization of internal pools of E-cadherin in HCC1937 breast cancer cells, with the most robust effects observed with the microtubule destabilizers eribulin and vinorelbine. Cortical E-cadherin localization was also promoted by the Src kinase inhibitor dasatinib or by siRNA-mediated depletion of the p130Cas scaffold. Mechanistic studies demonstrate that eribulin disrupts the interaction between p130Cas and Src, leading to decreased cortical Src phosphorylation that precedes the accumulation of cortical E-cadherin. These results suggest that microtubules can be actively co-opted by cancer cells to inhibit cortical E-cadherin localization, a hallmark of EMT, and provide a direct link between the initial disruption of the microtubule network and reversal of EMT phenotypes demonstrated by eribulin in long-term studies.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Breast Cancer Cell Lines Exhibit Differential Sensitivities to Microtubule-targeting Drugs Independent of Doubling Time.

BACKGROUND: Microtubule-targeting agents (MTAs) are a mainstay in breast cancer treatment, yet patient responses differ. The underlying mechanisms of these differences are unknown. While MTAs are mitotic inhibitors, recent evidence highlights that non-mitotic effects of these drugs can contribute to their anticancer effects. It is critical to identify the non-mitotic mechanisms that could contribute to differences among MTAs. However, it is not clear whether rapidly dividing cells in culture are optimal tools to address these mechanistic questions in interphase cells. MATERIALS AND METHODS: Detailed concentration response curves for five MTAs in a panel of diverse breast cancer cell lines were generated. RESULTS: Substantial differences among both drugs and cell lines, consistent with the clinical scenario, were observed. Importantly, these differences do not correlate with cell doubling time. CONCLUSION: The interphase actions of MTAs are critical to the full spectrum of their effects in cancer cells, even in cell culture models.

Eribulin mesylate: mechanism of action of a unique microtubule-targeting agent.

Eribulin mesylate (eribulin), an analogue of the marine natural product halichondrin B, is a microtubule-depolymerizing drug that has utility in the treatment of patients with breast cancer. Clinical trial results have demonstrated that eribulin treatment provides a survival advantage to patients with metastatic or locally advanced breast cancer previously treated with an anthracycline and a taxane. Furthermore, a pooled analysis of two pivotal phase III trials has demonstrated that eribulin also improves overall survival in several patient subgroups, including in women with HER2-negative disease and triple-negative breast cancer. This review covers the preclinical research that led to the clinical testing and approval of eribulin, as well as subsequent research that was prompted by distinct and unexpected effects of eribulin in the clinic. Initial studies with halichondrin B demonstrated unique effects on tubulin binding that resulted in distinct microtubule-dependent events and antitumor actions. Consistent with the actions of the natural product, eribulin has potent microtubule-depolymerizing activities and properties that distinguish it from other microtubule-targeting agents. Here, we review new results that further differentiate the effects of eribulin from other agents on peripheral nerves, angiogenesis, vascular remodeling, and epithelial-to-mesenchymal transition. Together, these data highlight the distinct properties of eribulin and begin to delineate the mechanisms behind the increased survival benefit provided by eribulin for patients.

Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9-Epipolygodial against Drug-Resistant Cancer Cells.

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1-agonist and anticancer activities. These experiments led to the identification of 9-epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9-Epipolygodial was found to maintain potency against apoptosis-resistant cancer cells as well as those displaying the multidrug-resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal-Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.

Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines.

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.

Mephedrone: Public health risk, mechanisms of action, and behavioral effects.

The recent shortage of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) has led to an increased demand for alternative amphetamine-like drugs such as the synthetic cathinone, 4-methylmethcathinone (mephedrone). Despite the re-classification of mephedrone as a Class B restricted substance by the United Kingdom and restrictive legislation by the United States, international policy regarding mephedrone control is still developing and interest in synthetic amphetamine-like drugs could drive the development of future mephedrone analogues. Currently, there is little literature investigating the mechanism of action and long-term effects of mephedrone. As such, we reviewed the current understanding of amphetamines, cathinones, and cocaine emphasizing the potentially translational aspects to mephedrone, as well as contrasting with the work that has been done specifically on mephedrone in order to present the current state of understanding of mephedrone in terms of its risks, mechanisms, and behavioral effects. Emerging research suggests that while there are structural and behavioral similarities of mephedrone with amphetamine-like compounds, it appears that serotonergic signaling may mediate more of mephedrone's effects unlike the more dopaminergic dependent effects observed in traditional amphetamine-like compounds. As new designer drugs are produced, current and continuing research on mephedrone and other synthetic cathinones should help inform policymakers' decisions regarding the regulation of novel 'legal highs.'