Cellular Antiproliferative Effects Research Articles

Discovery of a potent and selective JAK1-targeting PROTAC degrader with anti-tumor activities

Aberrant activation of the JAK-STAT pathway is evident in various human diseases including cancers. Proteolysis targeting chimeras (PROTACs) provide an attractive strategy for developing novel JAK-targeting drugs. Herein, a series of CRBN-directed JAK-targeting PROTACs were designed and synthesized utilizing a JAK1/JAK2 dual inhibitor—momelotinib as the warhead. The most promising compound 10c exhibited both good enzymatic potency and cellular antiproliferative effects. Western blot analysis revealed that compound 10c effectively and selectively degraded JAK1 in a proteasome-dependent manner (DC50 = 214 nM). Moreover, PROTAC 10c significantly suppressed JAK1 and its key downstream signaling. Together, compound 10c may serve as a novel lead compound for antitumor drug discovery.

Bioreducible Micelles Self-Assembled from Poly(ethylene glycol)-Cholesteryl Conjugate As a Drug Delivery Platform

The ability of polymeric micelles to self-assemble into nanosized particles has created interest in their application as potential anticancer drug delivery systems. A poly(ethylene glycol)-cholesteryl conjugate (Chol-ss-PEG-ss-Chol) connected by cleavable disulfide linkages was synthesized and used as a nanocarrier for in vitro release of doxorubicin (DOX). Owing to its amphiphilic structure, Chol-ss-PEG-ss-Chol was able to self-assemble into micelles with an average diameter 18.6 nm in aqueous solution. The micelles formed large aggregates due to the shedding of the PEG shell through cleavage of disulfide bonds in a reductive environment. The in vitro release studies revealed that Chol-ss-PEG-ss-Chol micelles released 80% and approximately 9% of the encapsulated DOX within 6 h under reductive and non-reductive conditions, respectively. The glutathione (GSH)-mediated intracellular drug delivery was investigated in a KB cell line. The cytotoxicity of DOX-loaded micelles indicated a higher cellular anti-proliferative effect against GSH-pretreated than untreated KB cells. Furthermore, confocal laser scanning microscopy (CLSM) measurement demonstrated that Chol-ss-PEG-ss-Chol micelles exhibited faster drug release in GSH-pretreated KB cells than untreated KB cells. These results suggest the potential usefulness of disulfide-based polymeric micelles as controlled drug delivery carriers.

Disruption of the Ikaros-Mediated Gene Expression Program in Multiple Myeloma with Immunomodulatory Agents

The close chemical analogues lenalidomide and pomalidomide are immunomodulating drugs (IMiDs) that possess antineoplastic activity in multiple myeloma (MM) and other hematologic malignancies. IMiDs exert intrinsic antiproliferative effects on MM cells at least in part through direct interaction with intracellular cereblon. Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex that also includes CUL4, RBX1, and DDB1 – together referred to as CRL4CRBN. Direct binding of IMiDs to a small hydrophobic pocket of the CRBN carboxy-terminal domain induces altered ubiquitinylation activity, including disrupted autoubiquitinylation of CRL4CRBN (Ito et al. Science, 2010; Fischer et al. Nature, 2014). Recently, we and others described the zinc finger transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) as endogenous targets of CRL4CRBN ubiquitinylation in MM cells (Lu et al. Science, 2014; Kronke et al. Science, 2014). We found that treatment of MM cells with lenalidomide (LEN) causes increased ubiquitinylation of IKZF1 and IKZF3 by CRL4CRBN, and targets them for degradation by the proteasome. This loss of IKZF1/3 was both necessary and sufficient to observe the cellular antiproliferative effects of LEN, suggesting that this a primary mechanism of IMiD activity in MM. However, it remains unclear how depletion of these transcription factors ultimately leads to reduced proliferation of myeloma cells. Several gene products are known to be perturbed in MM cells upon IMiD treatment, including transcriptional downregulation of the master regulator transcription factor IRF4 (Zhu et al. Blood, 2011; Lu et al. Science, 2014). Yet rescue experiments with exogenous expression of IRF4 cannot fully rescue effects of IMiD treatment in MM cells, suggesting that other IKZF1/3 target genes play a role in the antiproliferative effects of IMiDs. Here we use complementary gene expression and genomic approaches to discern the global effects of IMiD treatment on MM cells. Using chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we have determined the genome-wide binding profile of both IKZF1 and IKZF3 in MM cells. We find both IKZF1 and IKZF3 occupy genomic regions including promoters, gene bodies, and distal enhancer elements. In MM cells, IKZF1 and IKZF3 enrichment almost always co-occurs, corresponding to reports of IKZF1/3 heterodimers facilitating transcriptional programs in lymphoid cells (Morgan et al. EMBO, 1997). Treatment of MM cells with LEN results in a dramatic decrease of both IKZF1 and IKZF3 binding to the genome at promoters and enhancers. Additionally, we performed ChIP-seq on RNA polymerase II (RNAPII) after LEN treatment in order to determine how IMiD-mediated IKZF1/3 depletion affects cellular transcriptional activity. Loss of IKZF1/3 binding to target genes most often correlates with increased density of RNAPII in gene bodies, suggesting transcriptional derepression of IKZF1/3 target genes. This observation was confirmed with genome-wide expression analysis by microarray. Among the derepressed target genes were genes with known tumor suppressor activity including CDKN1A, KLF6, and TXNIP. The IRF4 locus was also found to be a direct target of IKZF1/3, including binding to a large distal enhancer region upstream of the IRF4 coding region. Yet unlike most other IKZF1/3 target genes, RNAPII density within the IRF4 gene body is significantly decreased upon LEN treatment, suggesting that unique transcriptional regulatory mechanisms function at this locus that are distinct from other IKZF1/3 targets. Knockdown of IKZF1 and IKZF3 expression by shRNAs results in increased TXNIP and decreased IRF4 mRNA and protein expression, further suggesting that IMiD-mediated degradation of IKZF1/3 leads to opposite effects on these genes. TXNIP encodes the thioredoxin binding protein, which increases cellular reactive oxygen species and promotes G0/G1 cell cycle arrest. Forced exogenous overexpression of TXNIP inhibits MM cell growth, indicating a potential additional mechanism of IMiD activity. These studies define the IKZF1/3-mediated transcription program in MM cells and detail its perturbation by IMiDs. Ultimately these data will be informative for understanding the downstream effectors of intrinsic IMiD activity in hematologic malignancies, and to further understand mechanisms for acquired or innate resistance to these therapies. DisclosuresNo relevant conflicts of interest to declare.

Dual-acting agents that possess reversing resistance and anticancer activities: Design, synthesis, MES-SA/Dx5 cell assay, and SAR of Benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6 H-imidazo[3′,4′:1,2]pyridin[3,4- b]indol-2-substitutedacetates

Based on the structural analysis of fumitremorgin C (FTC), imidazoline and β-carboline amino acid benzylester, 14 novel 2-substitutedtetracyclic derivatives of tetrahydrocarboline 4a– n were prepared. We demonstrated that the exposure of MES-SA/Dx5 cells to some of 4a– n resulted in significant reduction of resistance of the cells against doxorubicin. This reduced resistance was accompanied by lowering of IC 50 value to doxorubicin from 1.55 ± 0.26 μmol/L to 0.33 ± 0.05 μmol/L for 2-(2-butyl)-derivative 4c, to 1.03 ± 0.22 μmol/L for 2-methyl-derivative 4d, to 0.46 ± 0.04 μmol/L for 2-benzyl-derivative 4f, to 0.98 ± 0.25 μmol/L for 2-indole-3-yl-methyl-derivative 4h, to 0.36 ± 0.03 μmol/L for 2-benzyloxycarbonylmethyl-derivative 4i, to 0.77 ± 0.08 μmol/L for 2-benzyloxycarbonylethyl-derivative 4j, and to 0.77 ± 0.08 μmol/L for 2-benzyloxycarbonylamino- n-butyl-derivative 4l. Proliferation assays of 4a– n indicated 4c, f, i, j were able to inhibit the proliferation of doxorubicin resistant MES-SA/Dx5 cells. The SAR analysis revealed that the benzylester form and the tetracyclic structure of 4a– n were critical for both sensitizing doxorubicin and the cellular anti-proliferative effect.

Natriuretic Peptides

natriuretic effects. They were first identified in 1981 by the pivotal work of de Bold and colleagues. In the 25 years elapsed since the initial discovery, an impressive amount of research has been carried out to identify the structure of the active peptide and its receptors, to characterise the biological functions of these molecules and their involvement in the pathophysiology of diseases and, finally, to identify their direct contributory role in the pathogenesis of some cardiovascular disorders. NPs include different biologically active fragments: the atrial natriuretic peptide (ANP), the brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). Extensive research has identified some interesting features of ANP and BNP. In particular, it has been demonstrated that the ANP plays a role in the regulation of salt and water balance, as well as that of blood pressure homeostasis. In addition, ANP is involved in the pathophysiology of hypertension and heart failure, and it exerts a cellular antiproliferative effect in the cardiovascular system. More recently, a direct contributory role of ANP in the development of hypertension and cerebrovascular disorders has been demonstrated. On the other hand, BNP has a key role in congestive heart failure, particularly because of its distinct enhancement in terms of myocardial expression and production. These observations have progressively led researchers to consider ANP as a potential determinant of cardiovascular diseases and qualified BNP as a sensitive and specific marker of cardiac dysfunction. In this article, the most recently pathophysiological properties of NPs are reviewed, with particular regard to the most intriguing new hypothesis on their pathological and clinical significance in different cardiovascular

The atrial natriuretic peptide: a changing view

The atrial natriuretic peptide (ANP), a component of the natriuretic peptide family, was discovered in 1981 when de Bold and his coworkers observed a natriuretic effect induced by infusion of atrial extracts in rats. Subsequently, an impressive amount of research has been carried out in order to identify the structure of the active peptide and its receptors, to characterize the biological functions of ANP and its involvement in the pathophysiology of diseases and, finally, its direct contributory role in the pathogenesis of some cardiovascular disorders. ANP plays a key role in the regulation of salt and water balance, as well as of blood pressure homeostasis. In addition, ANP is involved in the pathophysiology of hypertension and heart failure, and exerts a cellular antiproliferative effect in the cardiovascular system. More recently, a direct contributory role of ANP in the development of hypertension and of cerebrovascular disorders has been suggested by the use of molecular genetic approaches. Therefore, our understanding of the pathophysiologic relevance of ANP has changed over time, finally leading to the identification of ANP as a potential determinant of cardiovascular diseases, rather than as a simple marker of cardiac and vascular dysfunctions. This novel view of ANP may open interesting research pathways.

Synergistic Inhibition of HIV-1 in Activated and Resting Peripheral Blood Mononuclear Cells, Monocyte-Derived Macrophages, and Selected Drug-Resistant Isolates With Nucleoside Analogues Combined With a Natural Product, Resveratrol

Resveratrol (trans-3,5,4′-trihydroxystilbene) is a phytoalexin present in grapes, wine, and certain plants, which has recently been reported to possess properties that may protect against atherosclerosis, certain cancers, and inflammation. We now report that resveratrol (RV) synergistically enhances the anti-HIV-1 activity of the nucleoside analogues zidovudine (AZT), zalcitabine (ddC), and didanosine (ddl). RV at 10 μM was not toxic to cells, and by itself reduced viral replication by 20% to 30%. In phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs) infected with HTLV-IIIB, 10 μM RV reduced the 90 % inhibitory concentration (IC90) of AZT, ddC, and ddI by 3.5-, 5.5-, and 17.8-fold, respectively. Similar antiviral activity was demonstrated when ddI was combined with 5 or 10 mM RV in PBMCs infected with clinical isolates of HIV-1. The addition of RV resulted in a > 10-fold augmentation of ddI-antiviral activity in infected monocyte-derived macrophages (MDMs). In a resting cell model of T lymphocytes which were infected with HTLV-IIIB, RV plus ddI in combination, but not individually, suppressed establishment of a productive viral infection. In addition, RV plus ddI markedly inhibited the replication of four ddI-resistant viral isolates, three of which presented mutations in the RT gene conferring RT-multidrug resistance. Finally, when compared with hydroxyurea (HU), both 100 mM HU and 10 mM RV showed similar enhancement of ddI-antiviral suppressive activity. However, RV was shown to have less of a cellular antiproliferative effect than HU.

Mechanistic Studies on the Inactivation of the Proteasome by Lactacystin in Cultured Cells

The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.