Novel selective inhibitors of macropinocytosis-dependent growth in pancreatic ductal carcinoma.

Macropinocytosis is a cellular process that enables cells to engulf extracellular material, such as nutrients, growth factors, and even whole cells. It is involved in several physiological functions as well as pathological conditions. In cancer cells, macropinocytosis plays a crucial role in promoting tumor growth and survival under nutrient-limited conditions. In particular KRAS mutations have been identified as main drivers of macropinocytosis in pancreatic, breast, and non-small cell lung cancers. We performed a high-content screening to identify inhibitors of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC)-derived cells, aiming to prevent nutrient scavenging of PDAC tumors. The screening campaign was conducted in a well-known pancreatic KRAS-mutated cell line (MIAPaCa-2) cultured under nutrient deprivation and using FITC-dextran to precisely quantify macropinocytosis. We assembled a collection of 3584 small molecules, including drugs approved by the Food and Drug Administration (FDA), drug-like molecules against molecular targets, kinase-targeted compounds, and molecules designed to hamper protein-protein interactions. We identified 28 molecules that inhibited macropinocytosis, with potency ranging from 0.4 to 29.9 µM (EC50). A few of them interfered with other endocytic pathways, while 11 compounds did not and were therefore considered specific "bona fide" macropinocytosis inhibitors and further characterized. Four compounds (Ivermectin, Tyrphostin A9, LY2090314, and Pyrvinium Pamoate) selectively hampered nutrient scavenging in KRAS-mutated cancer cells. Their ability to impair albumin-dependent proliferation was replicated both in different 2D cell culture systems and 3D organotypic models. These findings provide a new set of compounds specifically targeting macropinocytosis, which could have therapeutic applications in cancer and infectious diseases.

Discovery of Reversible Inhibitors of KDM1A Efficacious in Acute Myeloid Leukemia Models.

Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-b]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.

Novel potent inhibitors of the histone demethylase KDM1A (LSD1), orally active in a murine promyelocitic leukemia model.

BACKGROUND: Histone lysine demethylases (KDMs) are well-recognized targets in oncology drug discovery. They function at the post-translation level controlling chromatin conformation and gene transcription. KDM1A is a flavin adenine dinucleotide-dependent amine oxidase, overexpressed in several tumor types, including acute myeloid leukemia, neuroblastoma and non-small-cell lung cancer. Among the many known monoamine oxidase inhibitors screened for KDM1A inhibition, tranylcypromine emerged as a moderately active hit, which irreversibly binds to the flavin adenine dinucleotide cofactor. MATERIAL & METHODS: The KDM1A inhibitors 5a-w were synthesized and tested in vitro and in vivo. The biochemical potency was determined, modulation of target in cells was demonstrated on KDM1A-dependent genes and the anti-clonogenic activity was performed in murine acute promyelocytic Leukemia (APL) blasts. An in vivo efficacy experiment was conducted using an established murine promyelocytic leukemia model. RESULTS: We report a new series of tranylcypromine derivatives substituted on the cyclopropyl moiety, endowed with high potency in both biochemical and cellular assays. CONCLUSION: The most interesting derivative (5a) significantly improved survival rate after oral administration in a murine model of promyelocitic leukemia.

Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds.

Tumor resistance to antitubulin drugs resulting from P-glycoprotein (Pgp) drug-efflux activity, increased expression of the ßIII tubulin isotype, and alterations in the drug-binding sites are major obstacles in cancer therapy. Consequently, novel antitubulin drugs that overcome these challenges are of substantial interest. Here, we study a novel chemotype named furan metotica that localizes to the colchicine-binding site in ß-tubulin, inhibits tubulin polymerization, and is not antagonized by Pgp. To elucidate the structure-activity properties of this chiral chemotype, the enantiomers of its most potent member were separated and their absolute configurations determined by X-ray crystallography. Both isomers were active and inhibited all 60 primary cancer cell lines tested at the U.S. National Cancer Institute. They also efficiently killed drug-resistant cancer cells that overexpressed the Pgp drug-efflux pump 10(6)-fold. In vitro, the R-isomer inhibited tubulin polymerization at least 4-fold more potently than the S-isomer, whereas in human cells the difference was 30-fold. Molecular modeling showed that the two isomers bind to ß-tubulin in distinct manners: the R-isomer binds in a colchicine-like mode and the S-isomer in a podophyllotoxin-like fashion. In addition, the dynamic binding trajectory and occupancy state of the R-isomer were energetically more favorable then those of the S-isomer, explaining the observed differences in biologic activities. The ability of a racemic drug to assume the binding modes of two prototypical colchicine-site binders represents a novel mechanistic basis for antitubulin activity and paves the way toward a comprehensive design of novel anticancer agents.

JAM-A promotes neutrophil chemotaxis by controlling integrin internalization and recycling.

The membrane-associated adhesion molecule JAM-A is required for neutrophil infiltration in inflammatory or ischemic tissues. JAM-A expressed in both endothelial cells and neutrophils has such a role, but the mechanism of action remains elusive. Here we show that JAM-A has a cell-autonomous role in neutrophil chemotaxis both in vivo and in vitro, which is independent of the interaction of neutrophils with endothelial cells. On activated neutrophils, JAM-A concentrates in a polarized fashion at the leading edge and uropod. Surprisingly, a significant amount of this protein is internalized in intracellular endosomal-like vesicles where it codistributes with integrin beta1. Clustering of beta1 integrin leads to JAM-A co-clustering, whereas clustering of JAM-A does not induce integrin association. Neutrophils derived from JAM-A-null mice are unable to correctly internalize beta1 integrins upon chemotactic stimuli and this causes impaired uropod retraction and cell motility. Consistently, inhibition of integrin internalization upon treatment with BAPTA-AM induces a comparable phenotype. These data indicate that JAM-A is required for the correct internalization and recycling of integrins during cell migration and might explain why, in its absence, the directional migration of neutrophils towards an inflammatory stimulus is markedly impaired.

Unique role of junctional adhesion molecule-a in maintaining mucosal homeostasis in inflammatory bowel disease.

BACKGROUND & AIMS: Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. METHODS: Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. RESULTS: In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. CONCLUSIONS: Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Junctional adhesion molecule-A-deficient polymorphonuclear cells show reduced diapedesis in peritonitis and heart ischemia-reperfusion injury.

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. Here we report that JAM-A is required for the correct infiltration of polymorphonuclear leukocytes (PMN) into an inflamed peritoneum or in the heart upon ischemia-reperfusion injury. The defect was not observed in mice with an endothelium-restricted deficiency of the protein but was still detectable in mice transplanted with bone marrow from JAM-A(-/-) donors. Microscopic examination of mesenteric and heart microvasculature of JAM-A(-/-) mice showed high numbers of PMN adherent on the endothelium or entrapped between endothelial cells and the basement membrane. In vitro, in the absence of JAM-A, PMN adhered more efficiently to endothelial cells and basement membrane proteins, and their polarized movement was strongly reduced. This paper describes a nonredundant role of JAM-A in controlling PMN diapedesis through the vessel wall.