Hadas Reuveni

Lyn Is a Target Gene for Prostate Cancer Sequence-Based Inhibition Induces Regression of Human Tumor Xenografts

The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC50 values of 2–4 μm. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.

Therapeutic destruction of insulin receptor substrates for cancer treatment

Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from insulin-like growth factor 1 receptor (IGF-IR), insulin receptor (IR), and other oncoproteins. IRS1 plays a central role in cancer cell proliferation, its expression is increased in many human malignancies, and its upregulation mediates resistance to anticancer drugs. IRS2 is associated with cancer cell motility and metastasis. Currently, there are no anticancer agents that target IRS1/2. We present new IGF-IR/IRS-targeted agents (NT compounds) that promote inhibitory Ser-phosphorylation and degradation of IRS1 and IRS2. Elimination of IRS1/2 results in long-term inhibition of IRS1/2-mediated signaling. The therapeutic significance of this inhibition in cancer cells was shown while unraveling a novel mechanism of resistance to B-RAF(V600E/K) inhibitors. We found that IRS1 is upregulated in PLX4032-resistant melanoma cells and in cell lines derived from patients whose tumors developed PLX4032 resistance. In both settings, NT compounds led to the elimination of IRS proteins and evoked cell death. Treatment with NT compounds in vivo significantly inhibited the growth of PLX4032-resistant tumors and displayed potent antitumor effects in ovarian and prostate cancers. Our findings offer preclinical proof-of-concept for IRS1/2 inhibitors as cancer therapeutics including PLX4032-resistant melanoma. By the elimination of IRS proteins, such agents should prevent acquisition of resistance to mutated-B-RAF inhibitors and possibly restore drug sensitivity in resistant tumors.

A novel inhibitor of the insulin/IGF signaling pathway protects from age‐onset, neurodegeneration‐linked proteotoxicity

Aging manipulation is an emerging strategy aimed to postpone the manifestation of late‐onset neurodegenerative disorders such as Alzheimers (AD) and Huntingtons diseases (HD) and to slow their progression once emerged. Reducing the activity of the insulin/IGF signaling cascade (IIS), a prominent aging‐regulating pathway, protects worms from proteotoxicity of various aggregative proteins, including the AD‐associated peptide, Aβ‐ and the HD‐linked peptide, polyQ40. Similarly, IGF1 signaling reduction protects mice from AD‐like disease. These discoveries suggest that IIS inhibitors can serve as new drugs for the treatment of neurodegenerative maladies including AD and HD. Here, we report that NT219, a novel IIS inhibitor, mediates a long‐lasting, highly efficient inhibition of this signaling cascade by a dual mechanism; it reduces the autophosphorylation of the IGF1 receptor and directs the insulin receptor substrates 1 and 2 (IRS 1/2) for degradation. NT219 treatment promotes stress resistance and protects nematodes from AD‐ and HD‐associated proteotoxicity without affecting lifespan. Our discoveries strengthen the theme that IIS inhibition has a therapeutic potential as a cure for neurodegenerative maladies and point at NT219 as a promising compound for the treatment of these disorders through a selective manipulation of aging.

Prevention of insulin resistance and beta-cell loss by abrogating PKCε-induced serine phosphorylation of muscle IRS-1 in Psammomys obesus

Psammomys obesus gerbil exhibits PKCε over‐expression on high‐energy (HE) diet. Muscle insulin receptor (IR) signalling and tyrosine kinase activity are inhibited eliciting insulin resistance. We aimed at preventing diabetes by inhibiting PKCε‐induced serine phosphorylation of IRS‐1 with novel PKCε abrogating peptides.

The tyrphostin, NT157, suppresses insulin receptor substrates and augments therapeutic response of prostate cancer

Insulin-like growth factor (IGF) signaling is associated with castrate-resistant prostate cancer (CRPC) progression. Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from IGF1 receptor (IGF1R), insulin receptor, and other oncoproteins. This study demonstrates that IRS1/2 expression is increased in prostate cancer, and persists in CRPC. Furthermore, this study assesses the anticancer activity of NT157, a small molecule tyrphostin targeting IRS proteins, using androgen-responsive (LNCaP) and -independent (PC3) prostate cancer cells in vitro and in vivo. NT157 treatment resulted in dose-dependent inhibition of IGF1R activation, suppression of IRS protein expression, inhibition of IGF1-induced AKT activation, but increased ERK activation in NT157-treated cells in vitro. These effects were correlated with decreased proliferation and increasing apoptosis of LNCaP cells and increasing G2–M arrest in PC3 cells. NT157 also suppressed androgen-responsive growth, delayed CRPC progression of LNCaP xenografts, and suppressed PC3 tumor growth alone and in combination with docetaxel. This study reports the first preclinical proof-of-principle data that this novel small molecule tyrosine kinase inhibitor suppresses IRS1/2 expression, delays CRPC progression, and suppresses growth of CRPC tumors in vitro and in vivo. Demonstration that IRS expression can be increased in response to a variety of stressors that may lead to resistance or reduced effect of the therapies indicate that NT157-mediated IRS1/2 downregulation is a novel therapeutic approach for management of advanced prostate cancer. Mol Cancer Ther; 13(12); 2827–39. ©2014 AACR.

Targeting melanoma with NT157 by blocking Stat3 and IGF1R signaling.

It is well known that specific signal transduction inhibitors rarely suffice as anti-cancer agents. In most cases, tumors possess primary drug resistance due to their inherent heterogeneity, or acquire drug resistance due to genomic instability and acquisition of mutations. Here we expand our previous study of the novel compound, NT157, and show that it acts as a dual-targeting agent that invokes the blockage of two signal transduction pathways that are central to the development and maintenance of multiple human cancers. We show that NT157 targets not only IGF1R-IRS1/2, as previously reported, but also the Stat3 signaling pathway and demonstrates remarkable anti-cancer characteristics in A375 human melanoma cells and in a metastatic melanoma model in mice.

Synthesis and biological activity of semipeptoid farnesyltransferase inhibitors

Semipeptoids derived from the Ras farnesyl transferase inhibitor, CVFM, were synthesized by the Simultaneous Multiple Analogue Peptide Synthesis methodology. The semipeptoids were screened for their in vitro inhibition potency towards farnesyl transferase and geranylgeranyl transferase. Structure-activity relationship studies led to a potent and selective inhibitor, HR-11, which blocks Ras farnesylation in vitro with an IC50 of 1.2 nM. The cell permeable methyl ester derivative of HR-11, HR-12, inhibits Ras farnesylation in intact cells with an IC50 of 10 microM and with no detectable inhibition of Rap1A/K-rev geranyl-geranylation.

The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition

The discovery that the alteration of aging by reducing the activity of the insulin/IGF‐1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration‐linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF‐1 signaling inhibitor, protects model worms from the aggregation of amyloid b peptide and polyglutamine peptides that are linked to the manifestation of Alzheimers and Huntingtons diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as “aggresomes.” NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF‐1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.—Moll, L., Ben‐Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF‐1 signaling promotes proteostasis by enhancing protein aggregation and deposition. FASEB J. 30, 1656–1669 (2016). www.fasebj.org

α-Cyanocinnamide derivatives: a new family of non-peptide, non-sulfhydryl inhibitors of ras farnesylation

Farnesylation of Ras and other proteins is required for their membrane attachment and normal function. Here we report on the synthesis of alpha-cyanocinnamide derivatives, a new family of farnesyltransferase inhibitors. These compounds are nonpeptidic and do not contain sulfhydryl groups. The most potent compound is a pure competitive inhibitor with respect to the Ras protein and mixed competitive with respect to farnesyl diphosphate. Selectivity studies against geranylgeranyltransferase and biological activities of selected compounds are described.

Sequence-based protein kinase inhibition: applications for drug development

Nearly a century ago, Paul Ehrlich proposed his lock and key theory that has remained one of the cornerstones of rational drug development. Disrupting or enhancing the interaction between a ligand and its substrate is the basis for numerous drugs targeting signal transduction processes. Much of drug discovery is based on finding molecular mimics that resemble either the lock or key of a given ligand-substrate pair. The challenge of drug development is the rapid and efficient identification of mimics that are highly selective and specific for the targeted interaction. Scientists associated with Keryx Biopharmaceuticals (New York, NY, USA) have developed two drug discovery technologies, based on molecular mimicry, that combines rapid development of drug candidates and identification of potential biomarker targets for selected indications. KinAce™ is a platform technology for the protein-sequence-based development of small peptide-derived kinase inhibitors developed by Shmuel A. Ben-Sasson at the Hebrew University in Jerusalem and Keryx Biopharmaceuticals (1,2). KinScreen™ is a method for identifying novel target kinases utilizing peptides developed via the KinAce technology. Peptides directed against specific kinases are tested in a series of biomarker assays that serve as in vitro models for a variety of diseases or pathologic processes including cancer, diabetes, angiogenesis, and immune