Boehringer Ingelheim (Austria)

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.

The transcription factor NF-B is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-B in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-. We show here that the IKK-2/IB/NF-B pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-B signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-. Furthermore, inhibition of NF-B activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-B is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-B activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-B during distinct steps of breast cancer progression and suggest that the cooperation of Ras-and TGF--dependent signaling pathways in late-stage tumorigenesis depends critically on NF-B activity.

BACKGROUND: Efficient gene transfer is a major challenge for non-viral gene therapy. Understanding how non-viral vectors initiate gene expression could lead to the development of new future vectors with enhanced efficacy. METHODS: Linear or branched polyethylenimine (PEI)/DNA complexes were generated in varying salt conditions and their transfection efficiencies were compared in vitro and in vivo using reporter genes, luciferase and green fluorescent protein, and rhodamine labeled DNA (pGeneGrip). RESULTS: The transfection efficiency of linear PEI22/DNA in vitro was generally greater than that of branched PEI/DNA when complexes were generated in salt containing buffer. However, PEI complexes generated under salt-free conditions generally had low transfection activity in vitro. In contrast, PEI22/DNA salt-free complexes were highly active in vivo. Branched PEI/DNA and salt containing PEI22/DNA complexes were generally 10-100-fold less active than the salt-free PEI22/DNA complexes. Salt-free PEI22/DNA complexes were small, but subsequently grew into aggregates when salt was added. In contrast, PEI25/DNA complexes remained small even after salt was added under the same conditions. Furthermore, PEI22/pGeneGrips complexes formed large aggregates associated with the cell membrane, cytoplasm and nucleus, while branched PEI complexes remained as small distinct particles associated with the cell membrane or in the cytoplasm. CONCLUSIONS: Branched and linear PEI/DNA complexes differ in their ability to transfect cells. The greater efficiency of linear PEI might be due to an inherent kinetic instability under salt conditions. Understanding how to employ this kinetic instability of linear PEI could help in designing future vectors with greater flexibility and transfection efficiency in vivo.

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.

Abstract KRAS is one of the most commonly mutated proteins in cancer, and efforts to directly inhibit its function have been continuing for decades. The most successful of these has been the development of covalent allele-specific inhibitors that trap KRAS G12C in its inactive conformation and suppress tumour growth in patients 1–7 . Whether inactive-state selective inhibition can be used to therapeutically target non-G12C KRAS mutants remains under investigation. Here we report the discovery and characterization of a non-covalent inhibitor that binds preferentially and with high affinity to the inactive state of KRAS while sparing NRAS and HRAS. Although limited to only a few amino acids, the evolutionary divergence in the GTPase domain of RAS isoforms was sufficient to impart orthosteric and allosteric constraints for KRAS selectivity. The inhibitor blocked nucleotide exchange to prevent the activation of wild-type KRAS and a broad range of KRAS mutants, including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N and A146V/T. Inhibition of downstream signalling and proliferation was restricted to cancer cells harbouring mutant KRAS, and drug treatment suppressed KRAS mutant tumour growth in mice, without having a detrimental effect on animal weight. Our study suggests that most KRAS oncoproteins cycle between an active state and an inactive state in cancer cells and are dependent on nucleotide exchange for activation. Pan-KRAS inhibitors, such as the one described here, have broad therapeutic implications and merit clinical investigation in patients with KRAS-driven cancers.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

BACKGROUND: Catheter ablation of atrial fibrillation is typically performed with uninterrupted anticoagulation with warfarin or interrupted non-vitamin K antagonist oral anticoagulant therapy. Uninterrupted anticoagulation with a non-vitamin K antagonist oral anticoagulant, such as dabigatran, may be safer; however, controlled data are lacking. We investigated the safety of uninterrupted dabigatran versus warfarin in patients undergoing ablation of atrial fibrillation. METHODS: In this randomized, open-label, multicenter, controlled trial with blinded adjudicated end-point assessments, we randomly assigned patients scheduled for catheter ablation of paroxysmal or persistent atrial fibrillation to receive either dabigatran (150 mg twice daily) or warfarin (target international normalized ratio, 2.0 to 3.0). Ablation was performed after 4 to 8 weeks of uninterrupted anticoagulation, which was continued during and for 8 weeks after ablation. The primary end point was the incidence of major bleeding events during and up to 8 weeks after ablation; secondary end points included thromboembolic and other bleeding events. RESULTS: The trial enrolled 704 patients across 104 sites; 635 patients underwent ablation. Baseline characteristics were balanced between treatment groups. The incidence of major bleeding events during and up to 8 weeks after ablation was lower with dabigatran than with warfarin (5 patients [1.6%] vs. 22 patients [6.9%]; absolute risk difference, -5.3 percentage points; 95% confidence interval, -8.4 to -2.2; P<0.001). Dabigatran was associated with fewer periprocedural pericardial tamponades and groin hematomas than warfarin. The two treatment groups had a similar incidence of minor bleeding events. One thromboembolic event occurred in the warfarin group. CONCLUSIONS: In patients undergoing ablation for atrial fibrillation, anticoagulation with uninterrupted dabigatran was associated with fewer bleeding complications than uninterrupted warfarin. (Funded by Boehringer Ingelheim; RE-CIRCUIT ClinicalTrials.gov number, NCT02348723 .).

The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be “undruggable,” between switch I and II on RAS; 1 is mechanistically distinct from covalent KRAS G12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte function-associated Ag-1 (LFA-1), plays an important role in leukocyte-endothelial cell interactions. It is induced by proinflammatory cytokines such as IL-1, TNF-alpha, or IFN-gamma. However, little is known concerning the intracellular regulatory mechanisms which trigger ICAM-1 up-regulation. In order to study potential regulatory elements involved in ICAM-1 induction we have cloned the human ICAM-1 gene and 5 kb of its 5'-regulatory region. The sequence of the cDNA was found to be distributed over seven exons separated by six introns, whereby each of the five extracellular Ig-like domains of ICAM-1 is encoded by its own exon. The upstream sequence harbors a number of sequence motifs implicated in the regulation and expression of eukaryotic genes, including binding sites for the transcription factors SP-1, AP-1, and NF-kB. Primer extension and S1 nuclease analysis revealed two transcription initiation sites 319 bp and 41 bp upstream of the translation start site. Consensus TATA boxes were found at the expected positions about 25 bp upstream of both start sites. Reverse transcriptase polymerase chain reaction showed differential use of the two TATA boxes in A549 and HS913T cells. Both RNA seem to code for the same for of ICAM-1 protein. For regulation studies a 1.3-kb EcoRI/SalI fragment of the 5'-flanking region was used to promote transcription of a linked luciferase reporter gene in transient-transfection assays in A549 and HS913T cells. Treatment of A549 cells with IL-1 or TNF-alpha resulted in a two- or fourfold increase in luciferase activity. Furthermore, a sixfold induction could be achieved after treatment with the phorbol ester PMA. In contrast, agents that increase intracellular cAMP levels did not induce luciferase activity. Northern blot analysis was used to investigate the kinetics of ICAM-1 mRNA synthesis upon induction with TNF-alpha and PMA. These data suggest that the up-regulation of ICAM-1 by cytokines occurs at least partly at the transcriptional level. Deletion analysis of the 1.3-kb fragment of the 5'-flanking region revealed sequences responsible for promotion and inhibition of transcription. In particular, two functionally distinct regions have been characterized: a short fragment containing an NF-kB binding site has been shown to function as an activator, followed immediately downstream by a sequence acting as a silencer element. Therefore, ICAM-1 gene expression seems to be modulated by multiple cis-acting elements.

Abstract KRAS is the most frequently mutated driver of pancreatic, colorectal, and non–small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF–MEK–ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. Significance: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D. See related commentary by Zhao et al., p. 17. This article is highlighted in the In This Issue feature, p. 1

PURPOSE: Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. EXPERIMENTAL DESIGN: The first compound in this series, suitable for i.v. administration, has entered clinical development. To fully explore the potential of Polo-like kinase 1 inhibition in oncology, we have profiled additional compounds and now describe a novel clinical candidate. RESULTS: BI 6727 is a highly potent (enzyme IC(50) = 0.87 nmol/L, EC(50) = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (V(ss) = 7.6 L/kg, t(1/2) = 46 h) and rats (V(ss) = 22 L/kg, t(1/2) = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. CONCLUSION: These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated.

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and Brd4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

BACKGROUND: Efficient and target-specific in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological fluids and extracellular matrix, and binding to a broad variety of non-target cell types. METHODS: Polycation-based vectors, including adenovirus-enhanced transferrinfection (AVET) and transferrin-polyethylenimine (Tf-PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. RESULTS: Gene transfer after intratumoral application was 10-100 fold more efficient with Tf-PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf-PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. CONCLUSION: For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (1Damm K. Hemmann U. Garin-Chesa P. Hauel N. Kauffmann I. Priepke H. Niestroj C. Daiber C. Enenkel B. Guilliard B. Lauritsch I. Müller E. Pascolo E. Sauter G. Pantic M. Martens U.M. Wenz C. Lingner J. Kraut N. Rettig W.J. Schnapp A. EMBO J. 2001; 20: 6958-6968Crossref PubMed Scopus (359) Google Scholar). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase. Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (1Damm K. Hemmann U. Garin-Chesa P. Hauel N. Kauffmann I. Priepke H. Niestroj C. Daiber C. Enenkel B. Guilliard B. Lauritsch I. Müller E. Pascolo E. Sauter G. Pantic M. Martens U.M. Wenz C. Lingner J. Kraut N. Rettig W.J. Schnapp A. EMBO J. 2001; 20: 6958-6968Crossref PubMed Scopus (359) Google Scholar). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase. reverse transcriptase human telomerase reverse transcriptase human telomerase RNA human immunodeficiency virus 2-((E)-3-naphthalen-2-yl-but-2-enoylamino)-benzoic acid telomeric repeat amplification protocol The reactivation of telomerase is a key requisite for human cancer cells to attain an unlimited proliferation potential and is regarded as an essential alteration in the physiology of the tumor cell to acquire malignant growth. (2Hanahan D. Weinberg R.A. Cell. 2002; 100: 57-70Abstract Full Text Full Text PDF Scopus (22628) Google Scholar, 3Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4147) Google Scholar, 4Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. 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Science. 1998; 279: 349-352Crossref PubMed Scopus (4147) Google Scholar), confirming further the role of telomerase in the immortalization process. In contrast, inhibition of telomerase results in telomere-shortening, subsequent growth arrest, and senescence in a wide range of tumor cell lines. This has been demonstrated by expressing a dominant-negative form of telomerase in immortal tumor cell lines (7Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 13: 2388-2399Crossref PubMed Scopus (562) Google Scholar, 8Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (945) Google Scholar) and, pharmacologically, by the use of the small molecule telomerase inhibitor, BIBR1532 (1Damm K. Hemmann U. Garin-Chesa P. Hauel N. Kauffmann I. Priepke H. Niestroj C. Daiber C. Enenkel B. Guilliard B. Lauritsch I. Müller E. Pascolo E. Sauter G. Pantic M. Martens U.M. Wenz C. Lingner J. Kraut N. Rettig W.J. Schnapp A. EMBO J. 2001; 20: 6958-6968Crossref PubMed Scopus (359) Google Scholar). These data underscore that telomerase may represent a valuable target for novel antitumor therapies. Telomerase is a ribonucleoprotein that acts as a reverse transcriptase (RT)1 by using a small region of its RNA subunit, hTR, as a template for the synthesis of telomeric DNA (9Greider C.W. Blackburn E.H. Nature. 1989; 337: 331-337Crossref PubMed Scopus (1316) Google Scholar, 10Harrington L. Zhou W. McPhail T. Oulton R. Yeung D.S. Mar V. Bass M.B. Robinson M.O. Genes Dev. 1997; 11: 3109-3115Crossref PubMed Scopus (410) Google Scholar, 11Morin G.B. Cell. 1989; 59: 521-529Abstract Full Text PDF PubMed Scopus (1377) Google Scholar). Reverse transcription itself is catalyzed by the telomerase protein subunit, hTERT. Since catalytically active telomerase has been assembled from recombinant hTERT protein andin vitro transcribed hTR (12Beattie T.L. Zhou W. Robinson M.O. Harrington L. Curr. Biol. 1998; 8: 177-180Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 13Weinrich S.L. Ma R. Pruzan L. Ouellette M. Tesmer V.M. Holt S.E. Bodnar A.G. Lichtsteiner S. Kim N.W. Trager J.B. Taylor R.D. Carlos R. Andrews W.H. Wright W.E. Shay J.W. Harley C.B. Morin G.B. Nat. Genet. 1997; 17: 498-502Crossref PubMed Scopus (862) Google Scholar), these subunits are regarded as the telomerase core enzyme. In vivo, however, human telomerase exists as a high molecular weight complex with an estimated molecular mass of 1000 KDa (14Ford L.P. Suh J.M. Wright W.E. Shay J.W. Mol. Cell. Biol. 2000; 20: 9084-9091Crossref PubMed Scopus (69) Google Scholar, 15Holt S.E. Aisner D.L. Baur J. Dy V.M. Tesmer M. Ouellette M. Trager J.B. Morin G.B. Toft D.O. Shay J.W. Wright W.E. White M.A. Genes Dev. 1999; 13: 817-826Crossref PubMed Scopus (477) Google Scholar, 16Mitchell J.R. Wood E. Collins K. Nature. 1999; 402: 551-555Crossref PubMed Scopus (911) Google Scholar, 17Schnapp G. Rodi H.P. Rettig W.J. Schnapp A. Damm K. Nucleic Acids Res. 1998; 26: 3311-3313Crossref PubMed Scopus (91) Google Scholar). This large size may be due to the multimeric nature of human telomerase and the association of the telomerase core components, hTERT and hTR, with several telomerase-associated proteins. These diverse proteins may play an important role in telomerase biogenesis, regulation and stability, or may modulate the interaction with telomeres in vivo; however, they are not considered to exert a direct function in catalysis (12Beattie T.L. Zhou W. Robinson M.O. Harrington L. Curr. Biol. 1998; 8: 177-180Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 13Weinrich S.L. Ma R. Pruzan L. Ouellette M. Tesmer V.M. Holt S.E. Bodnar A.G. Lichtsteiner S. Kim N.W. Trager J.B. Taylor R.D. Carlos R. Andrews W.H. Wright W.E. Shay J.W. Harley C.B. Morin G.B. Nat. Genet. 1997; 17: 498-502Crossref PubMed Scopus (862) Google Scholar, 18Bachand F. Autexier C. J. Biol. Chem. 1999; 274: 38027-38031Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In vitro telomerase is able to elongate a short single-stranded DNA in a processive manner by adding multiple TTAGGG repeats to the 3′-end of a suitable DNA primer. Since the enzyme appears to pause after synthesis of each set of six nucleotides representing a single telomeric repeat, a typical pattern of product bands spaced at six-nucleotide intervals is observed. Once the 5′ boundary of the template is copied the DNA substrate is thought to either translocate during processive synthesis, or it may dissociate from the enzyme. Thus, to allow addition of multiple telomeric repeats, translocation and re-initiation must take place after each cycle of template copying. The mechanisms involved are not elucidated yet, but a critical factor could be the dimeric nature of human telomerase with two hTERT and two hTR molecules present per functional telomerase complex (19Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (134) Google Scholar, 20Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (120) Google Scholar). Because of the structural and mechanistic similarity between hTERT and reverse transcriptases, it has been hypothesized that known reverse transcriptase inhibitors may potently inhibit human telomerase. HIV1-RT has been successfully inhibited by nucleoside analogs, which bind to the dNTP binding site (21Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar) and by non-nucleoside inhibitors (NNRTI), which bind to a hydrophobic pocket near the catalytic center resulting in a distortion of the active site (22Gu Z. Li Y. Quan Z. Arts E.J. Wainberg M.A. J. Biol. Chem. 1995; 270: 31046-31051Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1763) Google Scholar, 24Esnouf R. Ren J. Ross C. Jones Y. Stammers D. Stuart D. Nat. Struct. Biol. 1995; 2: 303-308Crossref PubMed Scopus (450) Google Scholar). However, all NNRTI and nucleoside analog inhibitors of HIV1-RT tested were found to be inactive or to exhibit only weak inhibitory activity toward human telomerase (25Fletcher T.M. Salazar M. Chen S.F. Biochemistry. 1996; 35: 15611-15617Crossref PubMed Scopus (77) Google Scholar, 26Strahl C. Blackburn E.H. Mol. Cell. Biol. 1996; 16: 53-65Crossref PubMed Scopus (358) Google Scholar, 27Tendian S.W. Parker W.B. Mol. Pharmacol. 2000; 57: 695-699Crossref PubMed Scopus (38) Google Scholar), suggesting that structural differences between these two families of reverse transcriptases are sufficient to allow specificity of the inhibitors. Additional strategies for inhibition of telomerase have been explored, including antisense approaches directed against hTR (28Bisoffi M. Chakerian A.E. Fore M.L. Bryant J.E. Hernandez J.P. Moyzis R.K. Griffith J.K. Eur. J. Cancer. 1998; 34: 1242-1249Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29Norton J.C. Piatyszek M.A. Wright W.E. Shay J.W. Corey D.R. Nat. Biotechnol. 1996; 14: 615-619Crossref PubMed Scopus (349) Google Scholar), compounds targeting telomeric DNA (30Mergny J.L. Helene C. Nat. Med. 1998; 4: 1366-1367Crossref PubMed Scopus (381) Google Scholar, 31Sun D. Thompson B. Cathers B.E. Salazar M. Kerwin S.M. Trent J.O. Jenkins T.C. Neidle S. Hurley L.H. J. Med. Chem. 1997; 40: 2113-2116Crossref PubMed Scopus (739) Google Scholar), and small molecule drugs (32White L.K. Wright W.E. Shay J.W. Trends Biotechnol. 2001; 19: 114-120Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In the present study, we present the initial characterization of the mode of telomerase inhibition by BIBR1532, a synthetic small molecule inhibitor of human telomerase (1Damm K. Hemmann U. Garin-Chesa P. Hauel N. Kauffmann I. Priepke H. Niestroj C. Daiber C. Enenkel B. Guilliard B. Lauritsch I. Müller E. Pascolo E. Sauter G. Pantic M. Martens U.M. Wenz C. Lingner J. Kraut N. Rettig W.J. Schnapp A. EMBO J. 2001; 20: 6958-6968Crossref PubMed Scopus (359) Google Scholar). Deoxyribonucleotides were from Amersham Biosciences. 1,4-Dithiothreitol was from Roche Diagnostics, and phenylmethylsulfonyl fluoride was from Invitrogen. [α-33P]dCTP (1 mCi/100 μl) was purchased from Hartmann Analytic. The PCR primer forward (tea-fw: 5′-CAT ACT GGC GAC CAG AGT T-3′) and reverse (tea-rev: 5′-GGC GCG CCC TTA CCC TTA CCC TTA CCC TAA-3′) were from Carl Roth GmbH. Crude HeLa nuclear extracts (Computer Cell Culture Center, Seneffe, Belgium) were enriched for telomerase activity with a one-step chromatography on Q-Sepharose column (HiTrap Q HP, Amersham Biosciences). The buffer used was 20 mm Tris-Cl (pH 8.0), 100 μm EGTA, 100 μm EDTA, 1 mmMgCl2, 10% (w/v) glycerol, complemented with different concentrations of KCl (BCE100, 100 mm; BCE250, 250 mm; BCE500, 500 mm; BCE1000, 1 m). The 1-ml column was equilibrated in BCE100 buffer. The following steps were carried at 4 °C. 2 mg of HeLa nuclear extract was diluted in a large volume of BCE100 and loaded twice on the column at 0.5 ml/min. The column was washed at 0.5 ml/min with 4 volumes of BCE100 and 3 volumes of BCE250. Most of the proteins were eluted by washing with 4 volumes of BCE500. Telomerase activity was as 1-ml by with volumes of and against BCE100 500 μm and 250 μm phenylmethylsulfonyl The were for protein with a and for telomerase activity in the N.W. Piatyszek M.A. Harley C.B. Wright W.E. S.L. Shay J.W. Science. PubMed Scopus Google Scholar). Telomerase activity was with hTERT in cells andin vitro transcribed hTERT with hTR was as chromatography with an directed against the hTR (19Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (134) Google Scholar). the direct telomerase with the telomerase, of extract was with different concentrations of BIBR1532 in a volume of 20 on 20 of the was and the was by the to °C. The concentrations in the were mm Tris-Cl (pH 1 mm 1 mm EGTA, 1 mm 1 mm μm mm of and μm Biosciences). the recombinant of telomerase μm were in a volume of mm (pH 1 mm 1 mm mm 1 mm 1 mm μm of Amersham and μm The was by at for 2 and by addition of of in mm Tris-Cl (pH and 20 mm and for 20 at °C. were by adding of in mm Tris-Cl (pH and for a at °C. DNA was by and and the were on an or were to a and the results were a The was as G. Rodi H.P. Rettig W.J. Schnapp A. Damm K. Nucleic Acids Res. 1998; 26: 3311-3313Crossref PubMed Scopus (91) Google Scholar, N.W. Piatyszek M.A. Harley C.B. Wright W.E. S.L. Shay J.W. Science. PubMed Scopus Google Scholar). In BIBR1532 has inhibitory after the telomerase the were each present at μm and the primer at The telomerase and BIBR1532 were for on in buffer. addition of different concentrations of the the was by at for by at for and on with the PCR the was to a of μm for each dNTP and for the DNA primer. The PCR was and PCR was for at at at The of was either by after with acid or on a after on a the of BIBR1532 were carried in in the of concentrations of substrate and were at and the results of were used for further The were with on and the of was by the was against the were as the of substrate to The inhibition were from and and to the of the respectively. BIBR1532 has been identified as a potent and selective inhibitor of human telomerase (1Damm K. Hemmann U. Garin-Chesa P. Hauel N. Kauffmann I. Priepke H. Niestroj C. Daiber C. Enenkel B. Guilliard B. Lauritsch I. Müller E. Pascolo E. Sauter G. Pantic M. Martens U.M. Wenz C. Lingner J. Kraut N. Rettig W.J. Schnapp A. EMBO J. 2001; 20: 6958-6968Crossref PubMed Scopus (359) Google Scholar). a of the of action by compound, the mode of telomerase inhibition was in further using native enzyme enriched from HeLa cell nuclear extract as as recombinant enzyme from recombinant hTERT and in The of BIBR1532 on telomerase activity was using two a G.B. Cell. 1989; 59: 521-529Abstract Full Text PDF PubMed Scopus (1377) Google Scholar) on a direct of enzyme activity and the N.W. Piatyszek M.A. Harley C.B. Wright W.E. S.L. Shay J.W. Science. PubMed Scopus Google Scholar), which an amplification in the native enzyme in the concentrations of BIBR1532 inhibit in a manner of the by a of at concentrations of the inhibitor, the synthesis of appears to be the synthesis of of telomerase activity is in a with the of the primer the synthesis of is inhibited at concentrations of BIBR1532 allow a the of was by and were for each in the for the product which to the two of template is The of the two and is inhibited with of and 100 respectively. BIBR1532 on the telomerase core active telomerase was by cell recombinant hTERT vitro transcribed telomerase were by RNA and the eluted enzyme was tested in the primer in the enzyme catalyzed the of the repeat for human telomerase with the product bands to the of template copying. recombinant telomerase was inhibited by BIBR1532 for the native the of is the of the The which to the cycle of template is only inhibited at concentrations of BIBR1532 1 the of the and to and of template is inhibited with of in the of high concentrations of BIBR1532, the of were observed. Thus, BIBR1532 not inhibit the catalytic steps during a single of template copying. the mode of inhibition by BIBR1532 as a function of the for telomerase and a DNA primer, a of enzyme experiments were The for the of primer were in an initial in the of substrate concentrations and of native telomerase. in a between enzyme and the of telomerase was for for the subsequent experiments between 4 and were and were for different substrate concentrations in the or of in a was telomerase activity was as a function of the DNA primer in the of the inhibitor The was with primer concentrations In the of of BIBR1532, a in was a for a non-competitive the inhibitor tested (1 was by The data were used to the of the DNA primer in the and in the of BIBR1532 and to the binding of the inhibitor in the and in the of the DNA primer. in the and for the DNA primer were not suggesting that BIBR1532 not the binding of the DNA primer to the enzyme. However, a but of was a of BIBR1532 to the enzyme to the primer The and the resulting were for each of the deoxyribonucleotides in the or of BIBR1532 and in for each of the a was in the of BIBR1532, a non-competitive mode of inhibition for the deoxyribonucleotides The for each of the in the of the inhibitor 1 μm BIBR1532, for and to and suggesting for each dNTP a to the The of BIBR1532 were to be for the enzyme and a complex This a binding of the drug to the enzyme and a to each In the these in the of the and the the was is a mixed-type non-competitive inhibitor for the binding of of in of BIBR1532 in the or the of the The in the for the and a in a mixed-type with different binding sites for the and the inhibitor, but with between the binding of each W.C. and of and Scholar). The of the of enzyme by small molecule drug is of as for the drugs (22Gu Z. Li Y. Quan Z. Arts E.J. Wainberg M.A. J. Biol. Chem. 1995; 270: 31046-31051Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1763) Google Scholar). In we show that BIBR1532 telomerase core as telomerase from hTR and recombinant hTERT is inhibited by BIBR1532 with with the native enzyme from tumor In BIBR1532 a non-competitive mode of which is distinct from the inhibition using compounds or antisense for the native and the recombinant enzyme data show that BIBR1532 not but inhibits the of In the inhibitor to an in the of TTAGGG the of six however, is This that BIBR1532 not the catalytic steps involved in template but the of the DNA substrate after its to the of the Thus, BIBR1532 may translocation of the enzyme DNA substrate complex or may between DNA substrate and the enzyme of template copying. these steps are to telomerase, may the high of the In a the of inhibition by BIBR1532 were only a inhibition of the binding of the DNA primer in the of However, BIBR1532 the for binding of deoxyribonucleotides the of the enzyme for This inhibition to a mixed-type non-competitive in which the enzyme has but binding sites for deoxyribonucleotides and may an the binding of the substrate or the drug a of the enzyme interfering with the binding of the the binding site of the drug and the binding site of the deoxyribonucleotides are in or a for the binding The data not an inhibition and we the with telomerase on enzyme and function are known HIV1 reverse transcriptase. The of enzyme is as a the as and with the catalytic center the L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1763) Google Scholar). are important for HIV1-RT The non-nucleosidic drug inhibits the translocation of (22Gu Z. Li Y. Quan Z. Arts E.J. Wainberg M.A. J. Biol. Chem. 1995; 270: 31046-31051Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). experiments that a hydrophobic pocket between the and the of the but not with the DNA binding site L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1763) Google Scholar). of the drug may either of the catalytic or may to the catalytic the similarity between telomerase and HIV1 reverse transcriptase is key in and of catalysis it has been that in that are between and reverse transcriptases or activity in types of or similar on processivity S.L. Ma R. Pruzan L. Ouellette M. Tesmer V.M. Holt S.E. Bodnar A.G. 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Nintedanib (BIBF1120) is a potent, oral, small-molecule tyrosine kinase inhibitor, also known as a triple angiokinase inhibitor, inhibiting three major signaling pathways involved in angiogenesis. Nintedanib targets proangiogenic and pro-fibrotic pathways mediated by the VEGFR family, the fibroblast growth factor receptor (FGFR) family, the platelet-derived growth factor receptor (PDGFR) family, as well as Src and Flt-3 kinases. The compound was identified during a lead optimization program for small-molecule inhibitors of angiogenesis and has since undergone extensive clinical investigation for the treatment of various solid tumors, and in patients with the debilitating lung disease idiopathic pulmonary fibrosis (IPF). Recent clinical evidence from phase III studies has shown that nintedanib has significant efficacy in the treatment of NSCLC, ovarian cancer, and IPF. This review article provides a comprehensive summary of the preclinical and clinical research and development of nintedanib from the initial drug discovery process to the latest available clinical trial data.

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations.

Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol.