A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.

Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3.

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Enantioselective synthesis and biological investigation of tetrahydro-ß-carboline-based HDAC6 inhibitors with improved solubility.

Aberrant epigenetic changes in DNA methylation and histone modification by acetylation or deacetylation regulate the pathogenesis of many diseases. Especially selective inhibitors are getting more and more attention. We recently reported on a new class of potent and selective anti-inflammatory and antirheumatic histone deacetylase 6 (HDAC6) inhibitors (e.g., Marbostat-100). The attachment of a morpholinoethoxy part to the head group dramatically enhances the solubility, in particular the solubility in aqueous solutions, of the lead compound Marbostat-100. Here, we present the enantioselective synthesis of small-molecule compounds based on the tetrahydro-ß-carboline core system with improved solubility, and the influence of the stereochemistry on the biological activity. The enantiomers were synthesized in good enantiomeric excess (ee) purity and were potent and selective HDAC6 inhibitors, whereas the S-derivative S-21 is clearly the eutomer. The potency of our selective HDAC6 inhibitors is demonstrated by Ki values in the range of 0.5-2 nM toward HDAC6, and the selectivity was proved in cellular assays by Western blot analysis taking ac-tubulin as surrogate parameter.

Analysis of the interplay between all-trans retinoic acid and histone deacetylase inhibitors in leukemic cells.

The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.

Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones.

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure led to a series of novel compounds. These compounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in intact A431 cells was shown, with IC(50) values ranging form 0.3-1µM for compound 42, and 0.1-0.3µM for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel compounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib and erlotinib (Tarceva®).

Identification of a highly efficient dual type I/II FMS-like tyrosine kinase inhibitor that disrupts the growth of leukemic cells.

Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.

A series of novel aryl-methanone derivatives as inhibitors of FMS-like tyrosine kinase 3 (FLT3) in FLT3-ITD-positive acute myeloid leukemia.

Mutants of the FLT3 receptor tyrosine kinase (RTK) with duplications in the juxtamembrane domain (FLT3-ITD) act as drivers of acute myeloid leukemia (AML). Potent tyrosine kinase inhibitors (TKi) of FLT3-ITD entered clinical trials and showed a promising, but transient success due to the occurrence of secondary drug-resistant AML clones. A further caveat of drugs targeting FLT3-ITD is the co-targeting of other RTKs which are required for normal hematopoiesis. This is observed quite frequently. Therefore, novel drugs are necessary to treat AML effectively and safely. Recently bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. In order to optimize these agents we synthesized novel derivatives of these methanones with various substituents. Methanone 16 and its carbamate derivative 17b inhibit FLT3-ITD at least as potently as the TKi AC220 (quizartinib). Models indicate corresponding interactions of 16 and quizartinib with FLT3. The activity of 16 is accompanied by a high selectivity for FLT3-ITD.

HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of ß-catenin, MYC, and WT1.

Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors ß-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of ß-catenin. Indomethacin destabilizes ß-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of ß-catenin by WT1. In conclusion, reduced levels of ß-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.

Human platelet lysate as validated replacement for animal serum to assess chemosensitivity.

Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used media supplement worldwide. FCS contains a mixture of largely undefined growth factors and cytokines, which support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesired immune responses, possible contaminations, and because of its way of production an ethical concern. Thus, alternative, defined, valid, and reliable media supplements should be characterized in a large number of experiments. Human platelet lysate (hPL) is increasingly appreciated as an alternative to FCS. Since it is unclear whether cells respond differentially to clinically relevant chemotherapeutics inducing replicative stress and DNA damage (Hydroxyurea, Irinotecan), induction of reactive oxygen species (ROS), the tyrosine kinase inhibitor (TKi) Imatinib, and novel epigenetic modifiers belonging to the group of histone deacetylase inhibitors (HDACi), we investigated these issues. Here we show that cancer cells derived from leukemia and colon cancer grow very similarly in culture media with FCS or outdated hPL. Notably, cells have practically identical proteomes under both culture conditions. Moreover, cells grown with FCS or hPL respond equally to all types of drugs and stress conditions that we have tested. In addition, the transfection of blood cells by electroporation can be achieved under both conditions. Furthermore, we reveal that class I HDACs, but not HDAC6, are required for the expression of the pan-leukemic marker WT1 under various culture conditions. Hence, hPL is a moderately priced substitute for FCS in various experimental settings.

Inhibition of PDGFR tyrosine kinase activity by a series of novel N-(3-(4-(pyridin-3-yl)-1H-imidazol-2-ylamino)phenyl)amides: a SAR study on the bioisosterism of pyrimidine and imidazole.

A series of N-(3-(4-(pyridin-3-yl)-1H-imidazol-2-ylamino)phenyl)amides were synthesized and tested for inhibition of PDGFR and FLT3 autophosphorylation. The novel N-(3-(4-(pyridin-3-yl)-1H-imidazol-2-ylamino)phenyl)amides, obtained by replacement of the pyrimidine system in Imatinib (1) with an imidazole ring, exhibit potent inhibitory activity on PDGFR, similar to the parent compound (IC(50) (9e)=0.2 microM; IC(50) Imatinib (1)=0.3 microM). Selectivity hereby seems to be conserved, as shown by the lack of activity on FLT3, a closely related class III receptor tyrosine kinase, which is not affected by the parent compound Imatinib.

Antibacterial activity of a novel series of 3-bromo-4-(1H-3-indolyl)-2,5-dihydro-1H-2,5-pyrroledione derivatives--an extended structure-activity relationship study.

Compounds containing 3-bromo-2,5-dihydro-1H-2,5-pyrroledione and indole substructures were found to have antibacterial activity against resistant strains of Staphylococcus aureus and some other Gram positive bacteria. The investigated compounds exhibit minimal inhibition concentrations (MICs) lower than those of common antibiotics like vancomycin or ciprofloxacin. Activity against multiresistant strains suggests a mechanism of action different from common antibiotics. This might be important in circumventing existing resistance mechanisms. Here we report about the antibacterial activity in an extended structure-activity relationship study.

Design and biological evaluation of tetrahydro-ß-carboline derivatives as highly potent histone deacetylase 6 (HDAC6) inhibitors.

Various diseases are related to epigenetic modifications. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, and thus are involved in the regulation of gene expression. First generation histone deacetylase inhibitors (HDACi) are unselective, hinder all different kinds of zinc dependent HDACs and additionally cause several side effects. Subsequently, selective HDACi are gaining more and more interest. Especially, selective histone deacetylase 6 inhibitors (HDAC6i) are supposed to be less toxic. Here we present a successful optimization study of tubastatin A, the synthesis and biological evaluation of new inhibitors based on hydroxamic acids linked to various tetrahydro-ß-carboline derivatives. The potency of our selective HDAC6 inhibitors, exhibiting IC50 values in a range of 1-10 nM towards HDAC6, was evaluated with the help of a recombinant human HDAC6 enzyme assay. Selectivity was proofed in cellular assays by the hyperacetylation of surrogate parameter α-tubulin in the absence of acetylated histone H3 analyzed by Western Blot. We show that all synthesized compounds, with varies modifications of the rigid cap group, were selective and potent HDAC6 inhibitors.

Marbostat-100 Defines a New Class of Potent and Selective Antiinflammatory and Antirheumatic Histone Deacetylase 6 Inhibitors.

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.

2-aroylindoles and 2-aroylbenzofurans with N-hydroxyacrylamide substructures as a novel series of rationally designed histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors are considered to be drugs for targeted cancer therapy and second-generation HDIs are currently being tested in clinical trials. Here, we report on the synthesis and biological evaluation of a novel HDAC inhibitor scaffold with the hydroxamate Zn(2+) complexing headgroup, selected from the 2-aroylindol motif. Inhibition of nuclear extract HDAC and recombinant HDAC 1 as well as induction of histone H3K(9+14) hyperacetylation mediated by E-N-hydroxy-(2-aroylindole)acrylamides or E-N-hydroxy-(2-aroylbenzofuran)acrylamides were studied. Moreover, the cytotoxic activity, the effects on the cell cycle, and histone H3S(10) phosphorylation of selected compounds were determined. By use of a panel of 24 different human tumor cell lines, mean IC(50) values of the most potent analogues 6c and 7b were 0.75 and 0.65 microM, respectively. The novel compounds were shown to be no substrates of the P-glycoprotein drug transporter. Comparable to N(1)-hydroxy-N(8)-phenyloctanediamide "2 (SAHA)", cells in the S phase of the cell cycle are depleted, with partial arrest in G1 and G2/M and finally induction of massive apoptosis.

Generation and Assessment of Fusions Between HDACi and TKi.

Chimeric compounds combine the structural features of inhibitors of histone deacetylases (HDACi) and tyrosine kinase inhibitors (TKi), and therefore unite the effects of a dual-targeting strategy in one compound. Here, we describe the generation of such hybrid molecules. Small molecules, known as TKi, are combined with a Zn2+ chelating motive, preferentially a hydroxamic acid, in addition. The resulting small molecules also can inhibit histone deacetylases, which are dependent on the catalytically active Zn2+. Moreover, we summarize how the growth-inhibitory effects of these combined compounds can be determined with a simple proliferation assay with a leukemic cell line.

HSP90 is necessary for the ACK1-dependent phosphorylation of STAT1 and STAT3.

Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT1 and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90α/ß). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT1 and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis.

Class I histone deacetylases regulate p53/NF-κB crosstalk in cancer cells.

The transcription factors NF-κB and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytokines promote signaling cascades that lead to the co-regulation of p53 and NF-κB. Consequently, nuclear p53/NF-κB signaling complexes activate NF-κB-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-κB, we addressed whether clinically relevant HDACi affect the NF-κB/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-κB-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class IIb deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-κB. HDACi decrease the basal and stress-induced expression of p53 and block NF-κB-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53.

[4-(imidazol-1-yl)thiazol-2-yl]phenylamines. A novel class of highly potent colchicine site binding tubulin inhibitors: synthesis and cytotoxic activity on selected human cancer cell lines.

Synthesis and cytotoxic activity in the submicromolar range of a series of [4-(imidazol-1-yl)thiazol-2-yl]phenylamines are described. Cell cycle dependent cytotoxicity on RKO human colon carcinoma cells with inducible expression of p27(kip1) and the influence on microtubule formation were investigated. Considering the significant correlation between the IC(50) values of tubulin polymerization inhibition, [(3)H]colchicine competition, and cytotoxicity of the investigated compounds, tubulin is the main cellular target. The inhibition of microtubule formation was shown to be mediated by interference with the colchicine binding site of tubulin. In depth analysis of the investigated compounds allowed the identification of modifications that altered the pharmacological profile of the compounds from a mitosis-inducing phenotype to a G1 cell cycle arresting phenotype.

Novel bis(1H-indol-2-yl)methanones as potent inhibitors of FLT3 and platelet-derived growth factor receptor tyrosine kinase.

FLT3 receptor tyrosine kinase is aberrantly active in many cases of acute myeloid leukemia (AML). Recently, bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. To optimize FLT3 activity and selectivity, 35 novel derivatives were synthesized and tested for inhibition of FLT3 and PDGFR autophosphorylation. The most potent FLT3 inhibitors 98 and 102 show IC50 values of 0.06 and 0.04 microM, respectively, and 1 order of magnitude lower PDGFR inhibiting activity. The derivatives 76 and 82 are 20- to 40-fold PDGFR selective. Docking at the recent FLT3 structure suggests a bidentate binding mode with the backbone of Cys-694. Activity and selectivity can be related to interactions of one indole moiety with a hydrophobic pocket including Phe-691, the only different binding site residue (PDGFR Thr-681). Compound 102 inhibited the proliferation of 32D cells expressing wildtype FLT3 or FLT3-ITD similarly as FLT3 autophosphorylation, and induced apoptosis in primary AML patient blasts.