Thesis title: From Single to Multitargeting Epigenetic Inhibitors: Design, Synthesis and Biological Evaluation
During my PhD period, with the collaboration of Mai research group, I have developed eight main topics concerning the design, synthesis and biochemical/biological evaluation of single target inhibitors of epigenetic players such as DNA methyltransferases (DNMTs), histone methyltransferase (HMT) EZH2 (enhancer of zeste homologue 2), and lysine specific demethylase 1 (LSD1). Moreover, based on the emerging epi-polypharmacology approach in modern cancer therapy,  we designed and synthesized novel multitargeting agents inhibiting at the same time two targets: EZH2 and the histone deacetylases (HDACs), EZH2 and LSD1, the histone acetyltransferase (HAT) p300 and the protein arginine N-methyltransferase 4 (PRMT4), hydroxi-methyl-gluaryl-CoA reductase (HMG-Co-A reductase) and HDACs enzymes.
DNMTs play a relevant role in epigenetic control of cancer cell survival and proliferation.  To date, only two DNMT inhibitors have been approved for hematological cancers treatment and the development of novel potent and specific inhibitors is urgent. Starting from the template of SGI-1027, a non-nucleoside DNMTi effective against cancer we described the design, synthesis, and biological evaluation of a new series of SGI-1029 analogues acting at the same time as DNMTs (mainly DNMT3A) inhibitors and degraders.  From biochemical assay toward human DNMT1 and DNMT3A, bis-quinolines 2a-c and Z-containing 4b and 4c regioisomers emerged as remarkable potent inhibitors of DNMT3A. Tested against leukemic and solid cancer cell lines, 2a–c and 4a–c (the last only for leukemias) displayed up to submicromolar antiproliferative activities. In HCT116 cells, such compounds induced EGFP gene expression in a promoter demethylation assay, confirming their demethylating activity in cells. In the same cell line, 2b and 4c chosen as representative samples induced DNMT1 and DNMT3A protein degradation, suggesting for these compounds a double mechanism of DNMT3A inhibition and DNMT protein degradation.
LSD1 is a FAD-dependent lysine demethylase highly involved in the initiation and the development of cancer.  Several covalent and reversible LSD1 inhibitors are currently in clinical trials, including the well-characterized covalent inhibitor tranylcypromine (TCP). According to the crystal structure analysis, the design of TCP-analogues decorated with long and hindered substituents able to fill the large LSD1 catalytic cleft is an effective strategy to inhibit LSD1 more potently and selectively.  With this aim, we prepared three different series of TCP derivatives, bearing aroyl- and arylacetyl-amino (5a-h), Z-amino acylamino (6a-o), or double-substituted benzamide (7a-n) residues at C4 or C3 position of TCP. Furthermore, we chemically manipulated the TCP scaffold to obtain different fragments (compounds 8a-i) suitable for the design of novel LSD1 inhibitors.  When tested against LSD1, most of 5 and 7 exhibited IC50 values in the low nanomolar range, with 5e and 7a,d,f,g being the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells compounds 7 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines (7a) or against NB4 cells (7c). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 7a,7d and 7g.
Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults.  To date, the sole drug in use against GBM is the alkylating agent temozolomide (TMZ). Among the epigenetic players, EZH2 has been found overexpressed or mutated in gliomas, and its overexpression is associated with poor outcome in GBM.  Starting from the structures of known indole-based EZH2i (such as GSK126, EI1, and CPI-1205), we synthesized a series of pyrrole dimethylpyridone-containing molecules to be tested against EZH2. Among them, compounds 10a and 10b were tested in primary GBM cell cultures.  10a and 10b displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, 10a and 10b displayed stronger, but not additive, effects on cell viability. At the molecular level, 10a and 10b reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with 10a and 10b also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-β, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10.
Since the histone modifying enzymes EZH2 and HDACs synergistically control a number of epigenetic-dependent carcinogenic pathways, we synthesized the first-in-class dual EZH2/HDAC inhibitor 12 by merging the structure of an our optimized EZH2i containing the dimethylpyridone moiety, with the structural motif of the known HDAC inhibitor vorinostat.  In biochemical assay, 12 displayed (sub)micromolar inhibition against both targets. When tested in several cancer cell lines, the hybrid 12 impaired cell viability at low micromolar level and in leukemia U937 and rhabdomyosarcoma RH4 cells provided G1 arrest, apoptotic induction, and increased differentiation, associated with an increase of acetyl-H3 and acetyl-α-tubulin and a decrease of H3K27me3 levels. In glioblastoma U87 cells, 12 hampered epithelial to mesenchymal transition by increasing the E-cadherin expression, thus proposing itself as a useful candidate for anticancer therapy.
Driven by the promising results obtained with compound 12, we designed and synthesized other dual EZH2/HDACs and EZH2/LSD1 inhibitors. In details, we replaced the morpholine moiety of tazemetostat with the HDAC-inhibiting zinc-binding group (ZBG) hydroxamic acid (13a,c) and ortho-aminoanilide (13b) shown in known HDACi (e.g. panobinostat and entinostat, respectively) as well as with tranylcypromine structure (14a,b). Among the dual EZH2/HDAC inhibitors, 13a and 13b showed a 10-fold higher inhibition potency against EZH2 than the single EZH2 inhibitor tazemetostat (IC50 = 0.31 nM) with an IC50 value of 0.032 nM. Concerning the inhibition potency on HDAC isoforms, the most interesting profile was showed by 13a which selectively inhibited HDAC6 (IC50 = 0.016 μM). Biochemical analysis of compound 14a,b revealed IC50 values around low micromolar on both the enzymes EZH2 and LSD1.
Bis-(3-bromo-4-hydroxy)benzylidene cyclic compounds have been reported by us as epigenetic multiple ligands,  but different substitutions at the two wings provided analogues with selective inhibition. Starting from the evidence that the 1-benzyl-3,5-bis((E)-3-bromobenzylidene)piperidin-4-one 17 displayed dual p300/EZH2 inhibition and cancer-selective cell death,  we prepared a series of bis((E)-2-bromobenzylidene) cyclic compounds 18a-n to be tested in biochemical (p300, PCAF, SIRT1/2, EZH2, and CARM1) and cellular (NB4, U937, MCF-7, SH-SY5Y) assays.  The majority of 18a-n exhibited potent dual p300 and CARM1 inhibition, sometimes reaching the submicromolar level, and induction of apoptosis mainly in the tested leukemia cell lines. The most active compounds in both enzyme and cellular assays carried a 4-piperidone moiety and a methyl (18d), benzyl (18e), or acyl (18k-m) substituent at N1 position. Elongation of the benzyl portion to 2-phenylethyl (18f) and 3-phenylpropyl (18g) decreased the potency of compounds at both the enzymatic and cellular levels, but the activity was promptly restored by introduction of a ketone group into the phenylalkyl substituent (18h-j). Western blot analyses performed in NB4 and MCF-7 cells on selected compounds confirmed their inhibition of p300 and CARM1 through decrease of the levels of acetyl-H3 and acetyl-H4 marks for p300 inhibition, and of H3R17me2 mark for CARM1 inhibition.
Very recently, we developed other hybrid compounds. For the design of dual LSD1/HDAC inhibitors, we merged the structure of the LSD1i GSK-2879552 with the HDAC-inhibiting ortho-aminoanilide function (19a-c), also including a substitution at the para position respect to amino function aimed to obtain metabolic stability or selective inhibition for HDAC1 and HDAC2 isoforms.  Additionally, based on recent studies concerning the HMG-CoA reductase involvement in cancer providing the prenylation proteins implications in pro-apoptotic mechanisms,  we developed dual HMG-CoA reductase/HDAC inhibitors starting from the molecular scaffold of atorvastatin, a well-known HMG-CoA reductase inhibitor characterized by a penta-substituted pyrrole. Specifically, we connected the C4 of the pyrrole directly to the hydroxamic acid or the ortho-aminoanilide (“short” derivatives 20a and 20b, respectively), or by using the known spacers as for the derivatives 20c-e. In biochemical assays compounds 19a-c showed a submicromolar potency against LSD1 and a general preferred inhibition of class I HDACs. Among them, compound 19c exhibited selective inhibition against HDAC1 (IC50 = 0.048 µM; SI over HDAC3 = 364.6). Biochemical assays of HMG-CoA reductase revealed that the “short” derivatives 20a,b were completely inactive against HDACs. Differently, the derivative 20c showed an interesting selectivity against HDAC6 and -8, while 20d displayed a typical class I HDACs selectivity profile in single digit nanomolar range. The biochemical IC50 values against HMG-CoA reductase validated these molecules as HMG-CoA reductase inhibitors in the submicromolar range. To date, we have only preliminary biological data showing that the dual LSD1/HDACs inhibitor 19c produced a significant increase in the pre-G1 phase when tested on lung (A549), colon (HCT-116), and breast (MCF7) cancer cell lines. Given the hybrid nature of 19c, we demonstrated that after treatment of MCF7 cells, 19c induced a strong increase of ac-H3 levels (mark for HDAC inhibition), and an increase of H3K4me2 (mark for LSD1 inhibition). Finally, we detected the effects of 19c on ER-α expression levels. As expected, 19c proved a significant dose-dependent decrease of also ERα levels. Other investigations on 19c to better characterize the molecular mechanism of its anticancer activity are in progress.
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