Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors

Epigenetics in ovarian cancer: premise, properties, and perspectives.

Malignant ovarian tumors bear the highest mortality rate among all gynecological cancers. Both late tumor diagnosis and tolerance to available chemical therapy increase patient mortality. Therefore, it is both urgent and important to identify biomarkers facilitating early identification and novel agents preventing recurrence. Accumulating evidence demonstrates that epigenetic aberrations (particularly histone modifications) are crucial in tumor initiation and development. Histone acetylation and methylation are respectively regulated by acetyltransferases-deacetylases and methyltransferases-demethylases, both of which are implicated in ovarian cancer pathogenesis. In this review, we summarize the most recent discoveries pertaining to ovarian cancer development arising from the imbalance of histone acetylation and methylation, and provide insight into novel therapeutic interventions for the treatment of ovarian carcinoma

Epigenomic Regulation of Androgen Receptor Signaling: Potential Role in Prostate Cancer Therapy.

Androgen receptor (AR) signaling remains the major oncogenic pathway in prostate cancer (PCa). Androgen-deprivation therapy (ADT) is the principle treatment for locally advanced and metastatic disease. However, a significant number of patients acquire treatment resistance leading to castration resistant prostate cancer (CRPC). Epigenetics, the study of heritable and reversible changes in gene expression without alterations in DNA sequences, is a crucial regulatory step in AR signaling. We and others, recently described the technological advance Chem-seq, a method to identify the interaction between a drug and the genome. This has permitted better understanding of the underlying regulatory mechanisms of AR during carcinogenesis and revealed the importance of epigenetic modifiers. In screening for new epigenomic modifiying drugs, we identified SD-70, and found that this demethylase inhibitor is effective in CRPC cells in combination with current therapies. The aim of this review is to explore the role of epigenetic modifications as biomarkers for detection, prognosis, and risk evaluation of PCa. Furthermore, we also provide an update of the recent findings on the epigenetic key processes (DNA methylation, chromatin modifications and alterations in noncoding RNA profiles) involved in AR expression and their possible role as therapeutic targets

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapySiirretty Doriast

Doctor of Philosophy

dissertationCancer is a genomic disease driven by interplay between genetic and epigenetic factors. While genetic mutations are irreversible events, epigenetic regulation is dynamic and reversible, and small molecule blockade of the epigenetic machinery has shown clinical benefit in hematological malignancies. However, the promise of epigenetic therapy has yet to be realized in solid tumors due do limited efficacy and elevated risk of toxicity. Development of potent and specific inhibitors targeting the histone methylation machinery shows promise in tailoring epigenetic therapy for a specific malignancy and decreasing the risk of off-target effects. One such target of interest is the histone lysine-specific demethylase 1 (LSD1). Several solid malignancies show upregulation of LSD1 associated with an aggressive clinical course. Validation of LSD1 as a target has been limited by poorly potent and nonspecific tool compounds, hindering evaluation in in vivo models of disease. This work describes the discovery of a novel potent, specific, and reversible series of LSD1 inhibitors. The identified lead compound, HCI2509, is a noncompetitive inhibitor with nanomolar affinity for LSD1. HCI2509 impaired cell viability across several human cancer cell lines, with both Ewing sarcoma and endometrial cancers showing particularly potent responses. Ewing sarcoma is a rare and aggressive pediatric malignancy characterized by by the chromosomal translocation-derived EWS/ETS fusion proteins. EWS/ETS fusions act iv as oncogenic transcription factors and facilitate cellular reprogramming through the activation of oncogenes and repression of tumor suppressors. Treatment with HCI2509 reverses both EWS/ETS-mediated transcriptional activation and transcriptional repression, and leads to apoptotic cell death in Ewing sarcoma cells. Notably, HCI2509 shows single-agent efficacy in xenograft models of Ewing sarcoma and represents a new therapeutic strategy for this devastating disease. HCI2509 also shows single-agent efficacy in a xenograft model of Type II endometrial carcinoma. Cases of Type II endometrial carcinoma comprise 11% of the incidence and 48% of the deaths due to endometrial cancer annually, such that new therapies are needed for this aggressive subtype. Reversible LSD1 inhibition was associated with tumor regression in an orthotopic model of this disease. These results demonstrate the promise of targeting the histone methylation machinery, specifically LSD1, as a therapeutic strategy for solid tumors

Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors

Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(epsilon)-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(epsilon)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors

Kinetic Analysis and Inhibition Studies of Iron-Dependent Histone Demethylases

The research presented herein focuses on the kinetics and inhibition of the KDM4 subfamily of Jumonji C (JmjC) domain-containing histone demethylases (HDMs). Belonging to the larger class of alpha-ketoglutarate (alpha-KG)-dependent, non-heme iron monooxygenases, the JmjC-HDMs remove methyl groups from mono-, di-, and tri-methylated histone lysine residues through an Fe(IV)-oxo-catalyzed hydroxylation reaction. JmjC-HDMs have been found to play integral roles in the maintenance of genomic integrity as well as in the regulation of transcription. Three KDM4 members were studied: the mixed H3K9/H3K36 demethylases KDM4A and KDM4C, and the pure H3K9 demethylase KDM4E. KDM4C is a hypoxia-inducible factor 1 (HIF-1) target gene and a putative oncogene, while KDM4A is found to be overexpressed at the protein level in multiple cancers. KDM4E is closely related to KDM4D, which has been found to regulate the tumor suppressor p53. Three in vitro enzyme activity assays, including a novel continuous O2 consumption assay, were employed to determine the kinetics of the three HDMs. The kinetic parameters of the three substrates, with an H3K9me3 peptide in place of the full histone substrate, were determined. All three HDMs were found to have low apparent affinities for O2, suggesting that these enzymes may act as O2 sensors in vivo. Alpha-KG was found to inhibit KDM4C competitively with respect to O2, with KDM4C displaying optimal activity in vitro at alpha-KG concentrations similar to those found in cancer cells. Additionally, a 2.1 Å structure of KDM4A in complex with Ni(II) and alpha-KG was solved. Various avenues were explored in the study of JmjC-HDM inhibition. First, a small library of simple primary- and secondary- substrate analogs was synthesized and screened against KDM4E. From this screen, two small molecules were identified as promising candidates for modification in future KDM4 inhibitor design studies. The analogs contained carbon-carbon triple bonds or 1,2,3-triazole groups. We find the triazole-containing compounds to be stronger inhibitors of KDM4E. Several of the alkyne-containing compounds were tested in KDM4-templated Huisgen 1,3-dipolar cycloaddition studies, with no evidence of enzyme-driven triazole formation. Using a peptidomimetic approach, two novel, modified histone H3(7-11) peptides were synthesized and tested for inhibition against KDM4A and KDM4C. Lys residues were modified to incorporate two propargyl groups on the terminal nitrogen, with one of the peptides having an additional methyl group on the terminal nitrogen to make it quaternary. We find enhanced inhibition of KDM4A and KDM4C using the positively charged peptide vs. the neutral peptide. KDM4 members show no hydroxylase or demethylase activity toward the methylated peptide, suggesting that modification of this peptide could lead to enhanced specific inhibition of JmjC-HDMs. No evidence of HDM-driven triazole formation was found for either peptide in studies with KDM4A- and KDM4C- azide adducts. The inhibition of KDM4A with respect to O2 for the pan-selective JmjC-HDM inhibitor JIB-04 was explored. KDM4A inhibition by JIB-04 increases with decreasing O2 concentration, suggesting that JIB-04 may inhibit KDM4A in part by disrupting the binding of O2. JIB-04 isomers were modeled into the KDM4A active site, revealing the predicted basis for JIB-04 isomer-specific inhibition of JmjC-HDMs. Finally, a 3.1 Å structure of KDM4A in complex with JIB-04 was solved, revealing only a fragment of the inhibitor in the enzyme active site

Molecular design of inhibitors for RNA methylation regulating enzymes

The RNA m6A methylation plays crucial role in various physiological processes and therefore the development of chemical agents controlling this process has large biological and medical importance. In the present work, the results of the computational design of the inhibitory ligands for the enzymes regulating the RNA adenosine N6-methylation are presented. The structure of the enzyme-ligand complexes was established using the molecular docking and molecular dynamics approaches. The activity of the best predicted RNA m6A methyltransferase inhibitors was confirmed using enzyme inhibition and cell proliferation assays. These compounds are the first known RNA m6A methylation inhibitors and therefore of substantial interest for further biomedical studies